Janis M Burt

Interests

Research

Research Interests Research in my laboratory is focused on the mechanisms underlying coordinated function of the heart and blood vessels by connexin proteins. These proteins form channels between the interiors of neighboring cells (gap junction channels) in virtually all tissues of the body, thereby providing the cells a pathway for exchange of information (communication) that supports coordinated tissue function in health, disease and injury. Compromised function of this intercellular communication pathway can lead to fatal cardiac arrhythmias, failure to provide tissues with adequate blood flow, and loss of growth control (leading to cancer and poor tissue recovery from injury). In ongoing studies we are testing the general hypothesis that phosphorylation-dependent and connexin-specific differences in 1) the permselective and gating properties of gap junction channels and 2) interactions with other proteins provides cells of the CV system strategies for maintaining coordinated contraction/relaxation functions while simultaneously providing them a “molecular switch” that supports their transition between the growth and quiescent states necessary for tissue development and remodeling.

Courses

Scholarly Contributions

Journals/Publications

• Burt, J. M., Langlais, P. R., Pontifex, T. K., Jacobsen, N. L., & Taylor, S. Z. (2020). Phosphorylation at serine 319 of Cx37 is both necessary and sufficient to induce a protein conformation that arrests cell cycling. Journal of Cell Science.
• Burt, J. M., Taylor, S. Z., Jacobsen, N. L., Pontifex, T. K., & Langlais, P. R. (2020). Serine 319 phosphorylation is necessary and sufficient to induce a Cx37 conformation that leads to arrested cell cycling.. J Cell Sci., 133. doi:doi:10.1242/jcs.240721
• Pontifex, T. K., Fye, M., Burt, J. M., & Alamban, A. (2020). Alternately Translated Product of Cx37 Fails to Arrest Growth in Rin Cells but Alters Cx37 Hemichannel Function when Co‐Expressed. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.04153
• Taylor, S. Z., Jacobsen, N. L., Jacobsen, N. L., Pontifex, T. K., Burt, J. M., & Taylor, S. Z. (2020). Connexin 37 Growth Suppressive Phenotype is Regulated in a Site-Specific, Phosphorylation-Dependent Manner. Journal of Cell Science, 133.
• Burt, J. M., Langlais, P. R., Pontifex, T. K., & Jacobsen, N. L. (2019). Phosphorylation-dependent intra-domain interaction of the Cx37 carboxyl-terminus controls cell survival. Cancers.
• Cotter, M. L., Boitano, S., Lampe, P. D., Solan, J. L., Vagner, J., Ek-Vitorin, J. F., & Burt, J. M. (2019). The lipidated connexin mimetic peptide SRPTEKT- is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform. American journal of physiology. Cell physiology, 317(4), C825-C842.
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 µM, 1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl () lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca-wave propagation (IC 64.8 pM) and HCh-mediated dye uptake (IC 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT- inhibition of Ca-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT- potently inhibited GJCh-mediated Ca-wave propagation (IC 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca-wave propagation, dye coupling, and dye uptake were largely unaffected (IC 12.3 μM) by SRPTEKT- in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT- is a potent inhibitor of physiological Ca-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
• Cotter, M. L., Cotter, M. L., Boitano, S. A., Boitano, S. A., Lampe, P. D., Lampe, P. D., Solan, J. L., Solan, J. L., Vagner, J., Vagner, J., Ek Vitorin, J. F., Ek Vitorin, J. F., Burt, J. M., & Burt, J. M. (2019). The lipidated connexin mimetic peptide SRPTEKT-Hdc is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho-isoform. Am J Physiol Cell Physiol, 317(4), C825-C842.
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  Connexin (Cx) mimetic peptides derived from extracellular loop II sequences (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) have been used as reversible, Cx-specific blockers of hemichannel (HCh) and gap junction channel (GJCh) function. These blockers typically require high concentrations (~5 microM, 1 h for GJCh) to achieve inhibition. We have shown that addition of a hexadecyl (Hdc) lipid tail to the conserved SRPTEKT peptide sequence (SRPTEKT-Hdc) results in a novel, highly efficacious, and potent inhibitor of mechanically induced Ca(2+)-wave propagation (IC50 64.8 pM) and HCh-mediated dye uptake (IC50 45.0 pM) in Madin-Darby canine kidney cells expressing rat Cx43 (MDCK43). The lack of similar effect on dye coupling (NBD-MTMA) suggested channel conformation-specific inhibition. Here we report that SRPTEKT-Hdc inhibition of Ca(2+)-wave propagation, dye coupling, and dye uptake depended on the functional configuration of Cx43 as determined by phosphorylation at serine 368 (S368). Ca(2+)-wave propagation was enhanced in MDCK cells expressing single-site mutants of Cx43 that mimicked (MDCK43-S368D) or favored (MDCK43-S365A) phosphorylation at S368. Furthermore, SRPTEKT-Hdc potently inhibited GJCh-mediated Ca(2+)-wave propagation (IC50 230.4 pM), dye coupling, and HCh-mediated dye uptake in MDCK43-S368D and -S365A cells. In contrast, Ca(2+)-wave propagation, dye coupling, and dye uptake were largely unaffected (IC50 12.3 muM) by SRPTEKT-Hdc in MDCK43-S368A and -S365D cells, mutations that mimic or favor dephosphorylation at S368. Together, these data indicate that SRPTEKT-Hdc is a potent inhibitor of physiological Ca(2+)-wave signaling mediated specifically by the pS368 phosphorylated form of Cx43.
• Jacobsen, N. L., Pontifex, T. K., Langlais, P. R., & Burt, J. M. (2019). Phosphorylation-dependent intra-domain interaction of the Cx37 carboxyl-terminus controls cell survival. Cancers, 11(2). doi:10.3390/cancers11020188
• Cotter, M. L., Boitano, S., Vagner, J., & Burt, J. M. (2018). Lipidated connexin mimetic peptides potently inhibit gap junction-mediated Ca-wave propagation. American journal of physiology. Cell physiology, 315(2), C141-C154.
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  Connexin (Cx) mimetic peptides (e.g., Gap27: SRPTEKTIFII; Peptide5: VDCFLSRPTEKT) reversibly inhibit hemichannel (HCh) and gap junction channel (GJCh) function in a concentration- and time-dependent manner (HCh: ~5 µM, 1 h). We hypothesized that addition of a hexadecyl tail to SRPTEKT (SRPTEKT- Hdc) would improve its ability to concentrate in the plasma membrane and consequently increase its inhibitory efficacy. We show that SRPTEKT- Hdc inhibited intercellular Ca-wave propagation in Cx43-expressing MDCK and rabbit tracheal epithelial cells in a time (61-75 min)- and concentration (IC: 66 pM)-dependent manner, a concentration efficacy five orders of magnitude lower than observed for the nonlipidated Gap27. HCh-mediated dye uptake was inhibited by SRPTEKT- Hdc with similar efficacy. Following peptide washout, HCh-mediated dye uptake was restored to control levels, whereas Ca-wave propagation was only partially restored. Scrambled and reverse sequence lipidated peptides had no detectable inhibitory effect on Ca-wave propagation or dye uptake. Cx43 expression was unchanged by SRPTEKT- Hdc incubation; however, Triton-insoluble Cx43 was reduced by SRPTEKT- Hdc exposure and reversed following washout. In summary, our results show that SRPTEKT- Hdc blocked HCh function and intercellular Ca signaling at concentrations that minimally affected dye coupling. Selective inhibition of intercellular Ca signaling, likely indicative of channel conformation-specific SRPTEKT- Hdc binding, could contribute significantly to the protective effects of these mimetic peptides in settings of injury. Our data also demonstrate that lipidation represents a paradigm for development of highly potent, efficacious, and selective mimetic peptide inhibitors of hemichannel and gap junction channel-mediated signaling.
• Ek-Vitorín, J. F., Pontifex, T. K., & Burt, J. M. (2018). Cx43 Channel Gating and Permeation: Multiple Phosphorylation-Dependent Roles of the Carboxyl Terminus. International journal of molecular sciences, 19(6).
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  Connexin 43 (Cx43), a gap junction protein seemingly fit to support cardiac impulse propagation and synchronic contraction, is phosphorylated in normoxia by casein kinase 1 (CK1). However, during cardiac ischemia or pressure overload hypertrophy, this phosphorylation fades, Cx43 abundance decreases at intercalated disks and increases at myocytes' lateral borders, and the risk of arrhythmia rises. Studies in wild-type and transgenic mice indicate that enhanced CK1-phosphorylation of Cx43 protects from arrhythmia, while dephosphorylation precedes arrhythmia vulnerability. The mechanistic bases of these Cx43 (de)phosphoform-linked cardiac phenotypes are unknown. We used patch-clamp and dye injection techniques to study the channel function (gating, permeability) of Cx43 mutants wherein CK1-targeted serines were replaced by aspartate (Cx43-CK1-D) or alanine (Cx43-CK1-A) to emulate phosphorylation and dephosphorylation, respectively. Cx43-CK1-D, but not Cx43-CK1-A, displayed high Voltage-sensitivity and variable permselectivity. Both mutants showed multiple channel open states with overall increased conductivity, resistance to acidification-induced junctional uncoupling, and hemichannel openings in normal external calcium. Modest differences in the mutant channels' function and regulation imply the involvement of dissimilar structural conformations of the interacting domains of Cx43 in electrical and chemical gating that may contribute to the divergent phenotypes of CK1-(de)phospho-mimicking Cx43 transgenic mice and that may bear significance in arrhythmogenesis.
• Fang, J. S., Coon, B. G., Gillis, N., Chen, Z., Qiu, J., Chittenden, T. W., Burt, J. M., Schwartz, M. A., & Hirschi, K. K. (2018). Addendum: Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification. Nature communications, 9(1), 720.
• Fang, J. S., Coon, B. G., Gillis, N., Chen, Z., Qiu, J., Chittenden, T. W., Burt, J. M., Schwartz, M. A., & Hirschi, K. K. (2017). Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification. Nature communications, 8(1), 2149.
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  Establishment of a functional vascular network is rate-limiting in embryonic development, tissue repair and engineering. During blood vessel formation, newly generated endothelial cells rapidly expand into primitive plexi that undergo vascular remodeling into circulatory networks, requiring coordinated growth inhibition and arterial-venous specification. Whether the mechanisms controlling endothelial cell cycle arrest and acquisition of specialized phenotypes are interdependent is unknown. Here we demonstrate that fluid shear stress, at arterial flow magnitudes, maximally activates NOTCH signaling, which upregulates GJA4 (commonly, Cx37) and downstream cell cycle inhibitor CDKN1B (p27). Blockade of any of these steps causes hyperproliferation and loss of arterial specification. Re-expression of GJA4 or CDKN1B, or chemical cell cycle inhibition, restores endothelial growth control and arterial gene expression. Thus, we elucidate a mechanochemical pathway in which arterial shear activates a NOTCH-GJA4-CDKN1B axis that promotes endothelial cell cycle arrest to enable arterial gene expression. These insights will guide vascular regeneration and engineering.
• Jacobsen, N. L., Pontifex, T. K., Li, H., Solan, J. L., Lampe, P. D., Sorgen, P. L., & Burt, J. M. (2017). Regulation of Cx37 channel and growth-suppressive properties by phosphorylation. Journal of cell science, 130(19), 3308-3321.
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  Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.
• Li, H., Spagnol, G., Pontifex, T. K., Burt, J. M., & Sorgen, P. L. (2017). Chemical shift assignments of the connexin37 carboxyl terminal domain. Biomolecular NMR assignments, 11(2), 137-141.
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  Connexin37 (Cx37) is a gap junction protein involved in cell-to-cell communication in the vasculature and other tissues. Cx37 suppresses proliferation of vascular cells involved in tissue development and repair in vivo, as well as tumor cells. Global deletion of Cx37 in mice leads to enhanced vasculogenesis in development, as well as collateralgenesis and angiogenesis in response to injury, which together support improved tissue remodeling and recovery following ischemic injury. Here we report the 1H, 15N, and 13C resonance assignments for an important regulatory domain of Cx37, the carboxyl terminus (CT; C233-V333). The predicted secondary structure of the Cx37CT domain based on the chemical shifts is that of an intrinsically disordered protein. In the 1H-15N HSQC, N-terminal residues S254-Y259 displayed a second weaker peak and residues E261-Y266 had significant line broadening. These residues are flanked by prolines (P250, P258, P260, and P268), suggesting proline cis-trans isomerization. Overall, these assignments will be useful for identifying the binding sites for intra- and inter-molecular interactions that affect Cx37 channel activity.
• Ek Vitorín, J. F., Pontifex, T. K., & Burt, J. M. (2016). Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus. Biophysical journal, 110(1), 127-40.
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  Separate connexin domains partake in proposed gating mechanisms of gap junction channels. The amino-terminus (NT) domains, which contribute to voltage sensing, may line the channel's cytoplasmic-facing funnel surface, stabilize the channel's overall structure through interactions with the transmembrane domains and each other, and integrate to form a compound particle to gate the channel closed. Interactions of the carboxyl-terminus (CT) and cytoplasmic loop (CL) domains underlie voltage- and low pH-triggered channel closure. To elucidate potential cooperation of these gating mechanisms, we replaced the Cx43NT with the Cx37NT (chimera Cx43(∗)NT37), leaving the remainder of the Cx43 sequence, including the CT and CL, unchanged. Compared to wild-type Cx43 (Cx43WT), Cx43(∗)NT37 junctions exhibited several functional alterations: extreme resistance to halothane- and acidification-induced uncoupling, absence of voltage-dependent fast inactivation, longer channel open times, larger unitary channel conductances, low junctional dye permeability/permselectivity, and an overall cation selectivity more typical of Cx37WT than Cx43WT junctions. Together, these results suggest a cohesive model of channel function wherein: 1) channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the NT domains, and 2) transition between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains transduced by interactions of the pore-forming domains with either the NT, CT, or both, with the NT domains forming the gate of the completely closed channel.
• Dominguez Rieg, J. A., Burt, J. M., Ruth, P., & Rieg, T. (2015). P2Y₂ receptor activation decreases blood pressure via intermediate conductance potassium channels and connexin 37. Acta physiologica (Oxford, England), 213(3), 628-41.
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  Nucleotides are important paracrine regulators of vascular tone. We previously demonstrated that activation of P2Y₂ receptors causes an acute, NO-independent decrease in blood pressure, indicating this signalling pathway requires an endothelial-derived hyperpolarization (EDH) response. To define the mechanisms by which activation of P2Y₂ receptors initiates EDH and vasodilation, we studied intermediate-conductance (KCa3.1, expressed in endothelial cells) and big-conductance potassium channels (KCa1.1, expressed in smooth muscle cells) as well as components of the myoendothelial gap junction, connexins 37 and 40 (Cx37, Cx40), all hypothesized to be part of the EDH response.
• Good, M. E., Ek-Vitorín, J. F., & Burt, J. M. (2014). Structural determinants and proliferative consequences of connexin 37 hemichannel function in insulinoma cells. The Journal of biological chemistry, 289(44), 30379-86.
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  Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C6A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C6A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.
• Jacobsen, N. L., & Burt, J. M. (2014). Connexin 37-mediated growth regulation in primary endothelial cells (LB680). The FASEB Journal, 28.
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  We have previously shown that connexin 37 (Cx37) is growth suppressive in vivo, as suggested by increased in ischemia-induced angiogenesis and collateral vasculogenesis in Cx37-deficient mice (Am.J.Physiol. 301: H1872-H1881, 2011). To provide an in vitro system for studies of microvascular capillary growth control mediated by Cx37, we isolated primary skeletal muscle endothelial cells (ECs) from the hindlimb of both wild type (WT) and Cx37-/- mice. Using VE-cadherin as the positive selection marker and a live cell sorting strategy, we are able to culture primary ECs for 14-21 days before they lose endothelial character. Evidence for endothelial nature was provided by cell morphology and the continued presence of VE-cadherin. The growth of ECs was monitored and revealed that proliferation of ECs isolated from WT mice is slowed compared to ECs originating from Cx37-/- mice. Additionally, there is a 4-day difference between the time it takes to reach confluence of Cx37-/- EC cultures and WT ECs. These data i...
• Koval, M., Molina, S. A., & Burt, J. M. (2014). Mix and match: investigating heteromeric and heterotypic gap junction channels in model systems and native tissues. FEBS letters, 588(8), 1193-204.
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  This review is based in part on a roundtable discussion session: "Physiological roles for heterotypic/heteromeric channels" at the 2013 International Gap Junction Conference (IGJC 2013) in Charleston, South Carolina. It is well recognized that multiple connexins can specifically co-assemble to form mixed gap junction channels with unique properties as a means to regulate intercellular communication. Compatibility determinants for both heteromeric and heterotypic gap junction channel formation have been identified and associated with specific connexin amino acid motifs. Hetero-oligomerization is also a regulated process; differences in connexin quality control and monomer stability are likely to play integral roles to control interactions between compatible connexins. Gap junctions in oligodendrocyte:astrocyte communication and in the cardiovascular system have emerged as key systems where heterotypic and heteromeric channels have unique physiologic roles. There are several methodologies to study heteromeric and heterotypic channels that are best applied to either heterologous expression systems, native tissues or both. There remains a need to use and develop different experimental approaches in order to understand the prevalence and roles for mixed gap junction channels in human physiology.
• Lancaster, J. J., Juneman, E., Arnce, S. A., Johnson, N. M., Qin, Y., Witte, R., Thai, H., Kellar, R. S., Ek Vitorin, J., Burt, J., Gaballa, M. A., Bahl, J. J., & Goldman, S. (2014). An electrically coupled tissue-engineered cardiomyocyte scaffold improves cardiac function in rats with chronic heart failure. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 33(4), 438-45.
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  Varying strategies are currently being evaluated to develop tissue-engineered constructs for the treatment of ischemic heart disease. This study examines an angiogenic and biodegradable cardiac construct seeded with neonatal cardiomyocytes for the treatment of chronic heart failure (CHF).
• Oulton, J., Good, M. E., Burt, J. M., & Barendrick, L. (2014). Neutrophil depletion: beneficial or detrimental to post-ischemic hind limb recovery in Cx40-/- vs. WT mice? (670.8). The FASEB Journal, 28.
• Sharik, M., Ruth, P., Rieg, T., & Burt, J. M. (2014). Connexin 37 and KCa3.1 contribute to P2Y2 receptor initiated endothelial-derived hyperpolarization (1079.15). The FASEB Journal, 28.
• Burt, J., Ek-Vitorin, J. F., & Burt, J. M. (2013). Structural basis for the selective permeability of channels made of communicating junction proteins. Biochimica et biophysica acta, 1828(1).
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  The open state(s) of gap junction channels is evident from their permeation by small ions in response to an applied intercellular (transjunctional/transchannel) voltage gradient. That an open channel allows variable amounts of current to transit from cell-to-cell in the face of a constant intercellular voltage difference indicates channel open/closing can be complete or partial. The physiological significance of such open state options is, arguably, the main concern of junctional regulation. Because gap junctions are permeable to many substances, it is sensible to inquire whether and how each open state influences the intercellular diffusion of molecules as valuable as, but less readily detected than current-carrying ions. Presumably, structural changes perceived as shifts in channel conductivity would significantly alter the transjunctional diffusion of molecules whose limiting diameter approximates the pore's limiting diameter. Moreover, changes in junctional permeability to some molecules might occur without evident changes in conductivity, either at macroscopic or single channel level. Open gap junction channels allow the exchange of cytoplasmic permeants between contacting cells by simple diffusion. The identity of such permeants, and the functional circumstances and consequences of their junctional exchange presently constitute the most urgent (and demanding) themes of the field. Here, we consider the necessity for regulating this exchange, the possible mechanism(s) and structural elements likely involved in such regulation, and how regulatory phenomena could be perceived as changes in chemical vs. electrical coupling; an overall reflection on our collective knowledge of junctional communication is then applied to suggest new avenues of research. This article is part of a Special Issue entitled: The Communicating junctions, roles and dysfunctions.
• Burt, J., Fang, J. S., Angelov, S. N., Simon, A. M., & Burt, J. M. (2013). Compromised regulation of tissue perfusion and arteriogenesis limit, in an AT1R-independent fashion, recovery of ischemic tissue in Cx40(-/-) mice. American journal of physiology. Heart and circulatory physiology, 304(6).
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  Recently, we reported that recovery of tissue perfusion in the ischemic hindlimb was reduced, inflammatory response increased, and survival of distal limb tissue compromised in connexin 40 (Cx40)-deficient (Cx40(-/-)) mice. Here we evaluate whether genotype-specific differences in tissue perfusion, native vascular density, arteriogenesis, blood pressure, and chronic ANG II type 1 receptor (AT1R) activation contribute to poor recovery of ischemic hindlimb tissue in Cx40(-/-) mice. Hindlimb ischemia was induced in wild-type (WT), Cx40(-/-), and losartan-treated Cx40(-/-) mice by using surgical procedures that either maintained (mild surgery) or compromised (severe surgery) perfusion of major collateral vessels supplying the distal limb. Pre- and postsurgical hindlimb perfusion was evaluated, and tissue survival, microvascular density, and macrophage infiltration were documented during recovery. Hindlimb perfusion was compromised in presurgical Cx40(-/-) versus WT mice despite comparable native microvascular density. Hindlimb perfusion 24 h postsurgery in Cx40(-/-) and WT mice was comparable after mild surgery (collateral vessels maintained), but compromised arteriogenesis in Cx40(-/-) animals nevertheless limited subsequent recovery of tissue perfusion and compromised tissue survival. Prolonged pre- and postsurgical treatment of Cx40(-/-) mice with losartan (an AT1R antagonist) normalized blood pressure but did not improve tissue perfusion or survival, despite reduced macrophage infiltration. Thus it appears Cx40 is necessary for normal tissue perfusion and for recovery of perfusion, arteriogenesis, and tissue survival in the ischemic hindlimb. Our data suggest that Cx40(-/-) mice are at significantly greater risk for poor recovery from ischemic insult due to compromised regulation of tissue perfusion, vascular remodeling, and prolonged inflammatory response.
• Burt, J., Nelson, T. K., Sorgen, P. L., & Burt, J. M. (2013). The carboxyl terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37 mediated suppression of insulinoma cell proliferation. American journal of physiology. Cell physiology.
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  Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with non-functional channels but normally localized, wild-type carboxyl termini are not growth suppressive. Here we determined whether the carboxyl terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37-273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1 cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37-273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37.
• Fang, J. S., Dai, C., Kurjiaka, D. T., Burt, J. M., & Hirschi, K. K. (2013). Connexin45 regulates endothelial-induced mesenchymal cell differentiation toward a mural cell phenotype. Arteriosclerosis, thrombosis, and vascular biology, 33(2), 362-8.
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  The focus of this study was to investigate the role of connexin (Cx) 45 in endothelial-induced mural cell differentiation.
• Burt, J., Fang, J. S., Angelov, S. N., Simon, A. M., & Burt, J. M. (2012). Cx40 is required for, and cx37 limits, postischemic hindlimb perfusion, survival and recovery. Journal of vascular research, 49(1).
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  Ischemia induced by large-vessel obstruction or vascular injury induces a complex cascade of vasodilatory, remodeling and inflammatory pathways; coordination of these processes by vascular endothelium is likely to involve endothelial gap junctions. Vascular endothelium predominantly expresses two connexin (Cx) isoforms: Cx37 and Cx40. The relevance of these Cxs to postischemic limb recovery remains unclear.
• Burt, J., Good, M. E., Ek-Vitorín, J. F., & Burt, J. M. (2012). Extracellular loop cysteine mutant of cx37 fails to suppress proliferation of rat insulinoma cells. The Journal of membrane biology, 245(7).
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  Although a functional pore domain is required for connexin 37 (Cx37)-mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)--expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37's growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels.
• Gemel, J., Nelson, T. K., Burt, J. M., & Beyer, E. C. (2012). Inducible coexpression of connexin37 or connexin40 with connexin43 selectively affects intercellular molecular transfer. The Journal of membrane biology, 245(5-6), 231-41.
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  Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4 μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.
• Halidi, N., Alonso, F., Burt, J. M., Bény, J., Haefliger, J., & Meister, J. (2012). Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by connexin43. Cell communication & adhesion, 19(2), 25-37.
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  Intercellular Ca(2+) wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca(2+) wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca(2+) waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca(2+) wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca(2+) waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca(2+) waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.
• Burt, J., Fang, J. S., Angelov, S. N., Simon, A. M., & Burt, J. M. (2011). Cx37 deletion enhances vascular growth and facilitates ischemic limb recovery. American journal of physiology. Heart and circulatory physiology, 301(5).
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  The unique contributions of connexin (Cx)37 and Cx40, gap junction-forming proteins that are coexpressed in vascular endothelium, to the recovery of tissues from ischemic injury are unknown. We recently reported that Cx37-deficient (Cx37(-/-)) animals recovered ischemic hindlimb function more quickly and to a greater extent than wild-type (WT) or Cx40(-/-) animals, suggesting that Cx37 limits recovery in the WT animal. Here, we tested the hypothesis that enhanced angiogenesis, arteriogenesis, and vasculogenesis contribute to improved postischemic hindlimb recovery in Cx37(-/-) animals. Ischemia was induced unilaterally in the hindlimbs of WT or Cx37(-/-) mice (isoflurane anesthesia). Postsurgical limb appearance, use, and perfusion were documented during recovery, and the number (and size) of large and small vessels was determined. Native collateral number, predominantly established during embryonic development (vasculogenesis), was also determined in the pial circulation. Both microvascular density in the gastrocnemius of the ischemic limb (an angiogenic field) and the number and tortuosity of larger vessels in the gracilis vasculature (an arteriogenic field) were increased in Cx37(-/-) animals compared with WT animals. Cx37(-/-) mice also had an increased (vs. WT) number of collateral vessels in the pial circulation. These findings suggest that in Cx37(-/-) animals, improved recovery of the ischemic hindlimb involves enhanced vasculogenesis, resulting in increased numbers of collaterals in the hindlimb (and pial circulations) and more extensive collateral remodeling and angiogenesis. These results are consistent with Cx37 exerting a growth-suppressive effect in the vasculature that limits embryonic vasculogenesis as well as arteriogenic and angiogenic responses to ischemic injury in the adult animal.
• Burt, J., Good, M. E., Nelson, T. K., Simon, A. M., & Burt, J. M. (2011). A functional channel is necessary for growth suppression by Cx37. Journal of cell science, 124(Pt 14).
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  Connexin 37 (Cx37) profoundly suppresses the proliferation of rat insulinoma (Rin) cells by unknown mechanisms. To determine whether a functional pore domain is necessary for Cx37-mediated growth suppression, we introduced a mutation that converted threonine 154 into alanine (T154A). Like other connexins mutated at the homologous site, Cx37-T154A localized to appositional membrane but failed to form functional channels and exerted a dominant-negative effect on coexpressed wild-type Cx37 or Cx43. Unlike the wild-type protein, Cx37-T154A did not suppress the proliferation of Rin cells and did not, with serum deprivation, result in cell cycle arrest. Furthermore, progression through the cell cycle was unaffected by expression of Cx37-T154A. These results indicate that a pore-forming domain that is able to form functional channels is essential for the anti-proliferative, cell-cycle arrest and serum-sensitivity effects of Cx37, and furthermore that the normally localized C-terminal domain is not sufficient for these effects of Cx37.
• Liu, J., Ek Vitorin, J. F., Weintraub, S. T., Gu, S., Shi, Q., Burt, J. M., & Jiang, J. X. (2011). Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function. The Journal of biological chemistry, 286(19), 16914-28.
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  Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ∼200-pS open state while simultaneously reducing transitions between this open state and a ∼65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ∼200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.
• Vitorin, J. F., Vitorin, J. F., Shi, Q., Liu, J., Jiang, J. X., Gu, S., & Burt, J. M. (2011). In vivo Phosphorylation of Lens Connexin 50 by Protein Kinase A Enhances Gap Junction and Hemichannel Function. Investigative Ophthalmology & Visual Science, 52(14), 3929-3929.
• Juneman, E., Vitorin, J. F., Vitorin, J. F., Thai, H., Lancaster, J. J., Kellar, R. S., Juneman, E., Johnson, N. M., Goldman, S. A., Burt, J. M., Bahl, J. J., & Arnce, S. A. (2010). In Vivo Evaluation of a Biologically Active Cardiomyocyte Seeded Scaffold. Journal of Cardiac Failure, 16(8), S45. doi:10.1016/j.cardfail.2010.06.155
• Burt, J., Heyman, N. S., Kurjiaka, D. T., Ek Vitorin, J. F., & Burt, J. M. (2009). Regulation of gap junctional charge selectivity in cells coexpressing connexin 40 and connexin 43. American journal of physiology. Heart and circulatory physiology, 297(1).
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  Expression of connexin 40 (Cx40) and Cx43 in cardiovascular tissues varies as a function of age, injury, and development with unknown consequences on the selectivity of junctional communication and its acute regulation. We investigated the PKC-dependent regulation of charge selectivity in junctions composed of Cx43, Cx40, or both by simultaneous assessment of junctional permeance rate constants (B(dye)) for dyes of similar size but opposite charge, N,N,N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl)amino]ethanaminium (NBD-M-TMA; +1) and Alexa 350 (-1). The ratio of dye rate constants (B(NBD-M-TMA)/B(Alexa 350)) indicated that Cx40 junctions are cation selective (10.7 +/- 0.5), whereas Cx43 junction are nonselective (1.22 +/- 0.14). In coexpressing cells, a broad range of junctional selectivities was observed with mean cation selectivity increasing as the Cx40 to Cx43 expression ratio increased. PKC activation reduced or eliminated dye permeability of Cx43 junctions without altering their charge selectivity, had no effect on either permeability or charge selectivity of Cx40 junctions, and significantly increased the cation selectivity of junctions formed by coexpressing cells (approaching charge selectivity of Cx40 junctions). Junctions composed of Cx43 truncated at residue 257 (Cx43tr) were also not charge selective, but when Cx43tr was coexpressed with Cx40, a broad range of junctional selectivities that was unaffected by PKC activation was observed. Thus, whereas the charge selectivities of homomeric/homotypic Cx43 and Cx40 junctions appear invariant, the selectivities of junctions formed by cells coexpressing Cx40 and Cx43 vary considerably, reflecting both their relative expression levels and phosphorylation-dependent regulation. Such regulation could represent a mechanism by which coexpressing cells such as vascular endothelium and atrial cells regulate acutely the selective intercellular communication mediated by their gap junctions.
• Simon, A. M., Lampe, P. D., Burt, J. M., & Boitano, S. (2009). Abstracts of the 2009 International Gap Junction Conference in Sedona Arizona.. Cell communication & adhesion, 16(4), 39-112. doi:10.3109/15419060903344130
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  Recently approximately 220 scientists gathered at the International Gap Junction Conference 2009. The conference took place at the Sedona Hilton Resort in Arizona's high desert with a backdrop of m...
• Burt, J. M., Nelson, T. K., Simon, A. M., & Fang, J. S. (2008). Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells. American journal of physiology. Cell physiology, 295(5), C1103-12.
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  In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G(1)/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G(1). Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G(1) and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G(1)/S checkpoint.
• Burt, J., Heyman, N. S., & Burt, J. M. (2008). Hindered diffusion through an aqueous pore describes invariant dye selectivity of Cx43 junctions. Biophysical journal, 94(3).
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  The permselectivity (permeance/conductance) of Cx43-comprised gap junctions is a variable parameter of junctional function. To ascertain whether this variability in junctional permselectivity is explained by heterogeneous charge or size selectivity of the comprising channels, the permeance of individual Cx43 gap junctions to combinations of two dyes differing in either size or charge was determined in four cell types: Rin43, NRKe, HeLa43, and cardiac myocytes. The results show that Cx43 junctions are size- but not charge-selective and that both selectivities are constant parameters of junctional function. The consistency of dye selectivities indicates that the large continuum of measured junctional permselectivities cannot be ascribed to an equivalent continuum of individual channel selectivities. Further, the relative dye permeance sequence of NBD-M-TMA approximately Alexa 350 > Lucifer yellow > Alexa 488 > Alexa 594 (Stokes radii of 4.3 A, 4.4 A, 4.9 A, 5.8 A, and 7.4 A, respectively) and the conductance sequence of KCl > TEACl approximately Kglutamate are well described by hindered diffusion through an aqueous pore with radius approximately 10 A and length 160 A. The permselectivity and dye selectivity data suggest the variable presence in Cx43-comprised junctions of conductive channels that are either dye-impermeable or dye-permeable.
• Kurjiaka, D. T., & Burt, J. M. (2008). Effects of angiotensin II on resistance vessels in hamster hypertension. The FASEB Journal, 22.
• Kurjiaka, D. T., Hirschi, K. K., Burt, J. M., & Boitano, S. (2008). The role of calcium in endothelial cell induced mesenchymal cell differentiation. The FASEB Journal, 22.
• Segal, S. S., Kurjiaka, D. T., & Burt, J. M. (2008). Hypertension compromises functional hyperemia in hamster feed arteries. The FASEB Journal, 22(S2), 122-122. doi:10.1096/fasebj.22.2_supplement.122
• Simon, A. M., Fang, J. S., & Burt, J. M. (2008). Connexin37 Regulation of Vascular Remodeling Following Ischemic Injury. The FASEB Journal, 22.
• Simon, A. M., Parkin, E. C., Kurjiaka, D. T., & Burt, J. M. (2007). Coarctation induces increased Cx43 expression by aortic endothelium. The FASEB Journal, 21(6).
• Burt, J., Ek-Vitorin, J. F., King, T. J., Heyman, N. S., Lampe, P. D., & Burt, J. M. (2006). Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation. Circulation research, 98(12).
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  Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.
• Lampe, P. D., Cooper, C. D., King, T. J., & Burt, J. M. (2006). Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart. Journal of cell science, 119(Pt 16), 3435-42.
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  The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43(-/-) cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.
• Burt, J., Ek-Vitorín, J. F., & Burt, J. M. (2005). Quantification of gap junction selectivity. American journal of physiology. Cell physiology, 289(6).
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  Gap junctions, which are essential for functional coordination and homeostasis within tissues, permit the direct intercellular exchange of small molecules. The abundance and diversity of this exchange depends on the number and selectivity of the comprising channels and on the transjunctional gradient for and chemical character of the permeant molecules. Limited knowledge of functionally significant permeants and poor detectability of those few that are known have made it difficult to define channel selectivity. Presented herein is a multifaceted approach to the quantification of gap junction selectivity that includes determination of the rate constant for intercellular diffusion of a fluorescent probe (k2-DYE) and junctional conductance (gj) for each junction studied, such that the selective permeability (k2-DYE/gj) for dyes with differing chemical characteristics or junctions with differing connexin (Cx) compositions (or treatment conditions) can be compared. In addition, selective permeability can be correlated using single-channel conductance when this parameter is also measured. Our measurement strategy is capable of detecting 1) rate constants and selective permeabilities that differ across three orders of magnitude and 2) acute changes in that rate constant. Using this strategy, we have shown that 1) the selective permeability of Cx43 junctions to a small cationic dye varied across two orders of magnitude, consistent with the hypothesis that the various channel configurations adopted by Cx43 display different selective permeabilities; and 2) the selective permeability of Cx37 vs. Cx43 junctions was consistently and significantly lower.
• Cottrell, G. T., & Burt, J. M. (2005). Functional consequences of heterogeneous gap junction channel formation and its influence in health and disease. Biochimica et biophysica acta, 1711(2), 126-41.
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  The capacity of multiple connexins to hetero-oligomerize into functional heterogeneous gap junction channels has been demonstrated in vivo, in vitro, and in nonmammalian expression systems. These heterogeneous channels display gating activity, channel conductances, selectivity and regulatory behaviors that are sometimes not predicted by the behaviors of the corresponding homogeneous channels. Such observations suggest that heteromerization of gap junction proteins offers an efficient cellular strategy for finely regulating cell-to-cell communication. The available evidence strongly indicates that heterogeneous gap junction assembly is important to normal growth and differentiation, and may influence the appearance of several disease states. Definitive evidence that heterogeneous gap junction channels differentially regulate electrical conduction in excitable cells is absent. This review examines the prevalence, regulation, and implications of gap junction channel hetero-oligomerization.
• Burt, J., Cottrell, G. T., Lin, R., Warn-Cramer, B. J., Lau, A. F., & Burt, J. M. (2003). Mechanism of v-Src- and mitogen-activated protein kinase-induced reduction of gap junction communication. American journal of physiology. Cell physiology, 284(2).
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  Connexin (Cx)43 gap junction channels are phosphorylated by numerous protein kinases, with the net effect typically being a reduction in gap junction communication (GJC). This reduction must result from a decrease in channel open probability, unitary conductance, or permselectivity, because previous results suggest that channel number is unaffected. Coexpression of v-Src with wild-type Cx43 (Cx43-wt) but not Cx43 with tyrosine to phenylalanine substitutions at 247 and 265 (Cx43-Y247,265F) resulted in reduced electrical and dye coupling but no change in single-channel amplitudes. EGF treatment of cells expressing Cx43-wt but not Cx43 with serine to alanine substitutions at 255, 279, and 282 (Cx43-S255,279,282A) resulted in reduced GJC, also with no change in single-channel amplitude. Dye coupling was reduced to a far greater extent than electrical coupling, suggesting that channel selectivity was also altered but with minimal effect on unitary conductance. The absence of Src- and MAPK-induced reductions in single-channel amplitude suggests that the decreases in GJC induced by these kinases result from reduced channel open probability and possibly altered selectivity.
• Hirschi, K. D., Hirschi, K. K., Dai, C., & Burt, J. M. (2003). Gap junction communication mediates transforming growth factor-beta activation and endothelial-induced mural cell differentiation.. Circulation research, 93(5), 429-37. doi:10.1161/01.res.0000091259.84556.d5
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  During blood vessel assembly, endothelial cells recruit mesenchymal progenitors and induce their differentiation into mural cells via contact-dependent transforming growth factor-beta (TGF-beta) activation. We investigated whether gap junction channels are formed between endothelial cells and recruited mesenchymal progenitors and whether intercellular communication is necessary for endothelial-induced mural cell differentiation. Mesenchymal progenitors from Cx43-/- murine embryos and Cx43+/+ littermates were cocultured with prelabeled endothelial cells. Intracellular dye injection and dual whole-cell voltage clamp revealed that endothelial cells formed gap junction channels with Cx43+/+ but not Cx43-/- progenitors. In coculture with endothelial cells, Cx43-/- progenitors did not undergo mural cell differentiation as did Cx43+/+ cells. Stable reexpression of Cx43 in Cx43-/- cells (reCx43) restored their ability to form gap junctions with endothelial cells and undergo endothelial-induced mural cell differentiation. Cocultures of endothelial cells and either Cx43+/+ or reCx43 mesenchymal cells produced activated TGF-beta; endothelial-Cx43-/- cocultures did not. However, Cx43-/- cells did produce latent TGF-beta and undergo mural cell differentiation in response to exogenous TGF-beta1. These studies indicate that gap junction communication between endothelial and mesenchymal cells mediates TGF-beta activation and subsequent mural cell differentiation.
• Steele, T. D., & Burt, J. M. (2003). Selective effect of PDGF on connexin43 versus connexin40 comprised gap junction channels.. Cell communication & adhesion, 10(4-6), 287-91. doi:10.1080/cac.10.4-6.287.291
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  The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g(j) and gamma(j), respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g(j) in Cx40/Cx40 pairs, but decreased g(j) in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g(j) suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g(j) involved a decrease in both gamma(j) and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.
• Burt, J., Cottrell, G. T., Wu, Y., & Burt, J. M. (2002). Cx40 and Cx43 expression ratio influences heteromeric/ heterotypic gap junction channel properties. American journal of physiology. Cell physiology, 282(6).
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  In cells that coexpress connexin (Cx)40 and Cx43, the ratio of expression can vary depending on the cellular environment. We examined the effect of changing Cx40:Cx43 expression ratio on functional gap junction properties. Rin cells transfected with Cx40 or Cx43 (Rin40, Rin43) were cocultured with 6B5n, A7r5, A7r540C1, or A7r540C3 cells for electrophysiological and dye coupling analysis. Cx40:Cx43 expression ratio in 6B5n, A7r5, A7r540C1, and A7r540C3 cells was ~1:1, 3:1, 5:1, and 10:1, respectively. When Rin43 cells were paired with coexpressing cells, there was an increasing asymmetry of voltage-dependent gating and a shift toward smaller conductance events as Cx40:Cx43 ratio increased in the coexpressing cell. These observations could not be predicted by linear combinations of Cx40 and Cx43 properties in proportion to the expressed ratios of the two Cxs. When Rin40 cells were paired with coexpressing cells, the net voltage gating and single-channel conductance behavior were similar to those of Rin40/Rin40 cell pairs. Dye permeability properties of cell monolayers demonstrated that as Cx40:Cx43 expression ratio increased in coexpressing cells the charge and size selectivity of dye transfer reflected that of Rin40 cells, as would be predicted. These data indicate that the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents expressed in the cell; however, the dye permeability of these same channels can be predicted by the relative Cx contributions.
• Cottrell, G. T., & Burt, J. M. (2001). Heterotypic gap junction channel formation between heteromeric and homomeric Cx40 and Cx43 connexons.. American journal of physiology. Cell physiology, 281(5), C1559-67. doi:10.1152/ajpcell.2001.281.5.c1559
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  Recent evidence indicating formation of functional homomeric/heterotypic gap junction channels by connexin40 (Cx40) and connexin43 (Cx43) raises the question of whether data previously interpreted as support for heteromeric channel formation by these connexins might not instead reflect the activity of homomeric/heterotypic channels. To address this question and to further characterize the behavior of these channels, we used dual whole cell voltage-clamp techniques to examine the junctions formed between cells that express only Cx40 (Rin40) or Cx43 (Rin43) and compared the results with those obtained when either of these cell types was paired with cells that naturally express both connexins (A7r5 cells). Rin40/Rin43 cell pairs formed functional gap junctions that displayed a strongly asymmetric voltage-dependent gating response. Single-channel event amplitudes ranged between 34 and 150 pS, with 90- to 130-pS events predominating. A7r5/Rin43 and A7r5/Rin40 cell pairs had voltage-dependent gating responses that varied greatly, with most pairs demonstrating strong asymmetry. These cell pairs exhibited a variety of single-channel events that were not consistent with homomeric/homotypic Cx40 or Cx43 channels or homomeric/heterotypic Cx40/Cx43 channels. These data indicate that Cx40 and Cx43 form homomeric/heterotypic as well as heteromeric/heterotypic channels that display unique gating and conductance properties.
• Wu, Y., Cottrell, G. T., & Burt, J. M. (2001). Functional characteristics of heteromeric Cx40-Cx43 gap junction channel formation.. Cell communication & adhesion, 8(4-6), 193-7. doi:10.3109/15419060109080722
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  Cx40:Cx43 expression ratio in A7r5 cells is augmented in growth stimulated vs. growth arrested conditions. To determine the impact of changing Cx40:Cx43 expression ratio on gap junction function, we have developed A7r5 cell lines that display Cx40:Cx43 ratios of 1:1 (66B5n) and 10:1 (A7r540C3). When Rin43 cells were paired with these coexpressing cells, there was an increasing asymmetry of voltage dependent gating as the Cx40:Cx43 ratio increased in the coexpressing cell. This asymmetry was opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In addition, when Rin43 cells were paired with coexpressing cells there was a shift toward smaller single channel event amplitudes with increasing Cx40:Cx43 ratio in the coexpressing cell. Again, this is opposite to that which is predicted by Cx40/Cx43 heterotypic channels. In dye coupling experiments, 6B5N, A7r5, and A7r540C3 cells displayed charge and size selectivity that increased with increasing Cx40:Cx43 expression ratio. These data indicate that although the electrophysiological properties of heteromeric/heterotypic channels are not directly related to the proportions of Cx constituents that comprise the channel, the dye permeability data fit what would be predicted by an increase in Cx40:Cx43 ratio.
• Wu, Y., Steele, T. D., Kurjiaka, D. T., Fletcher, A. M., Cottrell, G. T., & Burt, J. M. (2001). Alteration of Cx43:Cx40 expression ratio in A7r5 cells.. American journal of physiology. Cell physiology, 280(3), C500-8. doi:10.1152/ajpcell.2001.280.3.c500
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  Connexins (Cx) 40 and 43 are coexpressed by several cell types at ratios that vary as a function of development, aging, and disease. Because these connexins form heteromeric channels, changes in expression ratio might be expected to significantly alter the connexin composition of the gap junction channel population and, therefore, gap junction function. To examine this possibility, we stably transfected A7r5 cells, which naturally coexpress Cx43 and Cx40, with a vector encoding antisense Cx43. Cx43 mRNA continued to be expressed in the antisense transfected clones, although levels were inversely related to the number of copies of antisense DNA incorporated into the genome. Protein levels, quantified in the clones with the highest and lowest Cx43:Cx40 mRNA ratios, were not well predicted by the mRNA levels, although the trends predicted by the Cx43:Cx40 mRNA ratio were preserved. Electrical coupling did not differ significantly between clones, but the clone with elevated Cx43:Cx40 protein expression ratio and unchanged Cx43 banding pattern was significantly better dye coupled than the parental A7r5 cells. These results suggest that as the Cx43:Cx40 ratio increases, provided alterations of Cx43 banding pattern (phosphorylation) have not occurred, permeability to large molecules increases even though electrical coupling remains nearly constant.
• He, D. S., & Burt, J. M. (2000). Mechanism and selectivity of the effects of halothane on gap junction channel function.. Circulation research, 86(11), E104-9. doi:10.1161/01.res.86.11.e104
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  Volatile anesthetics alter tissue excitability by decreasing the extent of gap junction-mediated cell-cell coupling and by altering the activity of the channels that underlie the action potential. In the present study, we demonstrate, using dual whole-cell voltage-clamp techniques, that coexpression of connexin (Cx) 40 and Cx43 rendered cells more sensitive to uncoupling by halothane than cells that express only Cx40 or only Cx43. The halothane-induced reduction in junctional conductance was caused by decreased channel mean open time and increased channel mean closed time. The magnitude of the effect of halothane on channel open time was least for Cx40-like channels and greatest for heteromeric channels. Thus, the data indicate that halothane gates gap junction channels to the closed state in a dose-dependent and connexin-specific manner. One consequence of the selectivity of halothane is that the profile of single-channel events observed in the presence of halothane may not be quantitatively representative of the population of channels contributing to macroscopic conductance in cells that express more than one connexin. In addition, in tissues that express multiple connexins, such as heart and blood vessels, the capacity of the gap junctions to transmit electrical and chemical signals in the presence of halothane could vary according to the pattern of connexin expression.
• Tenbroek, E. M., Lau, A. F., Lampe, P. D., Kurata, W. E., Johnson, R. G., & Burt, J. M. (2000). Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication.. The Journal of cell biology, 149(7), 1503-12. doi:10.1083/jcb.149.7.1503
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  Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.
• Taffet, S. M., Jiang, J. X., He, D. S., & Burt, J. M. (1999). Formation of heteromeric gap junction channels by connexins 40 and 43 in vascular smooth muscle cells.. Proceedings of the National Academy of Sciences of the United States of America, 96(11), 6495-500. doi:10.1073/pnas.96.11.6495
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  Connexin (Cx) 43 and Cx40 are coexpressed in several tissues, including cardiac atrial and ventricular myocytes and vascular smooth muscle. It has been shown that these Cxs form homomeric/homotypic channels with distinct permeability and gating properties but do not form functional homomeric/heterotypic channels. If these Cxs were to form heteromeric channels, they could display functional properties not well predicted by the homomeric forms. We assessed this possibility by using A7r5 cells, an embryonic rat aortic smooth muscle cell line that coexpresses Cxs 43 and 40. Connexons (hemichannels), which were isolated from these cells by density centrifugation and immunoprecipitated with antibody against Cx43, contained Cx40. Similarly, antibody against Cx40 coimmunoprecipitated Cx43 from the same connexon fraction but only Cx40 from Cx (monomer) fractions. These results indicate that heteromeric connexons are formed by these Cxs in the A7r5 cells. The gap junction channels formed in the A7r5 cells display many unitary conductances distinct from homomeric/homotypic Cx43 or Cx40 channels. Voltage-dependent gating parameters in the A7r5 cells are also quite variable compared with cells that express only Cx40 or Cx43. These data indicate that Cxs 43 and 40 form functional heteromeric channels with unique gating and conductance properties.
• Steele, T. D., Olsen, M. V., Kurjiaka, D. T., & Burt, J. M. (1998). Gap junction permeability is diminished in proliferating vascular smooth muscle cells.. The American journal of physiology, 275(6), C1674-82. doi:10.1152/ajpcell.1998.275.6.c1674
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  In atherosclerosis and hypertension, vascular smooth muscle cells (SMCs) are stimulated to proliferate and exhibit enhanced gap junction protein expression. Our goal was to determine whether gap junction function differs in proliferating vs. growth-arrested SMCs. A7r5 cells (embryonic rat aortic SMCs) did not proliferate in media with reduced serum ( approximately 90% of cells in G0/G1 phase after 48-96 h in 1% fetal bovine serum). Dye coupling was less but electrical coupling was comparable in proliferating vs. growth-arrested A7r5 cells, suggesting differences in junctional permselectivity. In growth-arrested cells, junctional conductances measured with potassium glutamate, tetraethylammonium chloride, and KCl were well predicted by the conductivities of these solutions. In contrast, junctional conductances measured with potassium glutamate and tetraethylammonium chloride in proliferating cells were significantly greater than predicted by the conductivities of these solutions. These results suggest that junctions between growth-arrested cells are permeated equally well and simultaneously by anions and cations, whereas junctions between proliferating cells are poorly permeated by large molecules of either charge and equally well but not simultaneously by small anions and cations. The data indicate that A7r5 cells regulate chemical coupling independent of electrical coupling, a capacity that could facilitate growth control while protecting vasomotor responsiveness of vessels.
• Warn-cramer, B. J., Cottrell, G. T., Burt, J. M., & Lau, A. F. (1998). Regulation of connexin-43 gap junctional intercellular communication by mitogen-activated protein kinase.. The Journal of biological chemistry, 273(15), 9188-96. doi:10.1074/jbc.273.15.9188
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  Activation of the Ras/Raf/mitogen-activated protein kinase kinase/mitogen-activated protein (MAP) kinase signaling cascade is initiated by activation of growth factor receptors and regulates many cellular events, including cell cycle control. Our previous studies suggested that the connexin-43 gap junction protein may be a target of activated MAP kinase and that MAP kinase may regulate connexin-43 function. We identified the sites of MAP kinase phosphorylation in in vitro studies as the consensus MAP kinase recognition sites in the cytoplasmic carboxyl tail of connexin-43, Ser255, Ser279, and Ser282. In this study, we demonstrate that activation of MAP kinase by ligand-induced activation of the epidermal growth factor (EGF) or lysophosphatidic acid receptors or by pervanadate-induced inhibition of tyrosine phosphatases results in increased phosphorylation on connexin-43. EGF and lysophosphatidic acid-induced phosphorylation on connexin-43 and the down-regulation of gap junctional communication in EGF-treated cells were blocked by a specific mitogen-activated protein kinase kinase inhibitor (PD98059) that prevented activation of MAP kinase. These studies confirm that connexin-43 is a MAP kinase substrate in vivo and that phosphorylation on Ser255, Ser279, and/or Ser282 initiates the down-regulation of gap junctional communication. Studies with connexin-43 mutants suggest that MAP kinase phosphorylation at one or more of the tandem Ser279/Ser282 sites is sufficient to disrupt gap junctional intercellular communication.
• Warn-cramer, B. J., Tenbroek, E. M., Martyn, K. D., Lau, A. F., Kurata, W. E., & Burt, J. M. (1997). Immortalized connexin43 knockout cell lines display a subset of biological properties associated with the transformed phenotype.. Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 8(9), 1015-27.
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  Immortalized cells from embryonic connexin43 knockout mice (Cx43-/-) and homozygous littermates (Cx43+/+) were cloned and characterized to determine whether the absence of Cx43 function would induce observable phenotypic changes. Cells of the Cx43+/+ clones expressed Cx43 and engaged in gap junctional communication with 10-12 neighboring cells. The Cx43-/- cells were devoid of Cx43 and communicated to less than 1 cell. Electrophysiological analysis indicated that the Cx43-/- cells communicated through Cx45 channels from 8-80-fold less than did the Cx43+/+ subclones, which seemed to communicate through Cx43 and Cx45 channels. The Cx43-/- clones grew at faster rates and to higher saturation densities, had a more spindly morphology, were more refractile, and adhered less well to the substratum than did the Cx43+/+ clones. Reintroducing the Cx43 gene into the Cx43-/- clones resulted in three subclones that communicated to 3-4 cells. Partial restoration of gap junctional communication in the three subclones was accompanied by reduced growth rates and saturation densities (2-fold compared to that of parental Cx43-/- clones) but no reversions in morphology or cell-substratum adhesion. The increased growth rates and saturation densities, altered morphology, and decreased cell adhesion displayed by the Cx43-/- clones reflect a subset of the properties of transformed cells. These studies advance the hypothesis that loss of Cx43 function during development may cause cells to acquire a preneoplastic condition.
• Moore, L. K., & Burt, J. M. (1995). Gap junction function in vascular smooth muscle: influence of serotonin.. The American journal of physiology, 269(4 Pt 2), H1481-9. doi:10.1152/ajpheart.1995.269.4.h1481
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  In this study we examined the effects of serotonin (5-hydroxytryptamine, 5-HT) on the function of gap junctions between smooth muscle cells isolated from human and pig coronary and rat mesentery arteries and between A7r5 cells (cell line derived from embryonic rat aorta). Mesentery and pig coronary cells expressed connexin (Cx) 43, and human coronary cells expressed Cx40. Mesentery and pig coronary cells each exhibited a single gap junction channel population with unitary conductances of 75 and 59 pS, respectively. Human coronary cells exhibited two channel populations with unitary conductances of 51 and 107 pS. The A7r5 cells express Cx40 and Cx43 and exhibit three channel populations with unitary conductances of 70, 108, and 141 pS. Under control conditions, junctional conductance between the four cell types ranged from 11 to 20 nS. During maximal stimulation with 5-HT (1-10 microM), junctional conductance increased (29-75%) in all four cell types. The unitary conductance profiles in the rat mesentery and pig coronary cells were unaffected by 5-HT, suggesting that the observed increase in macroscopic conductance reflects an increase in open probability. Unitary conductances were also unaffected in the human coronary and A7r5 cells. However, there was a reduced frequency of the 105-pS channel in the human coronary cells and of the 70- and 141-pS channels in the A7r5 cells. These changes in the relative frequency histograms suggest that the open probabilities of the various channel types are differentially affected by the 5-HT treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
• Moore, L. K., & Burt, J. M. (1994). Selective block of gap junction channel expression with connexin-specific antisense oligodeoxynucleotides.. The American journal of physiology, 267(5 Pt 1), C1371-80. doi:10.1152/ajpcell.1994.267.5.c1371
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  Gap junctions in vascular smooth muscle provide a cell-to-cell conduction pathway that may contribute to regulation and coordination of changes in vascular tone. A7r5 cells, a cell line derived from embryonic rat aorta, express connexin 43 (Cx43) and connexin 40 (Cx40). Gap junction channels with three distinct unitary conductances (70, 108, and 141 pS) were observed in these cells. Events of each channel type were equally common, with an approximate frequency of 30-35%; however, the frequency at which each channel type was observed in individual cell pairs ranged between 10 and 65%. Treatment of the cells for 24-72 h with an antisense oligodeoxynucleotide (ODN) to Cx43 reduced the relative frequency of the 108- and 141-pS channel events, whereas comparable treatment with antisense Cx40 ODN reduced the frequency at which 70-pS channel events were observed. The simplest explanation of these findings is that Cx43 forms the 108- and 141-pS channels, whereas Cx40 forms the 70-pS channels in A7r5 cells.
• Moore, L. K., Minnich, B. N., Hirschi, K. K., & Burt, J. M. (1993). Oleic acid differentially affects gap junction-mediated communication in heart and vascular smooth muscle cells.. The American journal of physiology, 265(6 Pt 1), C1517-26. doi:10.1152/ajpcell.1993.265.6.c1517
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  The effects of oleic acid (OA) on gap junction-mediated intercellular communication between A7r5 cells and neonatal rat cardiac myocytes were determined. In A7r5 cells the extent of dye coupling was influenced in a biphasic manner by increasing concentrations of OA. Low concentrations of OA (0.1-1 microM) reduced the incidence of dye coupling from 90% (in control cells) to approximately 50%. Further increases in OA concentration, up to 100 microM, had no further effect on extent of dye coupling. In contrast, dye coupling between cardiac myocytes was reduced to near zero levels in a linear fashion by 1-25 microM OA. Whereas high OA concentrations reduce junctional conductance (gj) between heart cells to zero [J. M. Burt, K. D. Massey, and B. N. Minnich. Am. J. Physiol. 260 (Cell Physiol. 29): C439-C448, 1991], gj between A7r5 cells was decreased by a maximum of 45% by OA. These differences in OA sensitivity between the two cell types were not explained by differences in the rate or magnitude of OA uptake by the cells or by differences in the fraction of incorporated OA accessible to albumin washout, i.e., the plasma membrane fraction. Instead, the activity of the individual channel types exhibited different sensitivities to OA. In the presence of increasing concentrations of OA, the activities of first the 70-pS channel population [composed of connexin40 (Cx40)] and then the 108-pS channel population (composed of Cx43) were diminished, leaving predominantly the 140-pS channels (composed of Cx43) at high OA concentrations. The uncoupling effects of OA in both cell types could be reversed by washout with albumin-containing solution; however, higher concentrations of albumin and more vigorous wash conditions were required for full recovery in the A7r5 cells. In addition, albumin also reversed the effects of OA on channel activity. These data suggest that OA binds with greater affinity to the 70- vs. 108- or 140-pS channels and associated with binding is reduced channel activity.
• Minnich, B. N., Massey, K. D., & Burt, J. M. (1992). Arachidonic acid and lipoxygenase metabolites uncouple neonatal rat cardiac myocyte pairs.. The American journal of physiology, 263(2 Pt 1), C494-501. doi:10.1152/ajpcell.1992.263.2.c494
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  The effects of arachidonic acid (AA) and its metabolites on the conductance (gj) of the gap junctions between neonatal rat myocardial cells was investigated. AA reduced gj in a dose- (2, 5, and 20 microM) and time-dependent fashion. Pretreatment of the cells with an inhibitor of the 5-lipoxygenase pathway, U-70344A, shifted the dose-response curve to the right; pretreatment with indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect. The mean time to uncoupling was 3.7 +/- 0.3, 3.8 +/- 0.9, and 4.6 +/- 0.6 min (means +/- SE, P less than 0.05) for 5 microM AA, 5 microM AA + indomethacin, and 5 microM AA + U-70344A, respectively. Incorporation of AA into membrane phospholipids was not affected by the inhibitor. These studies suggest that complete uncoupling of the cells occurred at membrane concentrations of 3-4 mol%. The data indicate that AA and a 5-lipoxygenase metabolite uncouple neonatal rat heart cells. The data are discussed with respect to the possible underlying mechanism of uncoupling and the potential role of gap junctions in arrhythmia formation in ischemic heart disease.
• Minnich, B. N., Massey, K. D., & Burt, J. M. (1991). Uncoupling of cardiac cells by fatty acids: structure-activity relationships.. The American journal of physiology, 260(3 Pt 1), C439-48. doi:10.1152/ajpcell.1991.260.3.c439
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  The permeability and conductance of gap junctions between pairs of neonatal rat heart cells were rapidly and reversibly decreased by oleic acid in a dose- and time-dependent manner. Other unsaturated fatty acids (C-18: cis 6, 9, or 11, and C-18, 16, and 14, cis 9), saturated fatty acids (C-10, 12, and 14), and saturated fatty alcohols (C-8, 10, and 12) also caused uncoupling. The most effective compounds of the unsaturated and saturated fatty acid and saturated fatty alcohol series caused essentially complete uncoupling at comparable aqueous concentrations. However, oleic acid uncoupled cells at membrane concentrations as low as 1 mol%, whereas decanoic acid required upwards of 35 mol%. The channels that support the action potential remained functional at these same membrane concentrations. The data are discussed in terms of the possible mechanism by which these compounds cause uncoupling and the possible role of uncoupling by nonesterified free fatty acids in the initiation of arrhythmias during and after ischemic insults.
• Moore, L. K., Burt, J. M., & Beyer, E. C. (1991). Characterization of gap junction channels in A7r5 vascular smooth muscle cells.. The American journal of physiology, 260(5 Pt 1), C975-81. doi:10.1152/ajpcell.1991.260.5.c975
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  Recent evidence suggest that coordination of blood flow in the microcirculation involves cell-to-cell coupling via gap junctions. In this study, using A7r5 cells as a model of vascular smooth muscle, we have characterized the gap junctions in terms of the unitary conductances of the observed channels, the responses to second messengers, and subunit protein composition. The cells were typically well coupled several hours after plating, with junctional conductances on the order 20-40 nS. Channels with mean conductances of 36 and 89 pS were observed in low-conductance cell pairs and in cell pairs whose macroscopic conductance was reduced by exposure to halothane. Connexin43 was the only known gap junction sequence detected by Northern blots (low and high stringency), immunoblots, or immunohistochemical studies. Junctional conductance was reduced 15% by 8-bromoadenosine 3',5'-cyclic monophosphate; 8-bromoguanosine 3',5'-cyclic monophosphate had no effect. The results suggest that connexin43 can form stable channels of at least two distinct conductances and gap junctions with differing responses to second messengers.
• Burt, J. M. (1989). Uncoupling of cardiac cells by doxyl stearic acids specificity and mechanism of action.. The American journal of physiology, 256(4 Pt 1), C913-24. doi:10.1152/ajpcell.1989.256.4.c913
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  The influence of doxyl stearic acids (DSAs) on gap junctional conductance (gj) between pairs of neonatal rat heart cells was studied. DSAs are spin probes that perturb the membrane at different depths depending on position of the doxyl group on the fatty acyl chain. 16-DSA and 12-DSA rapidly and reversibly reduced gj to unmeasureable levels in a dose- and time-dependent manner. Single channel events observed when gj was low were of the same unitary size as those observed under control conditions. The methyl esters of 16- and 12-DSA, stearic acid itself, and TEMPO, an analogue of the doxyl group that has no fatty acyl chain, had no effect on gj. Protonation of the carboxyl head group (by acidifying the solution) reduced the potency of 16- or 12-DSA. Spontaneous beating activity and action potentials were observed at concentrations of the DSAs 15-20 times that necessary for uncoupling. These results indicate that uncoupling by the DSAs requires the presence of the charged carboxyl group and localized perturbation of the channel at the lipid-channel interface by the doxyl group. Furthermore, they predict that unsaturated free fatty acids, which accumulate during ischemia, may exert their arrhythmogenic effect by reducing gj, and thereby slowing conduction.
• Spray, D. C., & Burt, J. M. (1989). Volatile anesthetics block intercellular communication between neonatal rat myocardial cells.. Circulation research, 65(3), 829-37. doi:10.1161/01.res.65.3.829
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  The effects of halothane and ethrane on gap junction-mediated intercellular communication and on membrane excitability were examined in cultured neonatal rat cardiac myocytes using whole-cell voltage-clamp and current-clamp techniques. Excitability was maintained at doses of both anesthetics that reversibly abolished current flow through junctional membranes. The degree of reduction of junctional conductance was a steep function of the dose of anesthetic; complete block occurred at lower aqueous concentrations of halothane than ethrane. The time course for loss of communication was rapid; 90% reduction of initial junctional conductance occurred in less than 15 seconds after exposure to 2 mM halothane or 4 mM ethrane. Recovery of junctional conductance and junctional permeability to intracellularly injected Lucifer yellow was rapid and complete on washout of the anesthetics. As junctional conductance was reduced by halothane or ethrane exposure, unitary conductance of the gap junctional channels remained constant at about 50 pS. Uncoupling by these anesthetics is thus attributable to a decrease in the number of conducting channels rather than to reduction of the channel's unitary conductance. The data are discussed with regard to the possible role of this intercellular communication pathway in the arrhythmias and alterations of conduction velocity and contractility produced by volatile anesthetics.
• Spray, D. C., & Burt, J. M. (1988). Inotropic agents modulate gap junctional conductance between cardiac myocytes.. The American journal of physiology, 254(6 Pt 2), H1206-10. doi:10.1152/ajpheart.1988.254.6.h1206
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  Cardiac gap junction channels mediate the intercellular exchange of second messenger and small molecules. Through effects on conduction velocity, enhanced junctional conductance (gj) facilitates rapid and synchronous activation of the contractile myocardium, whereas reduced gj slows activation and could contribute to arrhythmogenesis. We report here that gj is enhanced by agents that elevate intracellular adenosine 3',5'-cyclic monophosphate (cAMP; 8-bromo-cAMP or isoproterenol) and is depressed by agents that elevate intracellular guanosine 3',5'-cyclic monophosphate (cGMP; 8-bromo-cGMP or carbachol). Both effects occur with a time course comparable to the inotropic events mediated by these agents. The effect of cAMP on gj is not dependent on simultaneous changes in intracellular calcium; however, during calcium-overload conditions cAMP can precipitate calcium-dependent uncoupling. These results indicate that cyclic nucleotide-dependent changes in gj may contribute to the inotropic effects of these agents. Furthermore, the results suggest that the inotropic effect of cAMP includes a calcium-independent component.
• Spray, D. C., & Burt, J. M. (1988). Single-channel events and gating behavior of the cardiac gap junction channel.. Proceedings of the National Academy of Sciences of the United States of America, 85(10), 3431-4. doi:10.1073/pnas.85.10.3431
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  The activity of gap junction channels between pairs of neonatal rat heart cells in culture was studied under control conditions and during uncoupling procedures by using dual whole-cell voltage clamp techniques. Under control conditions gap junctional conductance ranged from 0.05 to 35 nS. In cell pairs exhibiting low gap junctional conductance (less than 500 pS), single-channel events with a unitary conductance of 53 +/- 2 pS (5 experiments; 186 events) were apparent. Event duration and open-time probability were estimated to be 0.95 sec and 0.17, respectively. When the junctional conductance in well-coupled cell pairs (with initial junctional conductance, greater than 5 nS) was reduced by cytoplasmic acidification or application of heptanol, single-channel events could be visualized. Compared to low-conductance controls, unitary channel conductance was unaltered (for acidification the conductance was 58 +/- 3 pS in 11 experiments with 253 events; for heptanol the conductance was 61 +/- 1 pS in 2 experiments with 171 events), while the probability of channels being open was decreased. The constancy of unitary channel conductance under control conditions and during uncoupling procedures suggests that opening and closing of the gap junction channel are all-or-none processes during which no stable subconductance states are formed.
• Burt, J. M. (1987). Block of intercellular communication: interaction of intracellular H+ and Ca2+.. The American journal of physiology, 253(4 Pt 1), C607-12. doi:10.1152/ajpcell.1987.253.4.c607
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  The influence of elevated intracellular levels of H+ and Ca2+ on intercellular communication between cultured neonatal rat myocardial cells was examined by quantifying the percent of primary neighboring cells to which intracellularly injected Lucifer yellow had spread within 10 s of injection. Partial acidosis was induced by incubation in and then removal of NH4Cl. Intracellular Ca2+ was raised through the use of treatments that are standard in studies of heart muscle: reduction of the Na+ gradient, addition of caffeine, and combinations of these interventions. Under control conditions and during application of NH4Cl, cells exhibited spontaneous electrical and contractile activity and were well coupled (dye detectable in 100% of primary neighbors). Sustained intracellular acidosis without simultaneous elevation of intracellular Ca2+ (NH4Cl exposure followed by zero Na+, zero Ca2+) reduced the incidence of dye transfer to 90%. Elevation of intracellular Ca2+ (exposure to zero Na+, Ca2+-containing solution, with or without 10 mM caffeine) had no effect on coupling. These same interventions, when employed together, reduced the incidence of dye coupling to 18%. The results are consistent with a synergism of action of Ca2+ and H+ in the regulation of junctional permeability.
• Copeland, J. G., & Burt, J. M. (1986). Myocardial function after preservation for 24 hours. The Journal of Thoracic and Cardiovascular Surgery, 92(2), 238-246. doi:10.1016/s0022-5223(19)35904-5
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  The orthotopically transplanted heart undergoes several steps between harvest from the donor and reperfusion in the recipient: (1) cardioplegic arrest and cooling, (2) ischemia during the operation, (3) in most cases a preservation or storage period of varying duration, and (4) reperfusion. Each of these steps represents a period during which damage to the heart can occur. In this study we have quantified the degree of damage sustained by a donor heart during each of these steps. This objective was achieved by evaluating the function of rabbit hearts via Langendorff procedures following (Group 1) cooling and reperfusion; (Group 2) cardioplegic arrest, cooling, and reperfusion; (Group 3) arrest, cooling, 1 hour of ischemia (5° or 25° C), and reperfusion; (Group 4) arrest, cooling, 24 hours of preservation, and reperfusion; and (Group 5) arrest, cooling, 24 hours of preservation, 1 hour of ischemia (25° C), and reperfusion. Comparisons were made between groups and to control hearts. Cooling and reperfusing the heart (Group 1) led to no loss of function, although recovery to precooling function levels required approximately 25 minutes. Hearts that were arrested before cooling (Group 2) regained full function without the slow recovery time. Hearts that were arrested, cooled, and made ischemic at 5° C (Group 3) recovered 95% of preischemic contractile function: maximum systolic pressure and the maximum positive derivative of the systolic pressure curve. No change in diastolic compliance was detected. Hearts that were arrested, cooled, and made ischemic at 25° C (Group 3) recovered 89% of preischemic contractile function (maximum systolic pressure and the maximum positive derivative of the systolic pressure curve). Again, no change in diastolic compliance was detected. Hearts that were arrested, cooled, preserved for 24 hours, and reperfused (Group 4) recovered 84% of control contractile function (maximum systolic pressure and the maximum positive derivative of the systolic pressure curve), whereas hearts that had the additional hour of ischemia at 25° C (Group 5) recovered only 75% of control contractile function. In the latter two groups diastolic compliance was also compromised. Group 4 had a 20% decrease in the volume required to reach 10 mm Hg, and Group 5 had a 26% decrease. Pressure-volume curves suggest a loss of contractility and a loss of compliance in these hearts. These data indicate that while significant damage occurred as a result of ischemia and reperfusion, this damage was masked by the larger decrease in function occurring as a result of the preservation period.

Presentations

• Burt, J. M. (2017, July 30, 2017). A Many-Faced Protein: The Yin and Yang of Connexins. International Gap Junction Conference , Keynote Address. Glasgow, Scotland.
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  Keynote Address
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2017, Summer). Differential roles of the carboxyl terminus in Cx43 channel gating and permeation. International Gap Junction Conference 2017. Glasgow: Gap Junction Interest Group.
• Burt, J. M. (2016, July). Connexin-specific differences in gap junction channel gating and permeations (as determinants of cell phenotype). Federation European Physiological Society and French Physiological Society. Paris, France: Federation European Physiological Society and French Physiological Society.
• Burt, J. M. (2015, November). Complementary Regulation of Cell Growth by Cx37 and Cx43. Seminar. Omaha, Nebraska: University of Nebraska Medical Ctr.

Poster Presentations

• Burt, J. M., Pontifex, T., Ek Vitorin, J., Cotter, M., Alamban, A., Fye, M., Jacobsen, N., Taylor, S., Lampe, P., Solan, J., & Boitano, S. (2019, July 27-31). 5 abstracts: 1 oral presentation 4 posters,. International Gap Junction Conference. Victoria, Canada: Independent Group.
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  S5.3 Intra‐Domain Interactions of the Cx37 Carboxyl‐Terminus Necessary for Growth Suppressive Function Alter Channel Gating Tasha Pontifex¹, Nicole Jacobsen², Samantha Taylor¹, Janis Burt¹ ¹University of Arizona, ²University of Missouri ORAL PRESENTATIONP‐B‐32 A mutant mimicking an ischemic preconditioned phospho‐form of Cx43 lacks Vj‐gating. Jose Ek‐Vitorin¹, Tasha Pontifex¹, Janis Burt¹ ¹University of ArizonaP‐C‐41 Connexin 37 growth suppressive phenotype is regulated in a site‐specific, phosphorylation-dependent manner. Samantha Taylor¹, Tasha Pontifex¹, Nicole Jacobsen¹, Janis Burt¹ ¹University of ArizonaP‐C‐44 Cx37‐13k fails to reproduce phenotypic effects of Cx37 in rat insulinoma cells but alters Cx37’s hemichannel function when co‐expressed Andrew John Alamban¹, Tasha Pontifex, Janis Burt ¹The University of ArizonaP‐J‐88 The lipidated connexin mimetic peptide, SRPTEKT‐Hdc, is a potent inhibitor of Cx43 channels with specificity for the pS368 phospho‐isoformMaura Cotter¹, Paul Lampe², Joell Solan², Josef Vagner¹, Jose Ek‐Vitorin¹, Boitano S, Janis Burt¹ ¹University of Arizona, ²Fred Hutchinson Cancer Research Center
• Delamere, N. A., Burt, J. M., Mandal, A., Shahidullah, M., & Ek Vitorin, J. F. (2018, Fall). Opening for common goals: the transient (calcium) connection of TRP and Cx channels. 2018 Arizona Physiological Society (AZPS) Annual Meeting. Tempe AZ: AZPS.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2017, Fall). Comparison of two phospho-mimic mutations of Cx43 with opposite arrhythmogenic potential. 2017 Arizona Physiological Society (AZPS) Annual Meeting. NAU, Flagstaff, Arizona.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2017, Fall). Comparison of two phospho-mimic mutations of Cx43 with opposite arrhythmogenic potential. Arizona Physiological Society. NAU, Flagstaff, Arizona.
• Burt, J. M., Kanady, J., & Jacobsen, N. (2016, April). Two posters presented at Experimental Biology meeting by co-authors listed below. Experimental Biology. San Diego.
• Burt, J. M., Cotter, M., Jacobsen, N., Pontifex, T., & Ek Vitorin, J. (2015, March). Co-author on posters presented by each of the persons listed as co-authors below. International Gap Junction Conference. Chile.