Jose F Ek Vitorin

Interests

Research

Cardiovascular Physiology. Gap Junction Physiology and Regulation. TRP channels and Connexin Hemichannels interaction.

Courses

No activities entered.

Scholarly Contributions

Chapters

• Ek Vitorin, J. F. (2017). What Do You Need to Measure Gap Junctional Permselectivity?. In Gap Junction Channels and Hemichannels(pp 114-147). Taylor & Francis Group.
• Delmar, M., Stergiopoulos, K., Homma, N., Calero, G., Calero, G., Ek-Vitorin, J. F., & Taffet, S. M. (1999). Chapter 11: A Molecular Model for the Chemical Regulation of Connexin43 Channels: The “Ball-and-Chain” Hypothesis. In Current Topics in Membranes. Gap Junctions — Molecular Basis of Cell Communication in Health and Disease(pp 223–248). Elsevier.
• Delmar, M., Morley, G. E., Ek-Vitorin, J. F., Francis, D., Homma, N., Stergiopoulos, K., Lau, A., & Taffet, S. M. (1998). Intercellular regulation of the cardiac gap junction channel connexin43. In Cardiac Electrophysiology: From Cell to Beside. W.B. Saunders Company.
• Delmar, M., Stergiopoulos, K., Homma, N., Ek-Vitorin, J. F., & Taffet, S. M. (1998). A ball-and-chain model for chemical regulation of connexin43. In Gap Junctions(pp 8-12). ISO Press.
• Delmar, M., Ek, J. F., Liu, S., & Taffet, S. M. (1995). Studies on structure/function relation of pH gating of Cx43. In Progress in Cell Research, Vol. 4.
• Delmar, M., Liu, S., Morley, G. E., Ek, J. F., Pertsova, R. N., Anumonwo, J. M., & Taffet, S. M. (1995). Toward a molecular model for the pH regulation of intercellular communication in the heart. In Cardiac Electrophysiology: From Cell to Bedside(pp 135-143). W.B. Saunders Company.

Journals/Publications

• Ek Vitorin, J. F., Shahidullah, M., Lopez Rosales, J. E., & Delamere, N. A. (2023). Patch clamp studies on TRPV4-dependent hemichannel activation in lens epithelium. Frontiers in Pharmacology, 14, 1101498. doi:doi: 10.3389/fphar.2023.110149
• Ek-Vitorin, J. F., & Jiang, J. X. (2023). The Role of Gap Junctions Dysfunction in the Development of Cataracts: From Loss of Cell-to-Cell Transfer to Blurred Vision-Review. Bioelectricity, 5(3), 164-172.
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  Mutations of lens connexins are linked to congenital cataracts. However, the role of connexin mutations in the development of age-related lens opacification remains largely unknown. Here, we present a focused review of the literature on lens organization and factors associated with cataract development. Several lines of evidence indicate that disturbances of the lens circulation by dysfunctional connexin channels, and/or accumulation of protein damage due to oxidative stress, are key factors in cataract development. Phosphorylation by protein kinase A improves the permeability of connexins channels to small molecules and mitigates the lens clouding induced by oxidative stress. We conclude (1) that connexin channels are central to the lens circulation and (2) that their permeability to antioxidant molecules contributes to the maintenance of lens transparency.
• Ek-Vitorin, J. F., Silva-Mendoza, D., Pontifex, T. K., & Burt, J. M. (2023). Channel Behavior and Voltage Gating of a Cx43 Mutant Simulating Preconditioning. Bioelectricity, 5(3), 181-187.
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  Ischemic preconditioning induces lateralization and dephosphorylation of Connexin 43 (Cx43). However, the Cx43 protein that remains at intercalated disks may be phosphorylated by casein kinase 1 (CK1) and protein kinase C (PKC), and both kinases provide cardioprotection from further ischemic injury. Here we explore the channel characteristics of a Cx43 mutant mimicking preconditioning by CK1 and PKC phosphorylation.
• Babicheva, A., Ayon, R. J., Zhao, T., Ek Vitorin, J. F., Pohl, N. M., Yamamura, A., Yamamura, H., Quinton, B. A., Ba, M., Wu, L., Ravellette, K. S., Rahimi, S., Balistrieri, F., Harrington, A., Vanderpool, R. R., Thistlethwaite, P. A., Makino, A., & Yuan, J. X. (2020). MicroRNA-mediated downregulation of K channels in pulmonary arterial hypertension. American journal of physiology. Lung cellular and molecular physiology, 318(1), L10-L26.
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  Downregulated expression of K channels and decreased K currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3'-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K currents and downregulated voltage-gated K channel 1.5 (K1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K channel function and K1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that K1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca-activated K (BK) channel currents and downregulated BK channel β1 subunit (BKβ1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BK channel activity and BKβ1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of K and BK channels in PASMC from patients with IPAH.
• Cotter, M. L., Boitano, S. A., Lampe, P. D., Solan, J. L., Vagner, J., Ek Vitorin, J. F., & 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.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2018). Cx43 channel gating and permeation: Differential roles of the carboxyl terminus. IJMS, 19(6), E1659. doi:10.3390/ijms19061659
• 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.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2016). Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus. Biophysical Journal, 110, 127-140. doi:doi: 10.1016/j.bpj.2015.10.054.
• 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.
• 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. doi:DOI: 10.1074/jbc.M114.583054
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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.
• 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. doi:DOI: 10.1016/j.healun.2013.12.004
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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).
• 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), 51-68. doi:DOI: 10.1016/j.bbamem.2012.02.003
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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.
• 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), 369-80. doi:DOI: 10.1007/s00232-012-9459-x
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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.
• 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. doi:DOI: 10.1074/jbc.M111.218735
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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
• 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), H450-9. doi:DOI: 10.1152/ajpheart.00287.2009
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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.
• 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), 1498-505. doi:DOI: 10.1161/01.RES.0000227572.45891.2c
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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.
• Ek-Vitorín, J. F., & Burt, J. M. (2005). Quantification of gap junction selectivity. American journal of physiology. Cell physiology, 289(6), C1535-46. doi:DOI: 10.1152/ajpcell.00182.2005
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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.
• Gu, H., Ek-Vitorin, J. F., Taffet, S. M., & Delmar, M. (2000). Coexpression of connexins 40 and 43 enhances the pH sensitivity of gap junctions: a model for synergistic interactions among connexins. Circulation research, 86(10), E98-E103.
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  Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype. Xenopus oocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from approximately 6.7 when cells expressed only Cx40 or only Cx43 to approximately 7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined.
• Yahuaca, P., Ek-Vitorin, J. F., Rush, P., Delmar, M., & Taffet, S. M. (2000). Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner. Brazilian journal of medical and biological research = Revista brasileira de pesquisas médicas e biológicas / Sociedade Brasileira de Biofísica ... [et al.], 33(4), 399-406.
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  The carboxyl-terminal (CT) domain of connexin43 (Cx43) has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST)-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.
• Delmar, M., Stergiopoulos, K., Homma, N., Calero, G., Morley, G. E., Ek-vitorin, J. F., & Taffet, S. M. (1999). Chapter 11: A Molecular Model for the Chemical Regulation of Connexin43 Channels: The "Ball-and-Chain" Hypothesis. Current Topics in Membranes, 49, 223-248. doi:10.1016/s0070-2161(08)61015-1
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  Publisher Summary It is the current view that gap junctions are not only passive conduits of electrical charge but also dynamic filters that modulate the passage of molecular messages within the cellular network; hence, not only the presence but also the regulation of connexins is important for synchronous tissue function. Research has focused on the regulation of connexins by two separate factors: intracellular pH and insulin (or IGF) exposure. Regulation of gap junction conductance by factors such as pH i and cytokines may be critical in the development of lethal cardiac arrhythmias during ischemia. Growth factor-mediated regulation of intercellular communication may also be important for normal cardiogenesis. Finally, changes in pH sensitivity after connexin processing may allow the preservation of cell–cell communication in the lens. Understanding the molecular events controlling connexin regulation and developing means to manipulate their occurrence allow learning more about the importance of intercellular communication both in health and disease.
• Francis, D., Stergiopoulos, K., Ek-Vitorín, J. F., Cao, F. L., Taffet, S. M., & Delmar, M. (1999). Connexin diversity and gap junction regulation by pHi. Developmental genetics, 24(1-2), 123-36.
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  The molecular mechanisms controlling pH-sensitivity of gap junctions formed of two different connexins are yet to be determined. We used a proton-sensitive fluorophore and electrophysiological techniques to correlate changes in intracellular pH (pHi) with electrical coupling between connexin-expressing Xenopus oocytes. The pH sensitivities of alpha 3 (connexin46), alpha 2 (connexin38), and alpha 1 (connexin43) were studied when these proteins were expressed as: 1) nonjunctional hemichannels (for alpha 3 and alpha 2), 2) homotypic gap junctions, and 3) heterotypic gap junctions. We found that alpha 3 hemichannels are sensitive to changes in pHi within a physiological range (pKa = 7.13 +/- 0.03; Hill coefficient = 3.25 +/- 1.73; n = 8; mean +/- SEM); an even more alkaline pKa was obtained for alpha 2 hemichannels (pKa = 7.50 +/- 0.03; Hill coefficient = 3.22 +/- 0.66; n = 13). The pH sensitivity curves of alpha 2 and alpha 3 homotypic junctions were indistinguishable from those recorded from hemichannels of the same connexin. Based on a comparison of pKa values, both alpha 3 and alpha 2 gap junctions were more pHi-dependent than alpha 1. The pH sensitivity of alpha 2-containing heterotypic junctions could not be predicted from the behavior of the two connexons in the pair. When alpha 2 was paired with alpha 3, the pH sensitivity curve was similar to that obtained from alpha 2 homotypic pairs. Yet, pairing alpha 2 with alpha 1 shifted the curve similar to homotypic alpha 1 channels. Pairing alpha 2 with a less pH sensitive mutant of alpha 1 (M257) yielded the same curve as when alpha 1 was used. However, the pH sensitivity curve of alpha 3/alpha 1 channels was similar to alpha 3/alpha 3, while alpha 3/M257 was indistinguishable from alpha 3/alpha 1. Our results could not be consistently predicted by a probabilistic model of two independent gates in series. The data show that dissimilarities in the pH regulation of gap junctions are due to differences in the primary sequence of connexins. Moreover, we found that pH regulation is an intrinsic property of the hemichannels, but pH sensitivity is modified by the interactions between connexons. These interactions should provide a higher level of functional diversity to gap junctions that are formed by more than one connexin.
• Stergiopoulos, K., Alvarado, J. L., Mastroianni, M., Ek-Vitorin, J. F., Taffet, S. M., & Delmar, M. (1999). Hetero-domain interactions as a mechanism for the regulation of connexin channels. Circulation research, 84(10), 1144-55.
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  Previous studies have shown that chemical regulation of connexin43 (Cx43) depends on the presence of the carboxyl terminal (CT) domain. A particle-receptor (or "ball-and-chain") model has been proposed to explain the mechanism of gating. We tested whether the CT region behaved as a functional domain for other members of the connexin family. The pH sensitivity of wild-type and Ct-truncated connexins was quantified by use of electrophysiological and optical techniques and the Xenopus oocyte system. The CT domain of Cx45 had no role in pH regulation, although a partial role was shown for Cx37 and Cx50. A prominent effect was observed for Cx40 and Cx43. In addition, we found that the CT domain of Cx40 that was expressed as a separate fragment rescued the pH sensitivity of the truncated Cx40 (Cx40tr), which was in agreement with a particle-receptor model. Because Cx40 and Cx43 often colocalize and possibly heteromerize, we tested the pH sensitivity of Cx40tr when coexpressed with the CT domain of Cx43 (hetero-domain interactions). We found that the CT domain of Cx43 enhanced the pH sensitivity of Cx40tr; similarly, the CT domain of Cx40 restored the pH sensitivity of the truncated Cx43. In addition, the CT domain of Cx43 granted insulin sensitivity to the otherwise insulin-insensitive Cx26 or Cx32 channels. These data show that the particle-receptor model is preserved in Cx40 and the regulatory domain of one connexin can specifically interact with a channel formed by another connexin. Hetero-domain interactions could be critical for the regulation of heteromeric channels.
• Morley, G. E., Ek-Vitorín, J. F., Taffet, S. M., & Delmar, M. (1997). Structure of connexin43 and its regulation by pHi. Journal of cardiovascular electrophysiology, 8(8), 939-51.
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  Electrical coupling in the heart provides an effective mechanism for propagating the cardiac action potential efficiently throughout the entire heart. Cells within the heart are electrically coupled through specialized membrane channels called gap junctions. Studies have shown that gap junctions are dynamic, carefully regulated channels that are important for normal cardiogenesis. We have recently been interested in the molecular mechanisms by which intracellular acidification leads to gap junction channel closure. Previous results in this lab have shown that truncation of the carboxyl terminal (CT) of connexin43 (Cx43) does not interfere with functional channel expression. Further, the pH-dependent closure of Cx43 channels is significantly impaired by removal of this region of the protein. Other studies have shown that the CT is capable of interacting with its receptor even when not covalently attached to the channel protein. From these data we have proposed a particle-receptor model to explain the pH-dependent closure of Cx43 gap junction channels. Detailed analysis of the CT has revealed interesting new information regarding its possible structure. Here we review the most recent studies that have contributed to our understanding of the molecular mechanisms of regulation of the cardiac gap protein Cx43.
• Ek-Vitorín, J. F., Calero, G., Morley, G. E., Coombs, W., Taffet, S. M., & Delmar, M. (1996). PH regulation of connexin43: molecular analysis of the gating particle. Biophysical journal, 71(3), 1273-84.
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  Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.
• Ek, J. F., Delmar, M., Perzova, R., & Taffet, S. M. (1994). Role of histidine 95 on pH gating of the cardiac gap junction protein connexin43. Circulation research, 74(6), 1058-64.
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  We have studied the role of histidine 95 (H95) on the pH gating of the cardiac gap junction protein connexin43 (Cx43). Wild-type and mutant rat cardiac Cx43 channels were expressed in antisense-injected Xenopus oocytes. Junctional conductance was measured using the dual voltage-clamp technique, and intracellular acidification was induced by superfusion with a sodium acetate-containing solution balanced at a pH of 6.2. H95 was substituted by other amino acids by use of oligonucleotide-directed site-specific mutagenesis. Replacing H95 for the hydrophobic residues methionine or phenylalanine, for the charged basic residue arginine, or for the noncharged residue glutamine (H95Q) yielded nonfunctional channels. Functional expression of H95Q was rescued by placing a histidine residue in position 93 (H95Q-L93H), 94 (H95Q-A94H), or 97 (H95Q-F97H) but not in position 96. Further experiments showed that replacing H95 with either aspartate (an acidic residue) or tyrosine (a polar uncharged residue) led to the expression of functional channels with a reduced susceptibility to acidification-induced uncoupling, whereas lysine (a basic residue) was more susceptible to uncoupling than the wild-type protein. The susceptibility to acidification-induced uncoupling was enhanced for the H95Q-A94H mutant when compared with the wild-type mutant, but it was significantly reduced when histidine was placed at position 93 (H95Q-L93H). Our data indicate that a properly placed histidine residue is an important structural element for functional expression as well as for pH regulation of Cx43. The results suggest that the importance of H95 on pH gating may be associated with a possible protonation of this residue on acidification of the intracellular environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Presentations

• 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.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2016, October). Channels made of a specific Cx43 phospho-isoform contribute to normal cardiac function. 2016 Arizona Physiological Society (AZPS) Annual Meeting. Tucson, AZ: AZPS.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2015, Spring). Connexin channel gating: evaluating triggers and mechanisms. International Gap Junction Conference. Valparaiso, Chile.
• Calero, G. A., Ek Vitorin, J. F., Taffet, S. M., & Delmar, M. (1996, Fall). Mutation of a single amino acid (Asp 379) prevents pH regulation of connexin43. Biophysical Society Annual Meeting.
• Ek Vitorin, J. F., Delmar, M., & Taffet, S. M. (1995, Fall). Two specific sites of the carboxyl terminal domain of rat cardiac connexin43 are essential for normal pH gating of gap junctions. International Gap Junction Conference. France.

Poster Presentations

• Ek Vitorin, J. F., Shahidullah, M., Lopez Rosales, J. E., & Delamere, N. A. (2022, Fall). Stimulation of the mechanosensor transient receptor potential vanilloid 4 (TRPV4) channels opens connexin hemichannels. Arizona Physiological Society. Scottsdale, Arizona: AZPS.
• Ek Vitorin, J. F., Shahidullah, M., Lopez Rosales, J. E., & Delamere, N. A. (2022, Summer). Stimulation of transient receptor potential vanilloid 4 (TRPV4) channels opens connexin hemichannels. International Gap Junction Conference. A Coruña, Spain: Gap Junction Community.
• Ek Vitorin, J. F., Pontifex, T. K., Silva-Mendoza, D., & Burt, J. M. (2019, November). A mutant mimicking an ischemic preconditioned phospho-form of Cx43 lacks Vj-gating. AZPS Annual Meeting. Tempe, AZ: Arizona State University.
• Ek Vitorin, J. F., Shahidullah, M., Mandal, A., Burt, J. M., & Delamere, N. A. (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. (2016, Winter). Channels made of a specific phospho-isoform contribute to normal cardiac function. 2016 Arizona Physiological Society (AZPS) Annual Meeting. Tucson, AZ: AZPS.
• Ek Vitorin, J. F., Pontifex, T. K., & Burt, J. M. (2015, Spring). Connexin channel gating: evaluating triggers and mechanisms. International Gap Junction Conference. Valparaiso, Chile.
• Ek Vitorin, J. F., Nelson, T., & Burt, J. M. (2013, Fall). Gating and permselectivity properties of Cx43 gap junctions channels are modified in Cx43*NT37 chimera. International Gap Junction Conference. Charleston, SC, USA.
• Good, M. E., Ek-Vitorin, J. F., & Burt, J. M. (2013, Fall). Extracellular loop structure is uniquely critical to Cx37 hemichannels as well as gap junction channel function. International Gap Junction Conference. Charleston, SC, USA.
• Ek Vitorin, J. F., & Burt, J. M. (2011, Fall). The dye permeability (PDYE) of junctions composed of truncated Cx43 is proportional to junctional conductance (gj) and less than predicted. International Gap Junction Conference. Ghent, Belgium.
• Ek Vitorin, J. F., & Burt, J. M. (2009, Summer). Probenecid decreases dye permeability without changing the charge-selectivity of Cx43 junctions. International Gap Junction Conference. Sedona, Arizona.
• Ek Vitorin, J. F., Nelson, T. K., & Burt, J. M. (2009, Summer). Cx37 carboxyl terminus confers on the pore forming domain of Cx43 growth-suppressive function. International Gap Junction Conference. Sedona, Arizona, USA.
• Gu, H., Ek-Vitorin, J. F., Stergiopoulos, K., Taffet, S. S., & Delmar, M. (1999, Fall). Coexpression of 2 connexins (Cx40 and Cx43) enhances pH sensitivity of gap-junctions. American Heart AssociationAmerican Heart Association.
• Ek Vitorin, J. F., Homma, N., Calero, G., Taffet, S. M., & Delmar, M. (1996, Fall). Characterization of the pHi sensitivity of connexin 46 gap junction channels. ASCB Annual Meeting.
• Ek Vitorin, J. F., Morley, G. E., Coombs, W., Taffet, S. M., & Delmar, M. (1996, Fall). Characterization of the carboxyl terminal regions involved in pH regulation of connexin43. Biophysical Society Annual Meeting.
• Ek, J. F., Taffet, S. M., & Delmar, M. (1994, Fall). Truncation of rat connexin43 at amino acid 361 impairs acidification-induced closure of gap junctional channels. ASCB Annual Meeting.
• Ek, J. F., Delmar, M., Pertsova, R., & Taffet, S. (1993, fall). Functional expression and pH sensitivity of connexin 43 cytoplasmic loop histidine mutants. International Meeting on Gap Junctions.
• Ek, J. F., Delmar, M., Pertsova, R., & Taffet, S. M. (1993, Fall). Role of the carboxyl tail, and of histidine 95, on the pH gating of the cardiac gap junction protein, connexin43 (Cx43). American Heart Association 66th Scientific Sessions.
• Morley, G. E., Perzova, R., Anumonwo, J., Ek, J. F., Taffet, S., & Jalife, J. (1993, Fall). Protein kinase C regulation of junctional conductance between cell pairs expressing exogenous connexin 43. Biophysical Society Annual Meeting.

Reviews

• Fang, J. S., & Ek Vitorin, J. F. (2023. Connexins: A Brief Overview(pp 153-163).
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  AbstractConnexins, elementary protein units of gap junctions, make intercellular and membrane channels that work as conduits for ions and larger molecules >1 kDa. Electrically, cells well coupled by gap junctions display a relatively uniform resting potential. In excitable tissues, gap junctions are the pathway for electrotonic propagation of action potentials. When hemichannels open in the membrane, connecting the cytoplasm and the extracellular space, the resting potential could collapse, and action potential propagation be impaired. Because connexin channels are permeated by large molecules, gap junctions are assumed to synchronize cellular functions by the sharing of second messengers, metabolites, and other substances. In turn, hemichannel opening could allow the escape of those same substances, and the uptake of extracellular molecules. Separately fromtheir channel function, parts of the connexin molecule can induce cellular changes that suggest protein-protein interactions with the cytoskeleton, regulatory pathways, and the genomic machinery of the cells. This minireview gives an overview of connexins and discusses some of the outstanding issues in the field.