Joao Luis Carvalho De Souza
- Assistant Research Professor, Anesthesiology
- Research Assistant Professor, Physiology
- Research Assistant Professor, Ophthalmology
Contact
- (520) 626-7221
- Arizona Health Sciences Center, Rm. 4401
- Tucson, AZ 85724
- jcds@arizona.edu
Degrees
- Ph.D.
- State University of Ceara, Fortaleza, Brazil
- M.S.
- State University of Ceara, Fortaleza, Brazil
- B.S.
- State University of Ceara, Fortaleza, Brazil
Interests
No activities entered.
Courses
2022-23 Courses
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Methods In Neuroscience
NRSC 700 (Fall 2022)
2019-20 Courses
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Directed Research
PSIO 492 (Spring 2020) -
Independent Study
PSIO 499 (Spring 2020) -
Directed Research
PSIO 492 (Fall 2019)
Scholarly Contributions
Chapters
- Treger, J. S., Pepperberg, D. R., Bezanilla, F., & De Souza, J. L. (2018). Optocapacitance Allows for Photostimulation of Neurons without Requiring Genetic Modification. In Use of Nanoparticles in Neuroscience(pp 1-13). Humana Press, New York, NY. doi:10.1007/978-1-4939-7584-6_1More infoOptocapacitance is a novel technique that combines much of the power of optogenetics without requiring any transfection or genome modification in the organism or tissue of interest. It functions via the same principles as infrared neural stimulation, but uses cell-targeted gold nanoparticles to transduce incident light into local heating that stimulates excitable cells by altering their membrane capacitance. We have demonstrated this technique in both isolated neurons and brain slices, and in this chapter we describe in detail the methods used in these studies. Overall, optocapacitance is a technique that should be widely applicable to many cell-types and tissues, it can be performed using commercially available preparations with no modification, and this can complement optogenetic techniques in situations where the cost and difficulty of genetic modification are not justified.
Journals/Publications
- Moreira-Junior, L., Leal-Cardoso, J. H., Cassola, A. C., & Carvalho-de-Souza, J. L. (2024). State-Dependent Blockade of Dorsal Root Ganglion Voltage-Gated Na Channels by Anethole. International journal of molecular sciences, 25(2).More infoAnethole is a phenolic compound synthesized by many aromatic plants. Anethole is a substance that humans can safely consume and has been studied for years as a biologically active molecule to treat a variety of conditions, including nerve damage, gastritis, inflammation, and nociception. Anethole is thought to carry out its biological activities through direct interaction with ion channels. Anethole is beneficial for neurodegenerative Alzheimer's and Parkinson's diseases. Nevertheless, nothing has been investigated regarding the effects of anethole on voltage-gated Na+ channels (VGSCs), which are major players in neuronal function. We used cultured dorsal root ganglion neurons from neonatal rats as a source of natively expressed VGSCs for electrophysiological studies using the whole-cell patch-clamp technique. Our data show that anethole interacts directly with VGSCs. Anethole quickly blocks and unblocks (when removed) voltage-activated Na+ currents in this preparation in a fully reversible manner. Anethole's binding affinity to these channels increases when the inactive states of these channels are populated, similar to lidocaine's effect on the same channels. Our data show that anethole inhibits neuronal activity by blocking VGSCs in a state-dependent manner. These findings relate to the putative anesthetic activity attributable to anethole, in addition to its potential benefit in neurodegenerative diseases.
- Rosales, J. L., De Souza, J. L., & Chignalia, A. Z. (2023). Glypican 1 and intracellular calcium levels in lung endothelial cells. ASIP 2023 Joint Meeting of ASMB, HCS, and ASIP.
- Thota, L., Potje, S., Bendhack, L., De Souza, J. L., & Chignalia, A. Z. (2023). New Insights Into The Role Of The Glycocalyx Components In Heart Failure Induced By Pressure Overload. Circulation Research, AP2048-AP2048.
- Flood, M. D., Veloz, H. L., Hattar, S., & Carvalho-de-Souza, J. L. (2022). Robust visual cortex evoked potentials (VEP) in Gnat1 and Gnat2 knockout mice. Frontiers in cellular neuroscience, 16, 1090037.More infoIntrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin, imparting to themselves the ability to respond to light in the absence of input from rod or cone photoreceptors. Since their discovery ipRGCs have been found to play a significant role in non-image-forming aspects of vision, including circadian photoentrainment, neuroendocrine regulation, and pupillary control. In the past decade it has become increasingly clear that some ipRGCs also contribute directly to pattern-forming vision, the ability to discriminate shapes and objects. However, the degree to which melanopsin-mediated phototransduction, versus that of rods and cones, contributes to this function is still largely unknown. Earlier attempts to quantify this contribution have relied on genetic knockout models that target key phototransductive proteins in rod and cone photoreceptors, ideally to isolate melanopsin-mediated responses. In this study we used the Gnat1; Gnat2 mouse model, which have global knockouts for the rod and cone α-transducin proteins. These genetic modifications completely abolish rod and cone photoresponses under light-adapted conditions, locking these cells into a "dark" state. We recorded visually evoked potentials in these animals and found that they still showed robust light responses, albeit with reduced light sensitivity, with similar magnitudes to control mice. These responses had characteristics that were in line with a melanopsin-mediated signal, including delayed kinetics and increased saturability. Additionally, we recorded electroretinograms in a sub-sample of these mice and were unable to find any characteristic waveform related the activation of photoreceptors or second-order retinal neurons, suggesting ipRGCs as the origin of light responses. Our results show a profound ability for melanopsin phototransduction to directly contribute to the primary pattern-forming visual pathway.
- Souza, D. S., Chignalia, A. Z., & Carvalho-de-Souza, J. L. (2022). Modulation of cardiac voltage-activated K currents by glypican 1 heparan sulfate proteoglycan. Life sciences, 308, 120916.More infoGlypican 1 (Gpc1) is a heparan sulfate proteoglycan attached to the cell membrane via a glycosylphosphatidylinositol anchor, where it holds glycosaminoglycans nearby. We have recently shown that Gpc1 knockout (Gpc1) mice feature decreased systemic blood pressure. To date, none has been reported regarding the role of Gpc1 on the electrical properties of the heart and specifically, in regard to a functional interaction between Gpc1 and voltage-gated K channels.
- Bassetto, C. A., Carvalho-de-Souza, J. L., & Bezanilla, F. (2021). Molecular basis for functional connectivity between the voltage sensor and the selectivity filter gate in K channels. eLife, 10.More infoIn K channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower charge-voltage (Q-V) curve that crosses the conductance-voltage (G-V) curve. In L366H:W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A, or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels.
- Carvalho De Souza, J. L., Saponaro, A., Bassetto Jr, C., Oliver, R., Schroeder, I., Franciolini, F., Catacuzzeno, L., Bezanilla, F., Thiel, G., & Moroni, A. (2021). Experimental challenges in ion channel research: Uncovering basic principles of permeation and gating in potassium channels. Advances in Physics: X, 7(1), 1978317.
- Potje, S. R., Isbatan, A., Tostes, R. C., Bendhack, L. M., Dull, R. O., Carvalho-de-Souza, J. L., & Chignalia, A. Z. (2021). Glypican 1 and syndecan 1 differently regulate noradrenergic hypertension development: Focus on IP3R and calcium. Pharmacological research, 172, 105813.More infoVascular dysfunction is a checkpoint to the development of hypertension. Heparan sulfate proteoglycans (HSPG) participate in nitric oxide (NO) and calcium signaling, key regulators of vascular function. The relationship between HSPG-mediated NO and calcium signaling and vascular dysfunction has not been explored. Likewise, the role of HSPG on the control of systemic blood arterial pressure is unknown. Herein, we sought to determine if the HSPG syndecan 1 and glypican 1 control systemic blood pressure and the progression of hypertension.
- Souza, D., Chignalia, A. Z., & Carvalho-de-souza, J. L. (2021). Modulation of Ventricular Cardiomyocytes Electrophysiology by Glypican 1 Heparan Sulfate Proteoglycan. Biophysical Journal, 120(3), 244a. doi:10.1016/j.bpj.2020.11.1598
- Bassetto, C. A., Carvalho-de-souza, J. L., & Bezanilla, F. (2020). Metal Bridge in S4 Segment Supports Helix Transition in Shaker Channel.. Biophysical journal, 118(4), 922-933. doi:10.1016/j.bpj.2019.08.035More infoVoltage-gated ion channels play important roles in physiological processes, especially in excitable cells, in which they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared; the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, the possible helix transition from α- to 310-helix in S4 during the activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i + 3 and i, i + 6 or i, i + 4 and i, i + 7 pairs, to favor 310- or α-helical conformations, respectively. We have taken advantage of the ability of His to coordinate Zn2+ to promote metal ion bridges, and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In the presence of 20 μM of Zn2+, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, and the voltage dependence is shifted by 10 mV toward depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation, a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.
- Cannon, M., Garcia, R., Garcia, R., Dull, R. O., Chignalia, A. Z., Dull, R. O., Carvalho-de-souza, J. L., Carvalho-de-souza, J. L., Cannon, M., & Chignalia, A. Z. (2020). Glypican‐1 Plays a Cardiac Protective Effect During Heart Failure Induced by Pressure Overload. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05845
- Carvalho-de-souza, J. L., Bezanilla, F., Jr, C. A., & Bassetto, C. A. (2020). Residues Connecting Voltage Sensor Domain to Pore Domain in Shaker K+ Channel by Noncanonical Coupling Mechanism. Biophysical Journal, 118(3), 333a. doi:10.1016/j.bpj.2019.11.1860
- Du, X., Carvalho-de-Souza, J. L., Wei, C., Carrasquel-Ursulaez, W., Lorenzo, Y., Gonzalez, N., Kubota, T., Staisch, J., Hain, T., Petrossian, N., Xu, M., Latorre, R., Bezanilla, F., & Gomez, C. M. (2020). Loss-of-function BK channel mutation causes impaired mitochondria and progressive cerebellar ataxia. Proceedings of the National Academy of Sciences of the United States of America, 117(11), 6023-6034.More infoDespite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene , encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BK) in , in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BK channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BK cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BK channel, but not the BK channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.
- Hansen, M., Garcia, R., Dull, R. O., Chignalia, A. Z., & Carvalho-de-souza, J. L. (2020). Glypican‐1 and Remodeling Cardiac Hypertrophy.. The FASEB Journal, 34(S1), 1-1. doi:10.1096/fasebj.2020.34.s1.05765
- Acarón Ledesma, H., Li, X., Carvalho-de-Souza, J. L., Wei, W., Bezanilla, F., & Tian, B. (2019). An atlas of nano-enabled neural interfaces. Nature nanotechnology, 14(7), 645-657.More infoAdvances in microscopy and molecular strategies have allowed researchers to gain insight into the intricate organization of the mammalian brain and the roles that neurons play in processing information. Despite vast progress, therapeutic strategies for neurological disorders remain limited, owing to a lack of biomaterials for sensing and modulating neuronal signalling in vivo. Therefore, there is a pressing need for developing material-based tools that can form seamless biointerfaces and interrogate the brain with unprecedented resolution. In this Review, we discuss important considerations in material design and implementation, highlight recent breakthroughs in neural sensing and modulation, and propose future directions in neurotechnology research. Our goal is to create an atlas for nano-enabled neural interfaces and to demonstrate how emerging nanotechnologies can interrogate neural systems spanning multiple biological length scales.
- Bassetto, C. A., Carvalho-de-Souza, J. L., & Bezanilla, F. (2019). Metal Bridge in S4 Segment Supports Helix Transition in Shaker Channel. Biophysical journal.More infoVoltage-gated ion channels play important roles in physiological processes, especially in excitable cells, in which they shape the action potential. In S4-based voltage sensors voltage-gated channels, a common feature is shared; the transmembrane segment 4 (S4) contains positively charged residues intercalated by hydrophobic residues. Although several advances have been made in understating how S4 moves through a hydrophobic plug upon voltage changes, the possible helix transition from α- to 3-helix in S4 during the activation process is still unresolved. Here, we have mutated several hydrophobic residues from I360 to F370 in the S4 segment into histidine, in i, i + 3 and i, i + 6 or i, i + 4 and i, i + 7 pairs, to favor 3- or α-helical conformations, respectively. We have taken advantage of the ability of His to coordinate Zn to promote metal ion bridges, and we have found that the histidine introduced at position 366 (L366H) can interact with the introduced histidine at position 370 (stabilizing that portion of the S4 segment in α-helical conformation). In the presence of 20 μM of Zn, the activation currents of L366H:F370H channels were slowed down by a factor of 3.5, and the voltage dependence is shifted by 10 mV toward depolarized potentials with no change on the deactivation time constant. Our data supports that by stabilizing a region of the S4 segment in α-helical conformation, a closed (resting or intermediate) state is stabilized rather than destabilizing the open (active) state. Taken together, our data indicates that S4 undergoes α-helical conformation to a short-lived different secondary structure transiently before reaching the active state in the activation process.
- Carvalho-de-Souza, J. L., & Bezanilla, F. (2019). Noncanonical mechanism of voltage sensor coupling to pore revealed by tandem dimers of Shaker. Nature communications, 10(1), 3584.More infoIn voltage-gated potassium channels (VGKC), voltage sensors (VSD) endow voltage-sensitivity to pore domains (PDs) through a not fully understood mechanism. Shaker-like VGKC show domain-swapped configuration: VSD of one subunit is covalently connected to its PD by the protein backbone (far connection) and non-covalently to the PD of the next subunit (near connection). VSD-to-PD coupling is not fully explained by far connection only, therefore an additional mechanistic component may be based on near connection. Using tandem dimers of Shaker channels we show functional data distinguishing VSD-to-PD far from near connections. Near connections influence both voltage-dependence of C-type inactivation at the selectivity filter and overall PD open probability. We speculate a conserved residue in S5 (S412 in Shaker), within van der Waals distance from next subunit S4 residues is key for the noncanonical VSD-to-PD coupling. Natural mutations of S412-homologous residues in brain and heart VGKC are related to neurological and cardiac diseases.
- Carvalho-de-Souza, J. L., Nag, O. K., Oh, E., Huston, A. L., Vurgaftman, I., Pepperberg, D. R., Bezanilla, F., & Delehanty, J. B. (2019). Cholesterol Functionalization of Gold Nanoparticles Enhances Photoactivation of Neural Activity. ACS chemical neuroscience, 10(3), 1478-1487.More infoGold nanoparticles (AuNPs) attached to the extracellular leaflet of the plasma membrane of neurons can enable the generation of action potentials (APs) in response to brief pulses of light. Recently described techniques to stably bind AuNP bioconjugates directly to membrane proteins (ion channels) in neurons enable robust AP generation mediated by the photoexcited conjugate. However, a strategy that binds the AuNP to the plasma membrane in a non protein-specific manner could represent a simple, single-step means of establishing light-responsiveness in multiple types of excitable neurons contained in the same tissue. On the basis of the ability of cholesterol to insert into the plasma membrane, here we test whether AuNP functionalization with linear dihydrolipoic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP generation in neurons. Dorsal root ganglion (DRG) neurons of rat were labeled with 20 nm diameter spherical AuNP-PEG-Chol conjugates wherein ∼30% of the surface ligands (DHLA-PEG-COOH) were conjugated to PEG-Chol. Voltage recordings under current-clamp conditions showed that DRG neurons labeled in this manner exhibited a capacity for AP generation in response to microsecond and millisecond pulses of 532 nm light, a property attributable to the close tethering of AuNP-PEG-Chol conjugates to the plasma membrane facilitated by the cholesterol moiety. Light-induced AP and subthreshold depolarizing responses of the DRG neurons were similar to those previously described for AuNP conjugates targeted to channel proteins using large, multicomponent immunoconjugates. This likely reflected the AuNP-PEG-Chol's ability, upon plasmonic light absorption and resultant slight and rapid heating of the plasma membrane, to induce a concomitant transmembrane depolarizing capacitive current. Notably, AuNP-PEG-Chol delivered to DRG neurons by inclusion in the buffer contained in the recording pipet/electrode enabled similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol bound to the inner (cytofacial) leaflet of the plasma membrane. Our results demonstrate the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct responsiveness to light in neurons. Further, this strategy represents a general approach for establishing excitable cell photosensitivity that could be of substantial advantage for exploring a given tissue's suitability for AuNP-mediated photocontrol of neural activity.
- Jiang, Y., Parameswaran, R., Li, X., Carvalho-de-Souza, J. L., Gao, X., Meng, L., Bezanilla, F., Shepherd, G. M., & Tian, B. (2019). Nongenetic optical neuromodulation with silicon-based materials. Nature protocols, 14(5), 1339-1376.More infoOptically controlled nongenetic neuromodulation represents a promising approach for the fundamental study of neural circuits and the clinical treatment of neurological disorders. Among the existing material candidates that can transduce light energy into biologically relevant cues, silicon (Si) is particularly advantageous due to its highly tunable electrical and optical properties, ease of fabrication into multiple forms, ability to absorb a broad spectrum of light, and biocompatibility. This protocol describes a rational design principle for Si-based structures, general procedures for material synthesis and device fabrication, a universal method for evaluating material photoresponses, detailed illustrations of all instrumentation used, and demonstrations of optically controlled nongenetic modulation of cellular calcium dynamics, neuronal excitability, neurotransmitter release from mouse brain slices, and brain activity in the mouse brain in vivo using the aforementioned Si materials. The entire procedure takes ~4-8 d in the hands of an experienced graduate student, depending on the specific biological targets. We anticipate that our approach can also be adapted in the future to study other systems, such as cardiovascular tissues and microbial communities.
- Alberto, C., Junior, Z. B., Carvalho-de-souza, J. L., & Bezanilla, F. (2018). Exploring Possible Conversion between Alpha- and 3 10 -Helix in S4 of Shaker Potassium Channel. Biophysical Journal, 114(3), 477a. doi:10.1016/j.bpj.2017.11.2623
- Carvalho-de-Souza, J. L., & Bezanilla, F. (2018). Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K channels. The Journal of general physiology, 150(2), 307-321.More infoVoltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters (, the voltage-dependent transition from the resting to intermediate state and , from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting , , and according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but is affected by the hydrophobicity of the AA in position 361, whereas and are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on and in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics.
- Carvalho-de-Souza, J. L., Pinto, B. I., Pepperberg, D. R., & Bezanilla, F. (2018). Optocapacitive Generation of Action Potentials by Microsecond Laser Pulses of Nanojoule Energy. Biophysical journal, 114(2), 283-288.More infoMillisecond pulses of laser light delivered to gold nanoparticles residing in close proximity to the surface membrane of neurons can induce membrane depolarization and initiate an action potential. An optocapacitance mechanism proposed as the basis of this effect posits that the membrane-interfaced particle photothermally induces a cell-depolarizing capacitive current, and predicts that delivering a given laser pulse energy within a shorter period should increase the pulse's action-potential-generating effectiveness by increasing the magnitude of this capacitive current. Experiments on dorsal root ganglion cells show that, for each of a group of interfaced gold nanoparticles and microscale carbon particles, reducing pulse duration from milliseconds to microseconds markedly decreases the minimal pulse energy required for AP generation, providing strong support for the optocapacitance mechanism hypothesis.
- Carvalho-de-souza, J. L., & Bezanilla, F. (2018). Key Residues in the Interface between Voltage Sensor and Pore Domain in Shaker Potassium Channels. Biophysical Journal, 114(3), 476a. doi:10.1016/j.bpj.2017.11.2619
- Parameswaran, R., Carvalho-de-souza, J. L., Jiang, Y., Burke, M. J., Zimmerman, J. F., Koehler, K., Philips, A. W., Yi, J., Bezanilla, F., Tian, B., & Adams, E. J. (2018). Photoelectrochemical Modulation of Neuronal Activity with Free-Standing Coaxial Silicon Nanowires. Biophysical Journal, 114(3), 393a. doi:10.1016/j.bpj.2017.11.2177
- Parameswaran, R., Carvalho-de-souza, J. L., Jiang, Y., Burke, M. J., Zimmerman, J. F., Koehler, K., Phillips, A. W., Yi, J., Adams, E. J., Bezanilla, F., & Tian, B. (2018). Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires.. Nature nanotechnology, 13(3), 260-266. doi:10.1038/s41565-017-0041-7More infoOptical methods for modulating cellular behaviour are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells or cannot provide subcellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires. We reveal the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single nanowires, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single nanowires. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process.
- Pinto, B. I., Carvalho-de-souza, J. L., & Bezanilla, F. (2018). Nanoparticle-Mediated Heating of Cellular Membrane Induces Changes in Membrane Capacitance and Ionic Conduction. Biophysical Journal, 114(3), 669a. doi:10.1016/j.bpj.2017.11.3611
- Tian, B., Xu, S., Rogers, J. A., Cestellos-Blanco, S., Yang, P., Carvalho-de-Souza, J. L., Bezanilla, F., Liu, J., Bao, Z., Hjort, M., Cao, Y., Melosh, N., Lanzani, G., Benfenati, F., Galli, G., Gygi, F., Kautz, R., Gorodetsky, A. A., Kim, S. S., , Lu, T. K., et al. (2018). Roadmap on semiconductor-cell biointerfaces. Physical biology, 15(3), 031002.More infoThis roadmap outlines the role semiconductor-based materials play in understanding the complex biophysical dynamics at multiple length scales, as well as the design and implementation of next-generation electronic, optoelectronic, and mechanical devices for biointerfaces. The roadmap emphasizes the advantages of semiconductor building blocks in interfacing, monitoring, and manipulating the activity of biological components, and discusses the possibility of using active semiconductor-cell interfaces for discovering new signaling processes in the biological world.
- Bezanilla, F., & Carvalho-de-souza, J. L. (2017). Non-Canonical Interactions between Voltage Sensors and Pore Domain in Shaker K+-Channel. Biophysical Journal, 112(3), 162a. doi:10.1016/j.bpj.2016.11.892More infoW434F mutation in the pore domain (PD) of Shaker K+-channels yields non-conductive channels, useful for gating currents studies. Comparisons of Q-Vs recorded with W434F with those recorded with absence of K+ show virtually indistinguishable data. When a mutation in the S3-S4 linker, L361R, is introduced, the Q-V recorded with W434F, and the curve of K+ conductance activation by voltage (G-V) are both strongly shifted to more negative voltages, and they unexpectedly cross each other. This effect is typical when the channel has more than one open state, which does not seem to be the case according to our single channels recordings. As expected, Q-V curve recorded in the conductive Shaker containing L361R mutation does not cross its G-V. This unprecedentedly showed that W434F-containing PD can potentially affect mutant VSDs although it is virtually silent for WT VSDs. To investigate this non-canonical coupling between VSD and PD, other than via S4-S5 linker, we produced dimers of Shaker that would show a mutant VSD (L361R) CLOSE, or FAR from a mutant PD (W434F). According to the Kv1.2 crystal structure, VSDs are near the PD from the neighbor subunit and we consider that would happen similarly in dimers of shaker. VSD mutations shift the slow-inactivation curves to more negative voltages, with the effect being more intense in CLOSE compared to FAR channels. Furthermore, current peaks progressively increase at +60 mV after 100-ms pre-pulses from −180 to −100 mV differently on CLOSE and FAR channels, indicating the VSD mutation interfere with the PD inactivation according to its relative position. Our data show that VSD and PD are in close communication beyond what is predicted by the S3-S4 connection, especially when mutations are present in both domains of the protein. Support: NIH-GM030376.
- Carvalho-de-souza, J. L., & Bezanilla, F. (2017). Voltage Dependence and Non-Sensor Residues. Biophysical Journal, 112(3), 246a. doi:10.1016/j.bpj.2016.11.1344More infoThe gating charges in S4 segment are highly conserved in voltage-gated K+ channels. However, a broad range of voltage dependence for charge movement and K+ conductance activation are found, suggesting these features are determined by other motifs. The extracellular S3-S4 linker shows family-related conservation. We studied the influence of residues 353-361 of that region on the voltage dependence of both gating charges movement (Q-Vs) and K+-conductance activation (G-Vs) in Shaker K+-channel. Remarkably, hydrophilic mutations in L358 and L361 produce strong shifts of both Q-Vs and G-Vs to more negative voltages. The Q-V is shifted more than −80-mV inL361R. We scanned with mutagenesis L358 (L358X) and L361 (L361X) with different amino acids (AA) and measured the mid-points of Q-V curves (Vmed) and G-V curves (V0.5). We plotted those values with several AA scales and took their coefficient of determination from a linear regression (R2). For L358X, Vmed were correlated with the residue tendency to be in a transmembrane segment (R2=0.72) and V0.5 by the hydrophobic surface area of the residue (R2=0.66). For L361X, Vmed were correlated with the residue tendency to be buried in the protein (R2=0.86) and V0.5 by the hydration potential of the residue (R2=0.66). By fitting Q-Vs to a three-state sequential model we find V0 and V1 as the voltage dependence of two simplified steps during VSD activation. The voltage sensor (VS) coupling to the pore domain (PD) was accessed by plotting V1, the last step, with V0.5 for L358X (R2=0.68) and L361X (R2=0.91). V0.5 changes in both cases are not well correlated with V0 (R2
- Carvalho-de-souza, J. L., Pepperberg, D. R., & Bezanilla, F. (2017). Optocapacitance-Mediated Responses of Excitable Cells: New Developments. Biophysical Journal, 112(3), 286a. doi:10.1016/j.bpj.2016.11.1546More infoOptocapacitance is a technique whereby flash illumination quickly changes membrane capacitance to elicit action potentials (APs) in unmodified (not genetically engineered) excitable cells. Spherical 20-nm gold nanoparticles (AuNPs) interfaced with the plasma membrane serve as light absorbers to induce AP generation in response to 1-ms, 100 μJ, 532-nm laser pulses. Light absorption by the AuNP produces localized heating that changes local temperature by
- Kubota, T., Dang, B., Carvalho-de-Souza, J. L., Correa, A. M., & Bezanilla, F. (2017). Nav channel binder containing a specific conjugation-site based on a low toxicity β-scorpion toxin. Scientific reports, 7(1), 16329.More infoVoltage-gated sodium (Nav) channels play a key role in generating action potentials which leads to physiological signaling in excitable cells. The availability of probes for functional studies of mammalian Nav is limited. Here, by introducing two amino acid substitutions into the beta scorpion toxin Ts1, we have chemically synthesized a novel binder [S14R, W50Pra]Ts1 for Nav with high affinity, low dissociation rate and reduced toxicity while retaining the capability of conjugating Ts1 with molecules of interests for different applications. Using the fluorescent-dye conjugate, [S14R, W50Pra(Bodipy)]Ts1, we confirmed its binding to Nav1.4 through Lanthanide-based Resonance Energy Transfer. Moreover, using the gold nanoparticle conjugate, [S14R, W50Pra(AuNP)]Ts1, we were able to optically stimulate dorsal root ganglia neurons and generate action potentials with visible light via the optocapacitive effect as previously reported. [S14R, W50Pra]Ts1 is a novel probe with great potential for wider applications in Nav-related neuroscience research.
- Carvalho-de-souza, J. L., Jiang, Y., Tian, B., Bezanilla, F., & Wong, R. C. (2016). Silicon Mesostructures for Phospholipid Based Bioelectric Device and Deterministic Neuromodulation. Biophysical Journal, 110(3), 147a. doi:10.1016/j.bpj.2015.11.830More infoSilicon is a widely used material in biomedical research because it is biocompatible and biodegradable, and it exhibits a spectrum of important electrical, optical, thermal and mechanical properties. However, the fundamental forms of silicon building blocks for biophysical and biomedical applications are few, and to date, researchers have focused primarily on crystalline and rigid structures. Natural biomaterials have remarkable diversity in structure and function and may guide the design of new silicon forms for subcellular interfaces and biophysical modulation. Nevertheless, reproducing heterogeneous features in silicon is difficult to achieve due to limited chemical or engineering processes. Here we introduce a multiscale, injectable, and ultra-soft silicon-based biomaterial with ‘brick-and-mortar’ mesostructures. It has an amorphous atomic structure, an ordered nanowire-based framework, and random micron-scale voids, exhibiting a high surface area and pore volume. Atom probe tomography (APT) and energy dispersive X-ray (EDX) mapping reveal an ordered and size-dependent oxygen distribution. The amorphous silicon-based mesostructures show reduced Young's moduli in air and in a buffered phosphate saline solution (2-3 orders of magnitude smaller than that of single crystalline silicon). Additionally, because the soft mesostructures exhibit a controllable photothermal effect in saline, we were able to design a remotely controlled bioelectric device by interfacing silicon particles with painted phospholipid bilayers, where the photothermal effect quickly changes the membrane capacitance, producing currents which are proportional to the time derivative of the temperature. By adding these silicon particles to dorsal root ganglion neurons we could reliably generate action potentials upon illumination with short pulses of 523 nm light. These results show that the mesostructured silicon particles permit non-genetic, fast, low power and sub-cellular optical control of electrophysiological activity. Supported by NIH: GM030376; AFOSR: FA9550-14-1-0175, FA9550-15-1-0285.
- Carvalho-de-souza, J. L., Kubota, T., Du, X., Latorre, R., Gomez, C. M., & Bezanilla, F. (2016). A Missense Mutation in the Selectivity Filter of BK Affects the Channel's Potassium Conductance. Biophysical Journal, 110(3), 449a. doi:10.1016/j.bpj.2015.11.2412More infoLarge-conductance Ca2+ activated potassium channels (BK), which are synergistically modulated by voltage and intracellular calcium, play important role in the control of the cell excitability. Here we report data from a de novo missense mutation in KCNMA1, encoding the BK channel α subunit found in a patient with progressive cerebellar ataxia. The mutation changes the residue of glycine 354, in the selectivity filter signature sequence 352-TVGYG-356 in potassium channels, into a serine. We expressed BK and G354S mutant BK (BK-G354S) channels in Xenopus oocytes and we analyzed macroscopic and on-cell single channel potassium currents recorded under voltage clamp. The macroscopic potassium currents are reduced in magnitude by about ten times and are slowly activated in oocytes expressing BK-G354S channels when compared with oocytes injected with cRNA for BK channels. The microscopic single channel conductance in BK-G354S is also depressed to about a tenth of the single channel conductance of BK channels, which suggest that the smaller macroscopic currents recorded with BK-G354S channels is not a mere problem of expression in the membrane. Actually, as revealed by expressing both proteins in mammalian cells and measuring their expression level using immunofluorescence techniques, BK-G354S channels are expressed in comparable levels with the expression of the wild-type BK channels. The mutation G354S in BK channel significantly decreases its potassium conductance, which importantly denotes a phenotype with loss of function. Biophysical data presented here provides crucial information that helps understanding the neurologic pathophysiology in the affected patient and it may also help in designing possible treatments for this and other similar cases. Supported by NIH GM030376, NS094665-01 and NS082788-02.
- Jiang, Y., Carvalho-de-Souza, J. L., Wong, R. C., Luo, Z., Isheim, D., Zuo, X., Nicholls, A. W., Jung, I. W., Yue, J., Liu, D. J., Wang, Y., De Andrade, V., Xiao, X., Navrazhnykh, L., Weiss, D. E., Wu, X., Seidman, D. N., Bezanilla, F., & Tian, B. (2016). Heterogeneous silicon mesostructures for lipid-supported bioelectric interfaces. Nature materials, 15(9), 1023-30.More infoSilicon-based materials have widespread application as biophysical tools and biomedical devices. Here we introduce a biocompatible and degradable mesostructured form of silicon with multi-scale structural and chemical heterogeneities. The material was synthesized using mesoporous silica as a template through a chemical vapour deposition process. It has an amorphous atomic structure, an ordered nanowire-based framework and random submicrometre voids, and shows an average Young's modulus that is 2-3 orders of magnitude smaller than that of single-crystalline silicon. In addition, we used the heterogeneous silicon mesostructures to design a lipid-bilayer-supported bioelectric interface that is remotely controlled and temporally transient, and that permits non-genetic and subcellular optical modulation of the electrophysiology dynamics in single dorsal root ganglia neurons. Our findings suggest that the biomimetic expansion of silicon into heterogeneous and deformable forms can open up opportunities in extracellular biomaterial or bioelectric systems.
- Kubota, T., Dang, B., Carvalho-de-souza, J. L., Kent, S. B., Correa, A. M., & Bezanilla, F. (2016). High Affinity Nav Channel Binder with Specific Conjugation Site Derived from Scorpion Toxin with Attenuated Toxicity. Biophysical Journal, 110(3), 113a. doi:10.1016/j.bpj.2015.11.664More infoVoltage-gated sodium channels (Nav) play a key role in generating action potential which leads to physiological signaling in excitable cells. However, the availability of probes for functional study of mammalian Nav is limited. Based on the beta-scorpion toxin Ts1, we have chemically synthesized a novel biologically friendly binder [S14R, W50Pra]Ts1 (Ts1-S14R) for Nav with high affinity and extremely low dissociation rate. This Ts1 analog has a S14R point mutation, corresponding to E15R in Css4, another beta-scorpion toxin, and also has a propargylglycine (Pra) mutation from W50 for bioconjugation. We identified the pharmacological properties of Ts1-S14R on rat skeletal muscle Nav channel (Nav1.4) using the cut-open oocyte voltage clamp technique and confirmed that its toxicity has been dramatically reduced. Chemical synthesis of this binder enabled us to conjugate with molecules of interest for various studies. Using the fluorescent-dye, BODIPY, conjugated binder, we examined its binding location in Nav1.4 using Lanthanide-based Resonance Energy Transfer technique (LRET). Our LRET study showed Ts1-S14R-BODIPY binds to Nav1.4 at virtually identical location as that of Ts1-BODIPY. In addition, LRET data showed that, once Ts1-S14R-BODIPY binds to Nav1.4, it stays in place for at least 40 min in toxin-free solution, indicating that Ts1-S14R-BODIPY can be employed as a stable binder for Nav1.4. Moreover, we generated Ts1-S14R conjugated with gold nanoparticle (Ts1-S14R-AuNP). We demonstrated this conjugate can induce action potentials by visible light stimulation through the opto-thermal-capacitive effect in rat dorsal root ganglia (DRG) neurons similarly to that previously reported (Carvalho-de-Souza JL et al. Neuron 2015). Ts1-S14R is therefore a novel probe with great potential for wider applications in Nav-related neuroscience research. Support: U54GM087519 and GM030376.
- Staisch, J., Du, X., Carvalho-de-souza, J. L., Kubota, T., Bezanilla, F., & Gomez, C. M. (2016). A Mutation Causing Reduced BK Channel Activity Leads to Cognitive Inpairment and Progressive Cerebellar Ataxia (P5.394). Neurology, 86.More infoObjective: Determine the functional consequences of a BK potassium channel mutation associated with cerebellar degeneration. Background: We described a 16 yo female with delayed milestones and ataxic gait since 18 months. She had cognitive delay, dysarthria, perioral dyskinesias, and nystagmus. MRI scan showed severe cerebellar atrophy. Exome sequencing revealed a de novo variant in one KCNMA1 gene predicting a G354S mutation in the BK channel α subunit. The only other reported BK channel mutation causes enhanced channel activity, epilepsy, and dyskinesias. Methods: Wild type and G354S mutant BK channel α subunits were expressed in cultured HEK293 cells using eukaryotic expression vectors and immuno-localized using anti-α subunit antibodies. Also, mRNAs encoding the WT and G354S mutant were injected into Xenopus oocytes for macroscopic voltage-activated K+ currrent recordings under voltage-clamp during standard step depolarization protocols. Results: There was no difference in cellular distribution between the WT and G354S mutant. In oocytes expressing the G354S mutant alone, macroscopic K+ currents were 11[percnt] of those detected in WT-expressing oocytes, and the kinetics of activation were signficantly slowed compared to WT. Co-injection of the WT and G354S mutant in equal amounts (imitating heterozygous condition) led to currents less than 35[percnt] of WT. K+ currents recorded from dual-injected oocytes showed slower activation than WT. Unitary single channels recordings showed circa 90[percnt] decrease in the G354S mutant channel conductance relative to WT Thus, co-injection of G354S mutant together with WT α subunit reduced the conductance and slowed activation of BK channels out of proportion to abundance of subunits. Conclusions: The BK channel mutant G354S has a dominant negative effect on channel function. Reduced BK channel activity and delayed activation lead to progressive cerebellar atrophy and ataxia, possibly through damaging delays in repolarization. Drugs that increase BK channel activity may be a rational approach for treating this disease. Disclosure: Dr. Staisch has nothing to disclose. Dr. Du has nothing to disclose. Dr. Carvalho-de-Souza has nothing to disclose. Dr. Kubota has nothing to disclose. Dr. Bezanilla has nothing to disclose. Dr. Gomez has nothing to disclose.
- Carvalho-de-Souza, J. L., Treger, J. S., Dang, B., Kent, S. B., Pepperberg, D. R., & Bezanilla, F. (2015). Photosensitivity of neurons enabled by cell-targeted gold nanoparticles. Neuron, 86(1), 207-17.More infoUnmodified neurons can be directly stimulated with light to produce action potentials, but such techniques have lacked localization of the delivered light energy. Here we show that gold nanoparticles can be conjugated to high-avidity ligands for a variety of cellular targets. Once bound to a neuron, these particles transduce millisecond pulses of light into heat, which changes membrane capacitance, depolarizing the cell and eliciting action potentials. Compared to non-functionalized nanoparticles, ligand-conjugated nanoparticles highly resist convective washout and enable photothermal stimulation with lower delivered energy and resulting temperature increase. Ligands targeting three different membrane proteins were tested; all showed similar activity and washout resistance. This suggests that many types of ligands can be bound to nanoparticles, preserving ligand and nanoparticle function, and that many different cell phenotypes can be targeted by appropriate choice of ligand. The findings have applications as an alternative to optogenetics and potentially for therapies involving neuronal photostimulation.
- Carvalho-de-souza, J. L., Treger, J. S., Dang, B., Pepperberg, D. R., Bezanilla, F., & Kent, S. B. (2015). Robust Optical Stimulation of Neuronal Activity using Functionalized Gold Nanoparticles. Biophysical Journal, 108(2), 207a. doi:10.1016/j.bpj.2014.11.1144More infoIt was recently shown that infrared light pulses can directly stimulate neuronal action potentials by quickly heating the cell membrane and inducing capacitive currents (Shapiro, et al., Nat Commun, 2012). While useful, this technique works by heating the entire aqueous environment around a cell, rendering it nonspecific and incapable of stimulating specific populations of cells. Furthermore, since water absorbs infrared, the light source must be near the target. Here, we sought to stimulate neurons with visible light, using the ability of 20 nm spherical gold nanoparticles (AuNPs) to absorb green light and convert it into heat. By applying the AuNPs to painted lipid bilayers, we first confirmed that, upon 532 nm pulse stimulation, the membrane capacitance increases following the rate of temperature increase. The resulting currents are well-behaved, being linear with respect to both laser power and membrane potential. We next applied AuNPs to cultured dorsal root ganglion (DRG) neurons and demonstrated action potential initiation with 532 nm pulses of ≤1 ms. Finally, we obtained a dramatic improvement of this technique by functionalizing AuNPs with high-avidity ligands for DRG neuron membrane proteins. Nonfunctionalized AuNPs are cleared away from DRG neurons within seconds of starting a perfusion wash, causing the cells to lose optical excitability. However, AuNPs functionalized with Ts1 neurotoxin, or with anti-TRPV1 or anti-P2X3 antibodies, showed substantial resistance to washout; DRG neurons labeled with these functionalized particles remained light-sensitive after more than 20 minutes of perfusion washing. Furthermore, these particles required a lower concentration and less laser power to confer optical excitability. By appropriate selection of AuNP-conjugated ligand, this technique may enable improved cell-type specificity of neuronal photostimulation, such as ganglion cells in diseased retina where photoreceptors are non-functional. Support: R21-EY023430 and The Beckman Initiative for Macular Research.
- Carvalho-de-souza, J. L., Lacroix, J. J., & Bezanilla, F. (2014). Effects of Decreased Hydrophobicity above R1 in S4-Based Voltage Sensors. Biophysical Journal, 106(2), 742a. doi:10.1016/j.bpj.2013.11.4088More infoMembrane proteins containing S4-based voltage sensors domains (VSD) respond to changes in the membrane potential by transferring electrically charged side chains (gating charges), mainly located in the S4 segment, across the membrane electric field. The region just above the first charge (R1) is generally thought to control the VSD voltage-dependence. Here, we decreased residue hydrophobicity between N353 to L361 just above R1 in the Shaker Kv channels and found that for the two positions L358 and L361, less hydrophobic residues dramatically shifted the Charge vs. Voltage (Q-V) curve to more negative voltages, up to −132 mV. Screening L358 with several amino acids revealed a linear correlation between the side chain hydrophobicity and the Q-V curve midpoint. Interestingly, structural models of the VSD place the L358 and L361 side chains towards the hydrophobic core of the lipid bilayer in a resting state and towards the aqueous solvent in the active state, suggesting that less hydrophobic mutants may destabilize the resting state and/or stabilize the active state. To explore the origins of these phenotypes, we co-mutated some hydrophilic point mutations at position 358 or 361 together with the ILT mutation, which uncouples VSD movement from pore opening. Our results indicate that most double mutants shifts further the Q-V curves compared to the point mutations, but did not shift the G-V curves compared to the ILT mutant, suggesting that hydrophilic substitutions at positions 358 and 361 most likely destabilized the VSD resting state. Support: NIH-GM030376.
- Carvalho-de-Souza, J. L., Varanda, W. A., Tostes, R. C., & Chignalia, A. Z. (2013). BK Channels in Cardiovascular Diseases and Aging. Aging and disease, 4(1), 38-49.More infoAging is a major risk factor for cardiovascular diseases, one of the main world-wide causes of death. Several structural and functional changes occur in the cardiovascular system during the aging process and the mechanisms involved in such alterations are yet to be completely described. BK channels are transmembrane proteins that play a key role in many physiological processes, including regulation of vascular tone. In vascular smooth muscle cells, BK opening and the consequent efflux of potassium (K(+)) leads to membrane hyperpolarization, which is followed by the closure of voltage-dependent Ca(2+) channels, reduction of Ca(2+) entry and vasodilatation. BK regulates nitric oxide-mediated vasodilatation and thus is crucial for normal endothelial function. Herein we will briefly review general structural properties of BK and focus on their function in the cardiovascular system emphasizing their role in cardiovascular aging and diseases.
- Carvalho-de-souza, J. L., Lacroix, J. J., & Bezanilla, F. (2013). Effects of Charged Residues Inserted above R1 in S4-Based Voltage Sensors. Biophysical Journal, 104(2), 197a. doi:10.1016/j.bpj.2012.11.1110More infoMembrane proteins containing S4-based voltage sensors domains (VSD) respond to changes in the membrane potential by transferring electrically charged side chains (gating charges), mainly located in the S4 segment, across the membrane electric field. Yet, in the archetypical Shaker Kv channel, not all positive S4 residues contribute to the gating charge. Moreover, the number and positions of charged residues near the external side of S4 is poorly conserved amongst VSDs, suggesting that some S4 charged residues may have a regulatory role. We investigated this hypothesis by individually inserting Arg residues at the top of the S4 segment of the Shaker channel, from positions 356 to 361, and determining the impact of the mutations on the channel's gating currents. Our results show a non-ambiguous periodicity of the phenotypes that correlate with the periodicity of the S4 alpha-helical structure: Arg inserted on the same side of the gating charge positively-shifted the charge vs. voltage (Q-V) curve, while Arg inserted on the opposite side negatively-shifted the Q-V curve. We propose two distinct mechanisms that account for these observations. Charged side chains pointing towards the gating charge pathway exert a local bias in the electric field sensed by the innermost native Arg. However, charged side chains located on the opposite side of the gating charge pathway tend to favor more depolarized conformations of the S4 helix to avoid being in proximity to the hydrophobic environment of the membrane lipids. We propose that naturally-occurring charged residues located at the top of S4 may help to finely tune the VSD biophysical properties. This mechanism is illustrated by a myasthenia-associated muscle sodium channel variant that introduced the V1442E mutation at the top of the S4 in domain IV. This work was supported by NIH grant GM030376.
- Carvalho-de-souza, J. L., & Cassola, A. C. (2010). Insights on the Mechanisms of the Fast Blockade of TTX-R Na+ Channels by Eugenol. Biophysical Journal, 98(3), 114a. doi:10.1016/j.bpj.2009.12.4213More infoOBJECTIVES. It was previously shown that eugenol, a phenylpropene, blocks fast and reversibly voltage-gated Na+ channels (NaV), but little concern was given to the blocker binding to different conformational states of channel molecule. Here we reported a detailed analysis of state-dependent effects of eugenol on tetrodotoxin-resistant (TTX-R) NaV isoforms, comparing them to those of lidocaine, a reference blocker.METHODS. TTX-R Na+ currents were recorded in dorsal root ganglia neurons from newborn Wistar rats with whole-cell configuration of patch clamp technique. Tetrodotoxin-sensitive Na+ currents were blocked by TTX 100nM in the extracellular solution.RESULTS and CONCLUSIONS. A dose-dependent fast blockade due to eugenol was observed in 0.2Hz time series depolarizations from a holding potential of −110 mV to a 0 mV pulse. This tonic blockage is due to eugenol binding to the closed state. The IC50 was 2.28±0.10mM for eugenol compared to 0.44±0.08mM for lidocaine. The tonic NaV blockade was more effective when the membrane was held at more depolarized, still sublimiar, holding potentials. This observation indicates a higher affinity of eugenol for closed substates dwelled at less hyperpolarized potentials. No consistent evidences for additional binding to open state were observed. A displacement of steady-state inactivation curve to more negative potentials, associated with a slower recovery from fast inactivation under eugenol indicates that this molecule also binds to fast inactivated state. For currents undergoing slow inactivation, a consistent reduction by eugenol indicates that the phenylpropene additionally binds to the slow inactivated state. A frequency-dependent blocking effect of eugenol on NaV was observed, but the effect is smaller than that induced by lidocaine. In conclusion, eugenol binds to several isoforms of TTX-R NaV and to the different states of the proteins, leading to a channel blockage.
- Moreira-Lobo, D. C., Linhares-Siqueira, E. D., Cruz, G. M., Cruz, J. S., Carvalho-de-Souza, J. L., Lahlou, S., Coelho-de-Souza, A. N., Barbosa, R., Magalhães, P. J., & Leal-Cardoso, J. H. (2010). Eugenol modifies the excitability of rat sciatic nerve and superior cervical ganglion neurons. Neuroscience letters, 472(3), 220-4.More infoEugenol is a phenylpropene obtained from the essential oils of plants such as clove and basil which has ample use in dentistry. Eugenol possesses analgesic effects that may be related to the inhibition of voltage-dependent Na+ channels and/or to the activation of TRPV1 receptors or both. In the present study, electrophysiological parameters were taken from the compound action potentials of the isolated rat sciatic nerve and from neurons of the superior cervical ganglion (SCG) impaled with sharp microelectrodes under current-clamp conditions. In the isolated rat sciatic nerve, eugenol inhibited the compound action potential in a concentration-dependent manner. Action potentials recorded from SCG neurons were inhibited by eugenol with an IC(50) of 0.31 mM. At high concentrations (2 mM), during brief applications, eugenol caused significant action potential blockade while it did not interfere with the resting membrane potential or the membrane input resistance. Surprisingly, however, at low eugenol concentrations (0.6 mM), during long time applications, a reversible reduction (by about 50%) in the input membrane resistance was observed, suggesting the possible involvement of a secondary delayed effect of eugenol to reduce neuronal excitability.
- Carvalho-de-souza, J. L., & Cassola, A. C. (2009). Eugenol Blocks Tetrodotoxin-Resistant Nav Channels. Biophysical Journal, 96(3), 251a. doi:10.1016/j.bpj.2008.12.1238More infoEugenol, a phenylpropene synthesized by many angiosperms, acts as atimicrobial toxin and has analgesic properties for humans. Here we show that EUG has a blocking action on tetrodotoxin-resistant voltage-gated sodium channels. The effects of eugenol and lidocaine were thoroughly compared. Currents were recorded in dorsal root ganglia neurons from newborn rats, with patch-clamp technique, whole-cell configuration. The experiments were done in the presence of 100 μM tetrodotoxin.Eugenol blocked tetrotoxin-resistant Nav channels fast and reversibly, in a concentration-dependent manner. The IC50 for eugenol was of 2.27±0.22 mM and 0.44±0.08 mM for lidocaine and inhibition is due mostly to binding to the channel resting state. Eugenol and lidocaine did not shift the steady-state activation curve along voltage axis. The steady-state inactivation curve was displaced to more negative voltages, reflecting some binding to the inactivated state, by both agents. Eugenol affects the kinetics of inactivation recovery, increasing the weight of the slow component from 21.3% to 27.8%. Eugenol effect is smaller than the lidocaine effect (from 18.0% to 30.7%). Both inhibitors prolonged the half-times of the slow component. In concentrations around IC50 the frequency-dependent blockade was less conspicuous for eugenol. The ratio of a remaining current peak for the 20th /1st pulse, frequency of 5 Hz, was 0,86 for eugenol and 0,58 for lidocaine. In conclusion, eugenol is a fast and reversible blocker of tetrodotoxin-resistant Na currents, with affinity 5 times lower than that of lidocaine for the same channel isoforms. Compared to lidocaine, eugenol has a higher relative affinity for the resting state and lower relative affinity for the open/inactive channel state, as unveiled by low dependence on voltage and frequency of the blocking action.
- Lima, C. C., Carvalho-de-Souza, J. L., Lima, A. A., & Leal-Cardoso, J. H. (2008). Ileal smooth muscle motility depression on rabbit induced by toxin A from Clostridium difficile. Digestive diseases and sciences, 53(6), 1636-43.More infoThis study is aimed at elucidating with in vitro experiments the time course of alteration of ileal motility caused by in vivo exposure of ligated loops of ileum to toxin A (1 microg/ligated loop) of Clostridium difficile. In the sham-operated animals no significant alteration of motility was observed. In ligated loops directly injected with toxin A and in loops neighboring those administered with this toxin, a biphasic time course of motility alterations was observed. There was initially (2 h after toxin administration) an increase in spontaneous motility and in the amplitude of maximal contraction induced by potassium and acetylcholine. Afterwards there was a progressive depression of motility, which was more severe in loops directly injected. These results suggested a significant progressive depression of rabbit ileal motility induced by toxin A from C. difficile.
- Rizzi, C. T., Carvalho-de-Souza, J. L., Schiavon, E., Cassola, A. C., Wanke, E., & Troncone, L. R. (2007). Crotamine inhibits preferentially fast-twitching muscles but is inactive on sodium channels. Toxicon : official journal of the International Society on Toxinology, 50(4), 553-62.More infoCrotamine is a peptide toxin from the venom of the rattlesnake Crotalus durissus terrificus that induces a typical hind-limb paralysis of unknown nature. Hind limbs have a predominance of fast-twitching muscles that bear a higher density of sodium channels believed until now to be the primary target of crotamine. Hypothetically, this makes these muscles more sensitive to crotamine and would explain such hind-limb paralysis. To challenge this hypothesis, we performed concentration vs. response curves on fast (extensor digitorum longus (EDL)) and slow (soleus) muscles of adult male rats. Crotamine was tested on various human Na+ channel isoforms (Na(v)1.1-Na(v)1.6 alpha-subunits) expressed in HEK293 cells in patch-clamp experiments, as well as in acutely dissociated dorsal root ganglion (DRG) neurons. Also, the behavioral effects of crotamine intoxication were compared with those of a muscle-selective sodium channel antagonist mu-CgTx-GIIIA, and other sodium-acting toxins such as tetrodotoxin alpha- and beta-pompilidotoxins, sea anemone toxin BcIII, spider toxin Tx2-6. Results pointed out that EDL was more susceptible to crotamine than soleus under direct electrical stimulation. Surprisingly, electrophysiological experiments in human Na(v)1.1 to Na(v)1.6 Na+ channels failed to show any significant change in channel characteristics, in a clear contrast with former studies. DRG neurons did not respond to crotamine. The behavioral effects of the toxins were described in detail and showed remarkable differences. We conclude that, although differences in the physiology of fast and slow muscles may cause the typical crotamine syndrome, sodium channels are not the primary target of crotamine and therefore, the real mechanism of action of this toxin is still unknown.
Proceedings Publications
- Thota, L. R., De Souza, J. L., & Chignalia, A. Z. (2022, Fall). Novel mechanisms underlying heart failure induced by pressure overload. In Free Radical Biology and Medicine, 192, 56-57.
Presentations
- De Souza, J. L. (2018). Non-canonical mechanisms of voltage dependence regulation in potassium channels . Department of Physiology at the University of Arizona Seminar Series. Tucson, AZ: The University of Arizona.
- De Souza, J. L. (2023). Gating mechanisms in heteromeric VGKCs. Journal Club – Neurophysiology, in the Department of Physiology at the University of Arizona. Tucson, AZ: The University of Arizona.
- De Souza, J. L. (2023). Heteromeric Kv channels and new opportunities to modulate neuronal activity. Seminar series in the School of Pharmacy at the University of Mississippi – Olemiss. Oxford, MS: The University of MIssissippi.
- De Souza, J. L. (2023). Heteromeric voltage-gated ion channels in health and diseases. Physiological Sciences Seminar. Superior Institute of Biomedical Sciences at the State University of Ceara. Fortaleza, CE, Brazil: State University of Ceara.
- De Souza, J. L. (2023). Mechanisms of expression and gating in heteromeric voltage-gated K+ channels. Department of Physiology and Biophysics - Seminar Series. Sao Paulo, SP, Brazil: University of Sao Paulo.
- De Souza, J. L. (2023). Optocapacitance: Making cells photoexcitable without genetic modification & Robust visual cortex evoked potentials (VEP) in Gnat1 and Gnat2 knockout mice. Journal Club – Neurophysiology, in the Department of Physiology at the University of Arizona. Tucson, AZ: The University of Arizona.
- De Souza, J. L. (2022). Modulation of ion channels: Lessons from coupling mechanisms. Department of Anesthesiology Grand Rounds. Tucson, AZ: The University of Arizona.
- De Souza, J. L. (2022). Photosensitivity of excitable cells enabled by gold-nanoparticles-assisted optocapacitance. Department of Anesthesiology Grand Rounds. Tucson, AZ: The University of Arizona.
- Flood, M. D., & De Souza, J. L. (2022, February). Mice with Disrupted Canonical Phototransductive Pathways Retain Robust VEP Responses under Light-Adapted Conditions. 66th Biophysical Society Annual Meeting. San Francisco CA: Biophysical Society.
- Black, S., Zemskov, E., De Souza, J. L., & Chignalia, A. Z. (2021). RhoA Mediates Pressure-Induced Endothelial Hyperpermeability. AHA Scientific Session. Virtual: AHA.More infoCirculation 144 (Suppl_1), A13551-A13551
- De Souza, J. L. (2020). Stoichiometry of heteromeric Kv channels. Journal Club – Neurophysiology, in the Department of Physiology at the University of Arizona. Tucson, AZ: The University of Arizona.
- De Souza, J. L. (2019). Optocapacitance: Making cells photoexcitable without genetic modification. Biomedical Sciences at Western University of Health Sciences - Seminar Series. Pomona, CA: Western University of Health Sciences.
- De Souza, J. L. (2018). Noncanonical coupling in voltage-gated potassium channels at . ChalkTalk seminars of the Department Biochemistry and Molecular Biology at The University of Chicago. Chicago IL: The University of Chicago.
- De Souza, J. L. (2017). Optocapacitance-Mediated Responses Of Excitable Cells: New Developments. ChalkTalk seminars of the Department of Neurobiology at The University of Chicago. Chicago IL: The University of Chicago.
Poster Presentations
- Chignalia, A. Z., De Souza, J. L., & Rosalez, J. L. (2023). Glypican 1 and intracellular calcium levels in lung endothelial cells. ASIP 2023 Joint Meeting of ASMB, HCS, and ASIP.
- Chignalia, A. Z., Potje, S., Bendhack, L., De Souza, J. L., & Thota, L. (2023). New Insights Into The Role Of The Glycocalyx Components In Heart Failure Induced By Pressure Overload.. Basic Cardiovascular Sciences, American Heart Association. Boston, USA.
- Rosales, J. L., De Souza, J. L., & Chignalia, A. Z. (2023, October/Fall).
Glypican 1 and intracellular calcium levels in lung endothelial cells
. ASIP 2023 Joint Meeting of ASMB, HCS, and ASIP. Salt Lake City. - Thota, L., Potje, S., Bendhack, L., De Souza, J. L., & Chignalia, A. Z. (2023, July/Summer). New Insights Into The Role Of The Glycocalyx Components In Heart Failure Induced By Pressure Overload. Basic Cardiovascular Sciences (BCVS). Boston.
- Black, S., Zemskov, E., De Souza, J. L., & Chignalia, A. Z. (2022). Lung endothelial transport in glypican 1 knockout mice. Scientific Sessions American heart Association.
- Chignalia, A. Z., De Souza, J. L., Zemskov, E., & Black, S. M. (2021, Nov / Fall). RhoA Mediates Pressure-Induced Endothelial Hyperpermeability. CirculationLippincott Williams & Wilkins.
- De Souza, J. L., & Flood, M. D. (2023, Feb). Modulation of Delayed Rectifying K+ Currents by Silent Kv Subunits. 67th Biophysical Society Annual Meeting. San Diego CA: Biophysical Society.
- Souza, D., Chignalia, A. Z., & De Souza, J. L. (2021, February/Winter). Modulation of ventricular cardiomyocytes electrophysiology by glypican 1 heparan sulfate proteoglycan.. 65th Biophysical Society Annual Meeting. on line: Biophysical Society.
- Thota, L. R., De Souza, J. L., & Chignalia, A. Z. (2022, November). Novel mechanisms underlying heart failure by pressure overload. SfRBM 29th Annual Conference. Orlando: SfRBM.
- Chignalia, A. Z., De Souza, J. L., & Dull, R. (2020, April/Spring). Glypican‐1 Plays a Cardiac Protective Effect During Heart Failure Induced by Pressure Overload. Experimental Biology.
- Dull, R., De Souza, J. L., & Chignalia, A. Z. (2020, April/Spring). Glypican‐1 and Remodeling Cardiac Hypertrophy.. Experimental Biology.
- Bassetto Jr, C., De Souza, J. L., & Bezanilla, F. (2019, Spring). Dynamics of Pore Domain Affected by Single Mutations in S4 Segment of Shaker Potassium Channel. 64th Annual Meeting of the Biophysical Society. Baltimore: Biophysical Society.
- De Souza, J. L., Bassetto Jr, C., Lee, E. E., & Bezanilla, F. (2019, Spring). Mobility of S3-S4 Linker Modulates Activation Process in Shaker Potassium Channels. 64th Annual Meeting of the Biophysical Society. Baltimore: Biophysical Society.
- De Souza, J. L., Nag, O. K., Huston, A., Vurgaftman, I., Pepperberg, D. R., Bezanilla, F., & Delehanty, J. B. (2019, Spring). Cholesterol Functionalization of Gold Nanoparticles Enables Neural Photo-Activation. 64th Annual Meeting of the Biophysical Society. Baltimore: Biophysical Society.
- Potje, S. R., Bendhack, L. M., Tostes, R., Dull, R., De Souza, J. L., & Chignalia, A. Z. (2019, Fall). Heparan sulfate proteoglycans modulate calcium storage and mobility in aortic rings. Society for Free Radicals Biology and Medicine. Las Vegas: Society for Free Radicals Biology and Medicine.