Publications by authors named "Vladimir Yarov-Yarovoy"

78 Publications

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core.

PLoS Pathog 2021 Apr 7;17(4):e1009440. Epub 2021 Apr 7.

Department of Microbiology, University of Washington, Seattle, Washington, United States of America.

Critical molecular events that control conformational transitions in most allosteric proteins are ill-defined. The mannose-specific FimH protein of Escherichia coli is a prototypic bacterial adhesin that switches from an 'inactive' low-affinity state (LAS) to an 'active' high-affinity state (HAS) conformation allosterically upon mannose binding and mediates shear-dependent catch bond adhesion. Here we identify a novel type of antibody that acts as a kinetic trap and prevents the transition between conformations in both directions. Disruption of the allosteric transitions significantly slows FimH's ability to associate with mannose and blocks bacterial adhesion under dynamic conditions. FimH residues critical for antibody binding form a compact epitope that is located away from the mannose-binding pocket and is structurally conserved in both states. A larger antibody-FimH contact area is identified by NMR and contains residues Leu-34 and Val-35 that move between core-buried and surface-exposed orientations in opposing directions during the transition. Replacement of Leu-34 with a charged glutamic acid stabilizes FimH in the LAS conformation and replacement of Val-35 with glutamic acid traps FimH in the HAS conformation. The antibody is unable to trap the conformations if Leu-34 and Val-35 are replaced with a less bulky alanine. We propose that these residues act as molecular toggle switches and that the bound antibody imposes a steric block to their reorientation in either direction, thereby restricting concerted repacking of side chains that must occur to enable the conformational transition. Residues homologous to the FimH toggle switches are highly conserved across a diverse family of fimbrial adhesins. Replacement of predicted switch residues reveals that another E. coli adhesin, galactose-specific FmlH, is allosteric and can shift from an inactive to an active state. Our study shows that allosteric transitions in bacterial adhesins depend on toggle switch residues and that an antibody that blocks the switch effectively disables adhesive protein function.
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http://dx.doi.org/10.1371/journal.ppat.1009440DOI Listing
April 2021

Directed Evolution of a Selective and Sensitive Serotonin Sensor via Machine Learning.

Cell 2020 Dec 16;183(7):1986-2002.e26. Epub 2020 Dec 16.

Departments of Biochemistry and Molecular Medicine, Chemistry, Statistics, Molecular and Cellular Biology, and Physiology and Membrane Biology, the Center for Neuroscience, and Graduate Programs in Molecular, Cellular, and Integrative Physiology, Biochemistry, Molecular, Cellular and Developmental Biology and Neuroscience, University of California, Davis, Davis, CA 95616, USA. Electronic address:

Serotonin plays a central role in cognition and is the target of most pharmaceuticals for psychiatric disorders. Existing drugs have limited efficacy; creation of improved versions will require better understanding of serotonergic circuitry, which has been hampered by our inability to monitor serotonin release and transport with high spatial and temporal resolution. We developed and applied a binding-pocket redesign strategy, guided by machine learning, to create a high-performance, soluble, fluorescent serotonin sensor (iSeroSnFR), enabling optical detection of millisecond-scale serotonin transients. We demonstrate that iSeroSnFR can be used to detect serotonin release in freely behaving mice during fear conditioning, social interaction, and sleep/wake transitions. We also developed a robust assay of serotonin transporter function and modulation by drugs. We expect that both machine-learning-guided binding-pocket redesign and iSeroSnFR will have broad utility for the development of other sensors and in vitro and in vivo serotonin detection, respectively.
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http://dx.doi.org/10.1016/j.cell.2020.11.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8025677PMC
December 2020

Different arrhythmia-associated calmodulin mutations have distinct effects on cardiac SK channel regulation.

J Gen Physiol 2020 12;152(12)

Division of Cardiovascular Medicine, Department of Internal Medicine, School of Medicine, University of California, Davis, Davis, CA.

Calmodulin (CaM) plays a critical role in intracellular signaling and regulation of Ca2+-dependent proteins and ion channels. Mutations in CaM cause life-threatening cardiac arrhythmias. Among the known CaM targets, small-conductance Ca2+-activated K+ (SK) channels are unique, since they are gated solely by beat-to-beat changes in intracellular Ca2+. However, the molecular mechanisms of how CaM mutations may affect the function of SK channels remain incompletely understood. To address the structural and functional effects of these mutations, we introduced prototypical human CaM mutations in human induced pluripotent stem cell-derived cardiomyocyte-like cells (hiPSC-CMs). Using structural modeling and molecular dynamics simulation, we demonstrate that human calmodulinopathy-associated CaM mutations disrupt cardiac SK channel function via distinct mechanisms. CaMD96V and CaMD130G mutants reduce SK currents through a dominant-negative fashion. By contrast, specific mutations replacing phenylalanine with leucine result in conformational changes that affect helix packing in the C-lobe, which disengage the interactions between apo-CaM and the CaM-binding domain of SK channels. Distinct mutant CaMs may result in a significant reduction in the activation of the SK channels, leading to a decrease in the key Ca2+-dependent repolarization currents these channels mediate. The findings in this study may be generalizable to other interactions of mutant CaMs with Ca2+-dependent proteins within cardiac myocytes.
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http://dx.doi.org/10.1085/jgp.202012667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7681919PMC
December 2020

New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket.

Elife 2020 11 9;9. Epub 2020 Nov 9.

Department of Physiology and Membrane Biology, University of California Davis, School of Medicine, Davis, United States.

The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally, they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation.
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http://dx.doi.org/10.7554/eLife.62039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671684PMC
November 2020

An Unorthodox Mechanism Underlying Voltage Sensitivity of TRPV1 Ion Channel.

Adv Sci (Weinh) 2020 Oct 21;7(20):2000575. Epub 2020 Sep 21.

Department of Physiology and Membrane Biology University of California, Davis One Shields Avenue Davis CA 95616 USA.

While the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal nociceptor for heat, capsaicin, and protons, the channel's responses to each of these stimuli are profoundly regulated by membrane potential, damping or even prohibiting its response at negative voltages and amplifying its response at positive voltages. Therefore, voltage sensitivity of TRPV1 is anticipated to play an important role in shaping pain responses. How voltage regulates TRPV1 activation remains unknown. Here, it is shown that voltage sensitivity does not originate from the S4 segment like classic voltage-gated ion channels; instead, outer pore acidic residues directly partake in voltage-sensitive activation, with their negative charges collectively constituting the observed gating charges. Outer pore gating-charge movement is titratable by extracellular pH and is allosterically coupled to channel activation, likely by influencing the upper gate in the ion selectivity filter. Elucidating this unorthodox voltage-gating process provides a mechanistic foundation for understanding TRPV1 polymodal gating and opens the door to novel approaches regulating channel activity for pain management.
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http://dx.doi.org/10.1002/advs.202000575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578911PMC
October 2020

Gating Properties of Mutant Sodium Channels and Responses to Sodium Current Inhibitors Predict Mexiletine-Sensitive Mutations of Long QT Syndrome 3.

Front Pharmacol 2020 4;11:1182. Epub 2020 Aug 4.

Department of Cardiology, Peking University First Hospital, Beijing, China.

Background: Long QT syndrome 3 (LQT3) is caused by mutations. Late sodium current (late ) inhibitors are current-specific to treat patients with LQT3, but the mechanisms underlying mexiletine (MEX) -sensitive (N1325S and R1623Q) and -insensitive (M1652R) mutations remains to be elucidated.

Methods: LQT3 patients with causative mutations were treated with oral MEX following i.v. lidocaine. Whole-cell patch-clamp techniques and molecular remodeling were used to determine the mechanisms underlying the sensitivity to MEX.

Results: Intravenous administration of lidocaine followed by MEX orally in LQT patients with N1325S and R1623Q sodium channel mutation shortened QTc interval, abolished arrhythmias, and completely normalized the ECG. In HEK293 cells, the steady-state inactivation curves of the M1652R channels were rightward shifted by 5.6 mV relative to the WT channel. In contrast, the R1623Q mutation caused a leftward shift of the steady-state inactivation curve by 15.2 mV compared with WT channel, and N1325S mutation did not affect steady-state inactivation (n = 5-13, < 0.05). The extent of the window current was expanded in all three mutant channels compared with WT. All three mutations increased late with the greatest amplitude in the M1652R channel (n = 9-15, < 0.05). MEX caused a hyperpolarizing shift of the steady-state inactivation and delayed the recovery of all three mutant channels. Furthermore, it suppressed late in N1325S and R1623Q to a greater extent compared to that of M1652R mutant channel. Mutations altered the sensitivity of Na1.5 to MEX through allosteric mechanisms by changing the conformation of Na1.5 to become more or less favorable for MEX binding. Late inhibitors suppressed late in N1325S and R1623Q to a greater extent than that in the M1652R mutation (n = 4-7, < 0.05).

Conclusion: The N1325S, R1623Q, and M1652R mutations are associated with a variable augmentation of late , which was reversed by MEX. M1652R mutation changes the conformation of Na1.5 that disrupt the inactivation of channel affecting MEX binding, corresponding to the poor response to MEX. The lidocaine test, molecular modeling, and drugs screening in cells expressing mutant channels are useful for predicting the effectiveness of late inhibitors.
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http://dx.doi.org/10.3389/fphar.2020.01182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417866PMC
August 2020

Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence.

ACS Chem Neurosci 2020 08 9;11(15):2316-2326. Epub 2020 Jul 9.

Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.
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http://dx.doi.org/10.1021/acschemneuro.0c00276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894042PMC
August 2020

Cooperativity of K7.4 channels confers ultrafast electromechanical sensitivity and emergent properties in cochlear outer hair cells.

Sci Adv 2020 04 8;6(15):eaba1104. Epub 2020 Apr 8.

Department of Physiology, School of Medicine, University of Nevada, Reno, Reno, NV 89557, USA.

The mammalian cochlea relies on active electromotility of outer hair cells (OHCs) to resolve sound frequencies. OHCs use ionic channels and somatic electromotility to achieve the process. It is unclear, though, how the kinetics of voltage-gated ionic channels operate to overcome extrinsic viscous drag on OHCs at high frequency. Here, we report ultrafast electromechanical gating of clustered K7.4 in OHCs. Increases in kinetics and sensitivity resulting from cooperativity among clustered-K7.4 were revealed, using optogenetics strategies. Upon clustering, the half-activation voltage shifted negative, and the speed of activation increased relative to solitary channels. Clustering also rendered K7.4 channels mechanically sensitive, confirmed in consolidated K7.4 channels at the base of OHCs. K7.4 clusters provide OHCs with ultrafast electromechanical channel gating, varying in magnitude and speed along the cochlea axis. Ultrafast K7.4 gating provides OHCs with a feedback mechanism that enables the cochlea to overcome viscous drag and resolve sounds at auditory frequencies.
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http://dx.doi.org/10.1126/sciadv.aba1104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141818PMC
April 2020

The MX-Helix of Muscle nAChR Subunits Regulates Receptor Assembly and Surface Trafficking.

Front Mol Neurosci 2020 24;13:48. Epub 2020 Mar 24.

Department of Physiology and Membrane Biology, University of California, Davis, Davis, CA, United States.

Nicotinic acetylcholine receptors (AChRs) are pentameric channels that mediate fast transmission at the neuromuscular junction (NMJ) and defects in receptor expression underlie neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndrome (CMS). Nicotinic receptor expression at the NMJ is tightly regulated and we previously identified novel Golgi-retention signals in the β and δ subunit cytoplasmic loops that regulate trafficking of the receptor to the cell surface. Here, we show that the Golgi retention motifs are localized in the MX-helix, a juxta-membrane alpha-helix present in the proximal cytoplasmic loop of receptor subunits, which was defined in recent crystal structures of cys-loop receptor family members. First, mutational analysis of CD4-MX-helix chimeric proteins showed that the Golgi retention signal was dependent on both the amphipathic nature of the MX-helix and on specific lysine residues (βK353 and δK351). Moreover, retention was associated with ubiquitination of the lysines, and βK353R and δK351R mutations reduced ubiquitination and increased surface expression of CD4-β and δ MX-helix chimeric proteins. Second, mutation of these lysines in intact β and δ subunits perturbed Golgi-based glycosylation and surface trafficking of the AChR. Notably, combined βK353R and δK351R mutations increased the amount of surface AChR with immature forms of glycosylation, consistent with decreased Golgi retention and processing. Third, we found that previously identified CMS mutations in the ε subunit MX-helix decreased receptor assembly and surface levels, as did an analogous mutation introduced into the β subunit MX-helix. Together, these findings indicate that the subunit MX-helix contributes to receptor assembly and is required for normal expression of the AChR and function of the NMJ. In addition, specific determinants in the β and δ subunit MX-helix contribute to quality control of AChR expression by intracellular retention and ubiquitination of unassembled subunits, and by facilitating the appropriate glycosylation of assembled surface AChR.
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http://dx.doi.org/10.3389/fnmol.2020.00048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105636PMC
March 2020

A Computational Pipeline to Predict Cardiotoxicity: From the Atom to the Rhythm.

Circ Res 2020 04 24;126(8):947-964. Epub 2020 Feb 24.

From the Department of Physiology and Membrane Biology (P.-C.Y., K.R.D., P.A., M.-T.J., J.R.D.D., V.Y.-Y., I.V., C.E.C.), University of California Davis.

Rationale: Drug-induced proarrhythmia is so tightly associated with prolongation of the QT interval that QT prolongation is an accepted surrogate marker for arrhythmia. But QT interval is too sensitive a marker and not selective, resulting in many useful drugs eliminated in drug discovery.

Objective: To predict the impact of a drug from the drug chemistry on the cardiac rhythm.

Methods And Results: In a new linkage, we connected atomistic scale information to protein, cell, and tissue scales by predicting drug-binding affinities and rates from simulation of ion channel and drug structure interactions and then used these values to model drug effects on the hERG channel. Model components were integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model framework validation and showed excellent agreement, demonstrating feasibility of a new approach for cardiotoxicity prediction.

Conclusions: We present a multiscale model framework to predict electrotoxicity in the heart from the atom to the rhythm. Novel mechanistic insights emerged at all scales of the system, from the specific nature of proarrhythmic drug interaction with the hERG channel, to the fundamental cellular and tissue-level arrhythmia mechanisms. Applications of machine learning indicate necessary and sufficient parameters that predict arrhythmia vulnerability. We expect that the model framework may be expanded to make an impact in drug discovery, drug safety screening for a variety of compounds and targets, and in a variety of regulatory processes.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155920PMC
April 2020

α-Actinin-1 promotes activity of the L-type Ca channel Ca 1.2.

EMBO J 2020 03 27;39(5):e102622. Epub 2020 Jan 27.

Department of Pharmacology, University of California, Davis, CA, USA.

The L-type Ca channel Ca 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of Ca 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts Ca 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The Ca 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits Ca 1.2 to defined surface regions and simultaneously boosts its open probability so that Ca 1.2 is mostly active when appropriately localized.
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http://dx.doi.org/10.15252/embj.2019102622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7049811PMC
March 2020

Veratridine: A Janus-Faced Modulator of Voltage-Gated Sodium Ion Channels.

ACS Chem Neurosci 2020 02 17;11(3):418-426. Epub 2020 Jan 17.

Department of Chemistry , Stanford University , Stanford , California 94305 , United States.

Voltage-gated sodium ion channels (Nas) are integral to both neuronal and muscular signaling and are a primary target for a number of proteinaceous and small molecule toxins. Included among these neurotoxins is veratridine (VTD), a C-nor-D homosteroidal alkaloid from the seeds of members of the genus. VTD binds to Na within the pore region, causing a hyperpolarizing shift in the activation threshold in addition to reducing peak current. We have characterized the activity of VTD against heterologously expressed rat Na1.4 and have demonstrated that VTD acts on the channel as either an agonist or antagonist depending on the nature of the electrophysiological stimulation protocol. Structure-activity studies with VTD and VTD derivatives against Na mutants show that the functional duality of VTD can be decoupled. These findings suggest that the dichotomous activity of VTD may derive from two distinct, use-dependent binding orientations of the toxin.
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http://dx.doi.org/10.1021/acschemneuro.9b00621DOI Listing
February 2020

The Trials and Tribulations of Structure Assisted Design of K Channel Activators.

Front Pharmacol 2019 20;10:972. Epub 2019 Sep 20.

Department of Pharmacology, School of Medicine, University of California, Davis, Davis, CA, United States.

Calcium-activated K channels constitute attractive targets for the treatment of neurological and cardiovascular diseases. To explain why certain 2-aminobenzothiazole/oxazole-type K activators (SKAs) are K3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of K3.1 and K2.3 in complex with CaM using Rosetta modeling software. We here attempted to employ this atomistic level understanding of K activator binding to switch selectivity around and design K2.2 selective activators as potential anticonvulsants. In this structure-based drug design approach we used RosettaLigand docking and carefully compared the binding poses of various SKA compounds in the K2.2 and K3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the K2.2 and K.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and K2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any K2.2 selective compounds. Based on the full-length K3.1 structure it was recently demonstrated that the C-terminal crystal dimer was an artefact and suggested that the "real" binding pocket for the K activators is located at the S4-S5 linker. We here confirmed this structural hypothesis through mutagenesis and now offer a new, corrected binding site model for the SKA-type K channel activators. SKA-111 (5-methylnaphtho[1,2-]thiazol-2-amine) is binding in the interface between the CaM N-lobe and the S4-S5 linker where it makes van der Waals contacts with S181 and L185 in the SA helix of K3.1.
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http://dx.doi.org/10.3389/fphar.2019.00972DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6764326PMC
September 2019

The Sodium Channel Voltage Sensor Slides to Rest.

Trends Pharmacol Sci 2019 10 5;40(10):718-720. Epub 2019 Sep 5.

Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA. Electronic address:

Voltage-gated sodium channels (Nas) initiate the action potential waveforms in excitable cells. The molecular mechanisms controlling this process have been actively debated. New prokaryotic Na structures by Wisedchaisri et al. have completed our understanding of the molecular conformations required for cellular electrical signaling, and provide key templates for research to examine eukaryotic Nas.
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http://dx.doi.org/10.1016/j.tips.2019.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7373202PMC
October 2019

Opening TRPP2 () requires the transfer of gating charges.

Proc Natl Acad Sci U S A 2019 07 17;116(31):15540-15549. Epub 2019 Jul 17.

Department of Pharmacology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611;

The opening of voltage-gated ion channels is initiated by transfer of gating charges that sense the electric field across the membrane. Although transient receptor potential ion channels (TRP) are members of this family, their opening is not intrinsically linked to membrane potential, and they are generally not considered voltage gated. Here we demonstrate that TRPP2, a member of the polycystin subfamily of TRP channels encoded by the gene, is an exception to this rule. TRPP2 borrows a biophysical riff from canonical voltage-gated ion channels, using 2 gating charges found in its fourth transmembrane segment (S4) to control its conductive state. Rosetta structural prediction demonstrates that the S4 undergoes ∼3- to 5-Å transitional and lateral movements during depolarization, which are coupled to opening of the channel pore. Here both gating charges form state-dependent cation-π interactions within the voltage sensor domain (VSD) during membrane depolarization. Our data demonstrate that the transfer of a single gating charge per channel subunit is requisite for voltage, temperature, and osmotic swell polymodal gating of TRPP2. Taken together, we find that irrespective of stimuli, TRPP2 channel opening is dependent on activation of its VSDs.
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http://dx.doi.org/10.1073/pnas.1902917116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681712PMC
July 2019

A distinct structural mechanism underlies TRPV1 activation by piperine.

Biochem Biophys Res Commun 2019 08 15;516(2):365-372. Epub 2019 Jun 15.

Department of Physiology and Membrane Biology, UC Davis School of Medicine, Davis, CA, 95616, USA. Electronic address:

Piperine, the principle pungent compound in black peppers, is known to activate the capsaicin receptor TRPV1 ion channel. How piperine interacts with the channel protein, however, remains unclear. Here we show that piperine binds to the same ligand-binding pocket as capsaicin but in different poses. There was no detectable detrimental effect when T551 and E571, two major sites known to form hydrogen bond with capsaicin, were mutated to a hydrophobic amino acid. Computational structural modeling suggested that piperine makes interactions with multiple amino acids within the ligand binding pocket, including T671 on the pore-forming S6 segment. Mutations of this residue could substantially reduce or even eliminate piperine-induced activation, confirming that T671 is an important site. Our results suggest that the bound piperine may directly interact with the pore-forming S6 segment to induce channel opening. These findings help to explain why piperine is a weak agonist, and may guide future efforts to develop novel pharmaceutical reagents targeting TRPV1.
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http://dx.doi.org/10.1016/j.bbrc.2019.06.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6626684PMC
August 2019

Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers.

Br J Pharmacol 2019 09 22;176(17):3364-3377. Epub 2019 Jul 22.

Department of Physiology and Membrane Biology, UC Davis School of Medicine, Davis, CA, USA.

Background And Purpose: Like chili peppers, gingers produce pungent stimuli by a group of vanilloid compounds that activate the nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel. How these compounds interact with TRPV1 remains unclear.

Experimental Approach: We used computational structural modelling, functional tests (electrophysiology and calcium imaging), and mutagenesis to investigate the structural mechanisms underlying ligand-channel interactions.

Key Results: The potency of three principal pungent compounds from ginger -shogaol, gingerol, and zingerone-depends on the same two residues in the TRPV1 channel that form a hydrogen bond with the chili pepper pungent compound, capsaicin. Computational modelling revealed binding poses of these ginger compounds similar to those of capsaicin, including a "head-down tail-up" orientation, two specific hydrogen bonds, and important contributions of van der Waals interactions by the aliphatic tail. Our study also identified a novel horizontal binding pose of zingerone that allows it to directly interact with the channel pore when bound inside the ligand-binding pocket. These observations offer a molecular level explanation for how unique structures in the ginger compounds affect their channel activation potency.

Conclusions And Implications: Mechanistic insights into the interactions of ginger compounds and the TRPV1 cation channel should help guide drug discovery efforts to modulate nociception.
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http://dx.doi.org/10.1111/bph.14766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692589PMC
September 2019

Pathogenic effects of agrin V1727F mutation are isoform specific and decrease its expression and affinity for HSPGs and LRP4.

Hum Mol Genet 2019 08;28(16):2648-2658

Department of Physiology and Membrane Biology, University of California Davis, Davis, CA, USA.

Agrin is a large extracellular matrix protein whose isoforms differ in their tissue distribution and function. Motoneuron-derived y+z+ agrin regulates the formation of the neuromuscular junction (NMJ), while y-z- agrin is widely expressed and has diverse functions. Previously we identified a missense mutation (V1727F) in the second laminin globular (LG2) domain of agrin that causes severe congenital myasthenic syndrome. Here, we define pathogenic effects of the agrin V1727F mutation that account for the profound dysfunction of the NMJ. First, by expressing agrin variants in heterologous cells, we show that the V1727F mutation reduces the secretion of y+z+ agrin compared to wild type, whereas it has no effect on the secretion of y-z- agrin. Second, we find that the V1727F mutation significantly impairs binding of y+z+ agrin to both heparin and the low-density lipoprotein receptor-related protein 4 (LRP4) coreceptor. Third, molecular modeling of the LG2 domain suggests that the V1727F mutation primarily disrupts the y splice insert, and consistent with this we find that it partially occludes the contribution of the y splice insert to agrin binding to heparin and LRP4. Together, these findings identify several pathogenic effects of the V1727F mutation that reduce its expression and ability to bind heparan sulfate proteoglycan and LRP4 coreceptors involved in the muscle-specific kinase signaling pathway. These defects primarily impair the function of neural y+z+ agrin and combine to cause a severe CMS phenotype, whereas y-z- agrin function in other tissues appears preserved.
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http://dx.doi.org/10.1093/hmg/ddz081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687949PMC
August 2019

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Microbiologyopen 2019 11 19;8(11):e827. Epub 2019 Mar 19.

Department of Food Science & Technology, University of California-Davis, Davis, California.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-β-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two β-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg , Co , Zn , or Cu . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC.
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http://dx.doi.org/10.1002/mbo3.827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854853PMC
November 2019

Editorial.

Neurosci Lett 2019 05 26;700:1-2. Epub 2019 Feb 26.

Department of Physiology and Membrane Biology, University of California, Davis, CA, 95616, USA. Electronic address:

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http://dx.doi.org/10.1016/j.neulet.2019.02.040DOI Listing
May 2019

Antibodies and venom peptides: new modalities for ion channels.

Nat Rev Drug Discov 2019 05;18(5):339-357

Department of Physiology & Membrane Biology, University of California Davis, Davis, CA, USA.

Ion channels play fundamental roles in both excitable and non-excitable tissues and therefore constitute attractive drug targets for myriad neurological, cardiovascular and metabolic diseases as well as for cancer and immunomodulation. However, achieving selectivity for specific ion channel subtypes with small-molecule drugs has been challenging, and there currently is a growing trend to target ion channels with biologics. One approach is to improve the pharmacokinetics of existing or novel venom-derived peptides. In parallel, after initial studies with polyclonal antibodies demonstrated the technical feasibility of inhibiting channel function with antibodies, multiple preclinical programmes are now using the full spectrum of available technologies to generate conventional monoclonal and engineered antibodies or nanobodies against extracellular loops of ion channels. After a summary of the current state of ion channel drug discovery, this Review discusses recent developments using the purinergic receptor channel P2X purinoceptor 7 (P2X7), the voltage-gated potassium channel K1.3 and the voltage-gated sodium channel Na1.7 as examples of targeting ion channels with biologics.
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http://dx.doi.org/10.1038/s41573-019-0013-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499689PMC
May 2019

Structural basis for antiarrhythmic drug interactions with the human cardiac sodium channel.

Proc Natl Acad Sci U S A 2019 02 6;116(8):2945-2954. Epub 2019 Feb 6.

Department of Physiology and Membrane Biology, University of California, Davis, CA 95616;

The human voltage-gated sodium channel, hNa1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNa1.5-targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNa1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNa1.5 and will be useful for rational design of novel therapeutics.
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http://dx.doi.org/10.1073/pnas.1817446116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386684PMC
February 2019

The conformational wave in capsaicin activation of transient receptor potential vanilloid 1 ion channel.

Nat Commun 2018 07 23;9(1):2879. Epub 2018 Jul 23.

Department of Physiology and Membrane Biology, University of California, Davis, CA, 95616, USA.

The capsaicin receptor TRPV1 has been intensively studied by cryo-electron microscopy and functional tests. However, though the apo and capsaicin-bound structural models are available, the dynamic process of capsaicin activation remains intangible, largely due to the lack of a capsaicin-induced open structural model and the low occupancy of the transition states. Here we report that reducing temperature toward the freezing point substantially increased channel closure events even in the presence of saturating capsaicin. We further used a combination of fluorescent unnatural amino acid (fUAA) incorporation, computational modeling, and rate-equilibrium linear free-energy relationships analysis (Φ-analysis) to derive the fully open capsaicin-bound state model, and reveal how the channel transits from the apo to the open state. We observed that capsaicin initiates a conformational wave that propagates through the S4-S5 linker towards the S6 bundle and finally reaching the selectivity filter. Our study provides a temporal mechanism for capsaicin activation of TRPV1.
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http://dx.doi.org/10.1038/s41467-018-05339-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6056546PMC
July 2018

Structural Determinants for the Selectivity of the Positive KCa3.1 Gating Modulator 5-Methylnaphtho[2,1-]oxazol-2-amine (SKA-121).

Mol Pharmacol 2017 10 31;92(4):469-480. Epub 2017 Jul 31.

Department of Pharmacology (B.M.B., H.S., H.W.), Department of Physiology and Membrane Biology (V.Y.-Y.), School of Medicine, and Department of Chemistry (H.S.), University of California, Davis, California; and Department of Biomedical and Pharmaceutical Sciences, Chapman University School of Pharmacy, Irvine, California (M.Z.)

Intermediate-conductance (K3.1) and small-conductance (K2) calcium-activated K channels are gated by calcium binding to calmodulin (CaM) molecules associated with the calmodulin-binding domain (CaM-BD) of these channels. The existing K activators, such as naphtho[1,2-]thiazol-2-ylamine (SKA-31), 6,7-dichloro-1-indole-2,3-dione 3-oxime (NS309), and 1-ethylbenzimidazolin-2-one (EBIO), activate both channel types with similar potencies. In a previous chemistry effort, we optimized the benzothiazole pharmacophore of SKA-31 toward K3.1 selectivity and identified 5-methylnaphtho[2,1-]oxazol-2-amine (SKA-121), which exhibits 40-fold selectivity for K3.1 over K2.3. To understand why introduction of a single CH group in five-position of the benzothiazole/oxazole system could achieve such a gain in selectivity for K3.1 over K2.3, we first localized the binding site of the benzothiazoles/oxazoles to the CaM-BD/CaM interface and then used computational modeling software to generate models of the K3.1 and K2.3 CaM-BD/CaM complexes with SKA-121. Based on a combination of mutagenesis and structural modeling, we suggest that all benzothiazole/oxazole-type K activators bind relatively "deep" in the CaM-BD/CaM interface and hydrogen bond with E54 on CaM. In K3.1, SKA-121 forms an additional hydrogen bond network with R362. In contrast, NS309 sits more "forward" and directly hydrogen bonds with R362 in K3.1. Mutating R362 to serine, the corresponding residue in K2.3 reduces the potency of SKA-121 by 7-fold, suggesting that R362 is responsible for the generally greater potency of K activators on K3.1. The increase in SKA-121's K3.1 selectivity compared with its parent, SKA-31, seems to be due to better overall shape complementarity and hydrophobic interactions with S372 and M368 on K3.1 and M72 on CaM at the K3.1-CaM-BD/CaM interface.
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http://dx.doi.org/10.1124/mol.117.109421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5588545PMC
October 2017

A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.

FASEB J 2017 07 11;31(7):3167-3178. Epub 2017 Apr 11.

The National and Local Joint Engineering Laboratory of Animal Peptide Drug Development, College of Life Sciences, Hunan Normal University, Changsha, China;

Voltage-gated sodium channels (Nas) are activated by transiting the voltage sensor from the deactivated to the activated state. The crystal structures of several bacterial Nas have captured the voltage sensor module (VSM) in an activated state, but structure of the deactivated voltage sensor remains elusive. In this study, we sought to identify peptide toxins stabilizing the deactivated VSM of bacterial Nas. We screened fractions from several venoms and characterized a cystine knot toxin called JZTx-27 from the venom of tarantula as a high-affinity antagonist of the prokaryotic Nas NsBa (nonselective voltage-gated ) and NaChBac (bacterial sodium channel from ) (IC = 112 nM and 30 nM, respectively). JZTx-27 was more efficacious at weaker depolarizing voltages and significantly slowed the activation but accelerated the deactivation of NsBa, whereas the local anesthetic drug lidocaine was shown to antagonize NsBa without affecting channel gating. Mutation analysis confirmed that JZTx-27 bound to S3-4 linker of NsBa, with F98 being the critical residue in determining toxin affinity. All electrophysiological data and analysis suggested that JZTx-27 trapped VSM of NsBa in one of the deactivated states. In mammalian Nas, JZTx-27 preferably inhibited the inactivation of Na1.5 by targeting the fourth transmembrane domain. To our knowledge, this is the first report of peptide antagonist for prokaryotic Nas. More important, we proposed that JZTx-27 stabilized the NsBa VSM in the deactivated state and may be used as a probe to determine the structure of the deactivated VSM of Nas.-Tang, C., Zhou, X., Nguyen, P. T., Zhang, Y., Hu, Z., Zhang, C., Yarov-Yarovoy, V., DeCaen, P. G., Liang, S., Liu, Z. A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel.
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http://dx.doi.org/10.1096/fj.201600882RDOI Listing
July 2017

Gain-of-function mutation of a voltage-gated sodium channel Na1.7 associated with peripheral pain and impaired limb development.

J Biol Chem 2017 06 5;292(22):9262-9272. Epub 2017 Apr 5.

From the Department of Neurology,

Dominant mutations in voltage-gated sodium channel Na1.7 cause inherited erythromelalgia, a debilitating pain disorder characterized by severe burning pain and redness of the distal extremities. Na1.7 is preferentially expressed within peripheral sensory and sympathetic neurons. Here, we describe a novel Na1.7 mutation in an 11-year-old male with underdevelopment of the limbs, recurrent attacks of burning pain with erythema, and swelling in his feet and hands. Frequency and duration of the episodes gradually increased with age, and relief by cooling became less effective. The patient's sister had short stature and reported similar complaints of erythema and burning pain, but with less intensity. Genetic analysis revealed a novel missense mutation in Na1.7 (2567G>C; p.Gly856Arg) in both siblings. The G856R mutation, located within the DII/S4-S5 linker of the channel, substitutes a highly conserved non-polar glycine by a positively charged arginine. Voltage-clamp analysis of G856R currents revealed that the mutation hyperpolarized (-11.2 mV) voltage dependence of activation and slowed deactivation but did not affect fast inactivation, compared with wild-type channels. A mutation of Gly-856 to aspartic acid was previously found in a family with limb pain and limb underdevelopment, and its functional assessment showed hyperpolarized activation, depolarized fast inactivation, and increased ramp current. Structural modeling using the Rosetta computational modeling suite provided structural clues to the divergent effects of the substitution of Gly-856 by arginine and aspartic acid. Although the proexcitatory changes in gating properties of G856R contribute to the pathophysiology of inherited erythromelalgia, the link to limb underdevelopment is not well understood.
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http://dx.doi.org/10.1074/jbc.M117.778779DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5454107PMC
June 2017

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

Proc Natl Acad Sci U S A 2017 04 20;114(14):E2937-E2946. Epub 2017 Mar 20.

EyePod Small Animal Imaging Facility, University of California, Davis, CA 95618;

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gα), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gα the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s Analyzing swelling as osmotically driven water influx, we find the HO membrane permeability of the rod OS to be (2.6 ± 0.4) × 10 cm⋅s, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher HO permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.
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http://dx.doi.org/10.1073/pnas.1620572114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389324PMC
April 2017

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore.

Mol Pharmacol 2017 04 26;91(4):392-402. Epub 2017 Jan 26.

Department of Pharmacology (H.M.N, V.S., B.P., D.P.J., H.W.) and Department of Physiology and Membrane Biology (V. Y.-Y.), School of Medicine, University of California at Davis, Davis, California

The intermediate-conductance Ca-activated K channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.
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http://dx.doi.org/10.1124/mol.116.108068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363711PMC
April 2017

Potassium channels in the heart: structure, function and regulation.

J Physiol 2017 04 13;595(7):2209-2228. Epub 2016 Nov 13.

Department of Physiology and Membrane Biology, University of California, Davis, CA, 95616, USA.

This paper is the outcome of the fourth UC Davis Systems Approach to Understanding Cardiac Excitation-Contraction Coupling and Arrhythmias Symposium, a biannual event that aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2016 symposium was 'K Channels and Regulation'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies and challenges on the topic of cardiac K channels. This paper summarizes the topics of formal presentations and informal discussions from the symposium on the structural basis of voltage-gated K channel function, as well as the mechanisms involved in regulation of K channel gating, expression and membrane localization. Given the critical role for K channels in determining the rate of cardiac repolarization, it is hardly surprising that essentially every aspect of K channel function is exquisitely regulated in cardiac myocytes. This regulation is complex and highly interrelated to other aspects of myocardial function. K channel regulatory mechanisms alter, and are altered by, physiological challenges, pathophysiological conditions, and pharmacological agents. An accompanying paper focuses on the integrative role of K channels in cardiac electrophysiology, i.e. how K currents shape the cardiac action potential, and how their dysfunction can lead to arrhythmias, and discusses K channel-based therapeutics. A fundamental understanding of K channel regulatory mechanisms and disease processes is fundamental to reveal new targets for human therapy.
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http://dx.doi.org/10.1113/JP272864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5374109PMC
April 2017