Journal of General Physiology

Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses [~]50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10-7 cm2/s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, [~]20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by {beta}-escin (80 {micro}M) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming 1S subunit and the auxiliary 2{delta}-1 and {gamma}1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range. We generated a new HEK293-cell line stably expressing 2{delta}-1, {beta}3, and STAC3. When the adult (CaV1.1a) and the embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage-dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further co-expressed the {gamma}1 subunit, the current density of CaV1.1a, but not of CaV1.1e, was reduced by >50 %. In addition, {gamma}1 caused a shift of the voltage-dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of {gamma}1, but not its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between oppositely charged residues in the IVS3-S4 loop and the {gamma}1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the {gamma}1-dependent reduction of current density, suggesting that structural rearrangements of CaV1.1a induced by inclusion of exon 29 allosterically empower the {gamma}1 subunit to exert its inhibitory action on CaV1.1 calcium currents. SummaryEl Ghaleb et al. analyzed the effects of the {gamma}1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that {gamma}1 reduces the current amplitude in a splicing-dependent manner.

Voltage-gated sodium channels initiate action potentials in prokaryotes and in many eukaryotic cells, including vertebrate nerve and muscle. Their activation is steeply voltage-dependent, but it is unclear how the voltage sensitivity is set or whether it can be broadly shifted to positive voltages. Here we show that the voltage dependence of activation (VA) of the ancestral bacterial sodium channel NaVAb can be progressively shifted from -118 mV to +35 mV in chimeras with increasing numbers of amino acid residues from the extracellular half of the voltage sensor of human NaV1.7 channels. In a minimal chimera in which only 32 residues were transferred, we analyzed the effects of six additional mutations of conserved amino acid residues singly, in pairs, and as triple mutations. The resulting chimeric mutants exhibited a broad range of voltage sensitivity from VA=-118 mV to VA=+120 mV. Three mutations (N48K, L112A, and M119V) shifted VA to +61 mV when substituted in NaVAb itself, and substitution of two additional Cys residues in the Cys-free background of NaVAb further shifted VA to +105 mV. In these mutants, measurement of gating currents revealed that the voltage dependence of gating charge movement (VQ) shifted to positive membrane potentials as much or more than VA, confirming that the gating charges are trapped in their resting positions by these VA-shifting mutations. Our results demonstrate broadband shifting of VA and VQ of a sodium channel across a range of 240 mV and provide a toolbox of methods and constructs to analyze sodium channel structure and function in the resting state at 0 mV and in activated states at positive membrane potentials. GRAPHICAL ABSTRACTThe complete range of broadband tuning of voltage-dependent activation of a sodium channel. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=59 SRC="FIGDIR/small/392571v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@147c265org.highwire.dtl.DTLVardef@2b8b15org.highwire.dtl.DTLVardef@b84b48org.highwire.dtl.DTLVardef@1569072_HPS_FORMAT_FIGEXP M_FIG C_FIG

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel expressed in cardiac myocytes and human gastrointestinal (GI) smooth muscle cells (SMCs). NaV1.5 contributes to electrical excitability in the heart and slow waves in the gut. NaV1.5 is also mechanosensitive, and mechanical force modulates several modes of NaV1.5s voltage-dependent function. NaV1.5 mutations in patients with cardiac arrhythmias and gastrointestinal diseases lead to abnormal mechano- and voltage-sensitivity. Membrane permeable amphipathic drugs that target NaV1.5 in the heart and GI tract alter NaV1.5 mechanosensitivity (MS), suggesting that amphipaths may be a viable therapeutic option for modulating NaV1.5 function. We therefore searched for membrane-permeable amphipathic agents that would modulate NaV1.5 MS with minimal effect on NaV1.5 voltage-gating intact to more selectively target mechanosensitivity. We used two methods to assess NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macropatch and whole-cell Na+ current parameters with and without force. The pressure- and shear-mediated peak current increase and acceleration were effectively abolished by capsaicin. Capsaicin abolished the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of MS makes capsaicin is a novel modulator of NaV1.5 MS and a promising therapeutic candidate.

The cardiac voltage-gated sodium channel, Nav1.5 initiates the cardiac action potential. Its dysfunction can lead to dangerous arrhythmias, sudden cardiac arrest, and death. The functional Nav1.5 core consists of four homologous repeats (I, II, III, and IV), each formed from a voltage sensing and a pore domain. The channel also contains three cytoplasmic linkers (I-II, II-III, and III-IV). While Nav1.5 structures have been published, the I-II and II-III linkers have remained absent, are predicted to be disordered, and their functional role is not well understood. We divided the I-II linker into eight regions ranging in size from 32 to 52 residues, chosen based on their distinct properties. Since these regions had unique sequence properties, we hypothesized that they may have distinct effects on channel function. We tested this hypothesis with experiments with individual Nav1.5 constructs with each region deleted. These deletions had small effects on channel gating, though two (430 - 457del and 556 - 607del) reduced peak current. Phylogenetic analysis of the I-II linker revealed five prolines (P627, P628, P637, P640, P648) that were conserved in mammals but absent from the Xenopus sequence. We created mutant channels, where these were replaced with their Xenopus counterparts. The only mutation that had a significant effect on channel gating was P627S, which depolarized channel activation (10.13 +/- 2.28 mV). Neither a phosphosilent (P627A) nor a phosphomimetic (P627E) mutation had a significant effect, suggesting that either phosphorylation or another specific serine property is required. Since deletion of large regions had little effect on channel gating while a point mutation had a conspicuous impact, the I-II linker role may be to facilitate interactions with other proteins. Variants may have a larger impact if they create or disrupt these interactions, which may be key in evaluating pathogenicity of variants.

The Transient Receptor Vanilloid 1 (TRPV1) is a non-selective ion channel, which is activated by several chemical ligands and heat. We have previously shown that activation of TRPV1 by different ligands result in single-channel openings with different conductance, suggesting that the selectivity filter is highly dynamic. TRPV1 is weakly voltage-dependent, here we sought to explore whether the permeation of different monovalent ions could influence the voltage-dependence of this ion channel. By using single-channel recordings, we show that TRPV1 channels undergo rapid transitions to closed states that are directly connected to the open state, which may result from structural fluctuations of their selectivity filters. Moreover, we demonstrate that the rates of these transitions are strongly influenced by the permeant ion, suggesting that ion permeation regulates the voltage dependence of these channels.

Opening of the cardiac voltage-gated Na+ channel (Nav1.5) is responsible for robust depolarization of the cardiac action potential, while inactivation, which rapidly follows, allows for repolarization. Regulation of both the voltage- and time-dependent kinetics of Nav1.5 inactivation can alter the ability of the heart to initiate and sustain a re-entrant arrhythmia. The C-terminal domain (CTD) of Nav1.5 has been shown to modulate fast inactivation of the channel, and multiple auxiliary proteins bind to the CTD, including calmodulin (CaM) and intracellular fibroblast growth factor 12A (FGF12A). Recently, a non-canonical CaM-binding site was also discovered on the N-terminal of A-splice variants of iFGFs. We performed cut-open Vaseline gap (COVG) voltage-clamp to test whether FGF12A with and without CaM regulates Nav1.5 gating. In WT Nav1.5 channels, FGF12A with and without CaM present had a minimal effect on the voltage dependence of both activation and inactivation. Conversely, when CaM is absent on the Nav1.5 CTD (IQ/AA), a dramatic shift in steady-state inactivation (SSI) occurred, regardless of whether CaM was present on FGF12A. These two distinct mechanisms are operative in Nav1.5 LQT3 mutations where FGF12A requires CaM to shift in the voltage-dependence of inactivation, but not to inhibit the persistent late current. We conclude that there are two distinct mechanisms by which FGF12A modulates the Nav1.5 channel: CaM-regulated alteration of the voltage dependence of inactivation and CaM-independent inhibition of persistent late current.

Voltage-gated Na+ (Nav) channels, including Nav1.5, are responsible for the initiation of cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD) including calmodulin (CaM) and intracellular fibroblast growth factors (iFGF). Previous results demonstrate that Ca2+-bound CaM preferentially binds to iFGF12A. The role of intracellular Ca2+ ([Ca2+]i) in regulating Nav1.5 gating, either directly or via auxiliary proteins like CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and iFGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed Fluorescence Resonance Energy Transfer (FRET) imaging in live cells to observe the interaction between the Nav1.5 alpha subunit, CaM and iFGF12A. At resting [Ca2+]i, a 2-fold difference between acceptor and donor FRET efficiency was observed, implying that a single CaM acceptor is present on the Nav1.5 CTD even in the presence of FGF12A. After increasing [Ca2+]i, the donor and acceptor FRET efficiencies equalize, suggesting a 2:1:1 ratio between CaM, FGF12A, and the Nav1.5 CTD. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A in the presence/absence of calcium. With low [Ca2+]i, the steady-state inactivation of Nav1.5 with FGF12A was significantly shifted toward hyperpolarized potential compared to resting [Ca2+]i. Thus, the FGF12A:CaM complex confers a Ca2+-dependent mechanism enabling FGF12A modulates the Nav1.5 steady-state inactivation. Additionally, the ability of multiple subunits to bring CaM to the Nav1.5 CTD implies biological redundancy to prevent major alteration to Nav1.5 inactivation in the absence of CaM.

Polyphenolic compounds are widely explored for health benefits, including hypertension, but their active ingredients, molecular targets, and mechanisms remain poorly defined. We identify the xanthone Mangostin from Garcinia mangostana as a potent modulator of several potassium channels, with large-conductance K+ (BK) channels as its primary target for vasorelaxation. Mangostin activated BK channels as subunits alone, in complexes with vascular {beta}1 subunits, and in reconstituted BK/{beta}1-Cav nanodomains. It shifted BK voltage activation to more negative potentials by antagonizing channel closure and promoting channel opening without markedly altering Ca{superscript 2} sensitivity. Docking, competition, single channel analysis and mutagenesis localized the binding site in the pore cavity below the SF, involving gating-critical S6 residues I308, L312, and A316, and suggest that Mangostin stays bound in closed and open states. These findings establish BK channel activation as the core molecular mechanism driving Mangostins vascular effects and define its structural mode of action, informing nutraceutical safety assessment and BK-targeted drug design.

The Ca2+ activated Cl- channel formed by transmembrane member 16A (TMEM16A) is broadly expressed and regulates diverse processes. In addition to Ca2+, TMEM16A channels require the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) to open. Like other channels regulated by PI(4,5)P2, TMEM16A-conducted currents recorded in excised patches slowly decay overtime. Here we assessed how intracellular Ca2+ alters the rate of this current rundown, using the channels endogenously expressed in oocytes from the African clawed frog, Xenopus laevis. We found that in excised, inside-out patches, the concentration of applied Ca2+ alters the rate of rundown, with high Ca2+ concentrations speeding rundown by activating membrane associated phospholipase C (PLC). Together, these results clarify our understanding of how Ca2+ regulates both TMEM16A directly, and targets PLC to regulate the membrane PI(4,5)P2 content.

Myosin binding protein-C (MyBP-C) consists of a family of regulatory proteins expressed in sarcomeres of cardiac, fast and slow twitch skeletal muscles. The 3 MyBP-C paralogs expressed in each muscle type are encoded by separate genes but maintain a similar structure. Given the overall similarity in structure and localization of each of paralog, it is assumed that MyBP-C expressed in different muscles have similar functional effects. Here we directly tested this assumption by making use of our cut and paste approach to remove and replace N-terminal regions of MyBP-C in sarcomeres of different muscle types. We found that the different MyBP-C paralogs similarly slowed cross-bridge cycling kinetics, increased Ca2+ sensitivity of tension, and damped force oscillations. However, responses to a rapid stretch in actively contracting fibers, taken as indices of cross-bridge detachment and attachment kinetics, differed in each muscle type and responses depended on the presence or absence of a given paralog of MyBP-C. Altered responses to stretch were most evident for fast MyBP-C where loss of MyBP-C in psoas muscle resulted in transient responses to stretch that resembled those found in cardiomyocytes. Replacement of cardiac MyBP-C with fast MyBP-C in cardiomyocytes led to responses similar to psoas muscle. In separate X-ray diffraction experiments we also found that loss of MyBP-C in Ca2+-activated psoas muscle increased lattice disorder, reduced the ordering of myosin heads, and decreased thin filament length. Taken together, these results indicate that the different MyBP-C paralogs exert both common and unique effects on myosin cross-bridge kinetics. Significance StatementMyBP-C is a family of regulatory proteins found in muscle sarcomeres, where they regulate contraction and relaxation. Mutations in all MyBP-C paralogs cause disease in skeletal and cardiac muscles. We used a powerful "cut and paste" strategy to selectively remove MyBP-C from slow-twitch, fast-twitch, and cardiac muscle to show that each MyBP-C effects cross-bridge behavior similarly, though to varying degrees. Each MyBP-C had a notable effect on transient responses to rapid stretch, where MyBP-C was found to limit strain-induced cross-bridge detachment, especially in fast-twitch muscles. Strain-induced cross-bridge detachment is critical for rapid filling of the left ventricle in diastole and for sustained contraction in skeletal muscle. MyBP-C paralogs appear adapted to meet the mechanical demands of each muscle type.

Voltage clamp fluorometry was used to probe the S4 helix movement in the voltage sensing domain of the sea urchin HCN channel expressed in Xenopus oocytes. Markedly different fluorescence responses were obtained with either ALEXA-488 or MTS-TAMRA covalently linked to Cys332 at the N-terminal end of S4. With hyperpolarizing steps, ALEXA-488 fluorescence increased rapidly showing characteristics consistent with it reporting the initial inward movement of S4 in agreement with previous studies. In contrast, MTS-TAMRA fluorescence was slower and correlated with the early phase of channel opening. In addition, a slow fluorescence component was resolved with both labels that tracked the development of the mode shift or channel hysteresis. This was quantitated as an increased deactivation tail current delay with concomitantly longer activation periods and was found to depend strongly on the presence of K+ ions in the pore. This indicated that the microenvironment of the fluorescent probes attached to Cys332 was strongly influenced by conformational changes in the pore domain. Collisional quenching experiments established that ALEXA-488 was more exposed to solvent than MTS-TAMRA. This was supported by structural predictions based on homology modelling of spHCN in the closed and open conformations with covalently linked fluorophores. This study demonstrates that components of S4 movement during channel activation can be kinetically resolved using different fluorescent probes to reveal three distinct biophysical properties: voltage-sensor movement, early channel opening and mode-shift. These data support the use of different labelling probes to interrogate distinct biophysical aspects of voltage-gated membrane proteins. SummaryVoltage clamp fluorometry was used to probe the S4 helix movement in the voltage sensing domain of the spHCN channel expressed in Xenopus oocytes, labeled with either ALEXA-488 or MTS-TAMRA. Each fluorophore reported different components of S4 movement.

Two-pore voltage-gated CLC chloride channels control neuronal and muscle excitability. They share a dimeric structure but their activation mechanism remains unresolved. Here we determine the step-by-step activation mechanism of the broadly expressed CLC-2 channel using homology modelling, molecular dynamic simulations and functional studies. We establish that a two-leaf gate formed by Tyr561-H2O-Glu213 flanked by Lys568/Glu174 and Lys212 closes the canonical pore. Activation begins when a hyperpolarization-propelled intracellular chloride occupies the pore and splits Tyr561-H2O-Glu213 by electrostatic/steric repulsion. Unrestrained Glu213 rotates outwardly to bind Lys212 but the pore remains closed. Protonation breaks the Glu213-Lys212 interaction while another chloride occupies the pore thus catalysing chloride exit via Lys212. Also, we found that the canonical pore is uncoupled from a cytosolic cavity by a Tyr561-containing hydrophobic gate that prevents Glu213 protonation by intracellular protons. Our data provide atomistic details about CLC-2 activation but this mechanism might be common to other CLC channels.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Voltage-gated sodium (Nav) channels mediate rapid millisecond electrical signaling in excitable cells. Auxiliary subunits, {beta}1-{beta}4, are thought to regulate Nav channel function through covalent and/or polar interactions with the channel s voltage-sensing domains. How these interactions translate into the diverse and variable regulatory effects of {beta}-subunits remains unclear. Here, we find that the intrinsic movement order of the voltage-sensing domains during channel gating is unexpectedly variable across Nav channel isoforms. This movement order dictates the channel s propensity for closed-state inactivation, which in turn modulates the actions of {beta}1 and {beta}3. We show that the differential regulation of skeletal muscle, cardiac, and neuronal Nav channels is explained by their variable levels of closed-state inactivation. Together, this study provides a unified mechanism for the regulation of all Nav channel isoforms by {beta}1 and {beta}3, which explains how the fixed structural interactions of auxiliary subunits can paradoxically exert variable effects on channel function.

The human voltage-gated sodium channel hNaV1.5 is essential for cardiac excitability. Though underrecognized, NaV1.5 is also expressed in multiple cancers, promoting cell migration and malignancy. hNaV1.5 is a therapeutic target but limited isoform specificity presents a risk of side effects via neuronal and skeletal muscle NaV channels. Here we identify Mg2+-protoporphyrin IX (MgPpIX), a Mg-containing tetrapyrrole and intermediate in chlorophyll biosynthesis, as inhibitor of hNaV1.5 (IC50 of 1 nM). The activity profile of various metal protoporphyrins correlates with the electrostatic potential at the metal center of the compounds. MgPpIX is specific to hNaV1.5, as no inhibition of other hNaV isoforms (hNaV1.2, 1.4, 1.7, 1.8) was detected. A mutagenesis study and structural modeling reveals that MgPpIX stabilizes the domain-II voltage sensor in the deactivated conformation, with residues E795 and N803 being relevant determinants. MgPpIX also inhibits native NaV channels in breast cancer MDA-MB-231 and colorectal carcinoma SW480 cell lines, and suppresses cell migration. MgPpIX is an exceptionally potent and specific inhibitor of hNaV1.5 and may serve as a lead compound in anti-cancer drug development.

In cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) ensures repolarization of action potential during beta-adrenergic activation or when other repolarizing K+ currents fail. As a key factor of cardiac repolarization IKs should be present in model species used for cardiovascular drug screening, preferably with pharmacological characteristics similar to those of the human IKs. To this end, we investigated the effects of inhibitors and activators of the IKs on KCNQ1 and KCNQ1+KCNE1 channels of the zebrafish, an important model species, in Chinese hamster ovary cells. Inhibitors of IKs, chromanol 293B and HMR-1556, inhibited zebrafish IKs channels with approximately similar potency as that of mammalian IKs. Chromanol 293B concentration for half-maximal inhibition (IC50) of zebrafish IKs was at 13.1{+/-}5.8 and 13.4{+/-}2.8 M for KCNQ1 and KCNQ1+KCNE1 channels, respectively. HMR-1556 was a more potent inhibitor of zebrafish IKs with IC50=0.1{+/-}0.1 M and 1.5{+/-}0.8 M for KCNQ1 and KCNQ1+KCNE1 channels, respectively. R-L3 and mefenamic acid, generally identified as IKs activators, both inhibited zebrafish IKs. R-L3 almost completely inhibited zebrafish IKs generated by KCNQ1 and KCNQ1+KCNE1 channels with similar affinity (IC50 1.1{+/-}0.4 and 1.0{+/-}0.4 M, respectively). Mefenamic acid partially blocked zebrafish KCNQ1 (IC50=9.5{+/-}4.8 M) and completely blocked KCNQ1+KCNE1 channels (IC50=3.3{+/-}1.8 M). Although zebrafish IKs responds to IKs inhibitors in the same way as mammalian IKs, its response to activators is atypical, probably due to the differences in the binding domain of KCNE1 to KCNQ1. Therefore, care must be taken when translating the results from zebrafish to humans.

Voltage-gated calcium channels communicate electrical signals in membranes of excitable cells into cellular responses like secretion of hormones and neurotransmitters, or the contraction of heart and skeletal muscle cells. Their activation properties are tuned to match their specific functions. Consequently, the different members of the calcium channel family activate over a wide range of voltages and with greatly differing speeds. The skeletal muscle CaV1.1 and the cardiac/neuronal CaV1.2 represent two structurally closely related channels with particularly slow and fast activation kinetics, respectively. Both channel paralogs associate with the auxiliary calcium channel subunit 2{delta}-1, which is a known regulator of activation properties. By expressing CaV1.1 and CaV1.2 with and without 2{delta}-1 in a new double-knockout muscle cell line, we demonstrate that 2{delta}-1 regulates activation kinetics of the two channels in opposite directions. Molecular dynamics simulation revealed a string of charged amino acids connecting 2{delta}-1 to the intrinsic speed-control mechanism of voltage-sensing domain I (VSD I) in CaV1.1. Charge-neutralizing mutations of any of these charged amino acids abolished the 2{delta}-1 modulation and accelerated current kinetics. Together, these results reveal the molecular mechanism by which the 2{delta}-1 subunit regulates the intrinsic speed-control mechanism in the VSD I of CaV1.1 calcium channels.

Two-pore domain potassium (K2P) channels play a central role in modulating cellular excitability and neuronal function. The unique structure of the selectivity filter in K2P and other potassium channels determines their ability to allow the selective passage of potassium ions across cell membranes. The nematode C. elegans has one of the largest K2P families, with 47 subunit-coding genes. This remarkable expansion has been accompanied by the evolution of atypical selectivity filter sequences that diverge from the canonical TxGYG motif. Whether and how this sequence variation may impact the function of K2P channels has not been investigated so far. Here we show that the UNC-58 K2P channel is constitutively permeable to sodium ions and that a cysteine residue in its selectivity filter is responsible for this atypical behavior. Indeed, by performing in vivo electrophysiological recordings and Ca2+ imaging experiments, we demonstrate that UNC-58 has a depolarizing effect in muscles and sensory neurons. Consistently, unc-58 gain-of-function mutants are hypercontracted, unlike the relaxed phenotype observed in hyperactive mutants of many neuromuscular K2P channels. Finally, by combining molecular dynamics simulations with functional studies in Xenopus laevis oocytes, we show that the atypical cysteine residue plays a key role in the unconventional sodium permeability of UNC-58. As predicting the consequences of selectivity filter sequence variations in silico remains a major challenge, our study illustrates how functional experiments are essential to determine the contribution of such unusual potassium channels to the electrical profile of excitable cells. SIGNIFICANCEPotassium channels play a central role in modulating cellular excitability, particularly of neuronal cells. Their unique structure determines their ability to let ions pass selectively through cell membranes. The impact of pathological or evolutionary variations in this selectivity filter remains difficult to predict. Here, we reveal that UNC-58, a member of the two-pore domain potassium channel family of C. elegans, exhibits an unusual sodium permeability due to a unique cysteine residue in its selectivity filter. Our findings underscore the importance of functional studies to determine how sequence variation in potassium channel selectivity filters can shape the electrical profiles of excitable cells.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-coordinated excitation-contraction coupling (ECC) and, thus, homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarisation is active (local generation of action potentials; AP) or passive (following depolarisation of the outer cell surface sarcolemma; SS) has not been experimentally assessed in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesise that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were electrically insulated from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTSO_LICardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, ensuring synchronous and uniform cell contraction. C_LIO_LIIn this study, we investigate whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. C_LIO_LIFor this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of sub-cellular membrane domains. C_LIO_LIOur findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. C_LIO_LIThis active electrical capability may serve as a protective mechanism against voltage drops occurring within the T-tubular network. C_LI

Mavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards ordered off states close to the thick filament backbone. It remains unresolved whether mavacamten permanently sequesters these myosin heads in the off state(s) or whether these heads can be recruited in response to physiological stimuli when required to boost cardiac output. We show that cardiac myosins stabilized in these off state(s) by mavacamten are recruitable by Ca2+, increased heart rate, stretch, and {beta}-adrenergic ({beta}-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that, in presence of mavacamten, Ca2+ increases myosin ATPase activity by shifting myosin heads from the reserve super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+ and passive lengthening can shift ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments in the presence of mavacamten. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in telemetered rats, where left-ventricular dP/dtmax, an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that {beta}-AR stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Our data demonstrate that the mavacamten-promoted off states of myosin in the thick filament are activable, at least partially, thus leading to preservation of cardiac reserve mechanisms. Significance statementMavacamten is the first myosin-targeted small molecule inhibitor approved by the FDA to treat obstructive hypertrophic cardiomyopathy by attenuating myocardial hyperdynamic contraction. The recruitment of cardiac contractility is, however, vital to ensure sufficient cardiac output during increased physiological demand. Here we show that major inotropic effectors are at least partially preserved in the setting of mavacamten, resulting in maintenance of cardiac reserve mechanisms. These results not only suggest an alternative mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinically observed increase in peak oxygen uptake with exercise in HCM patients receiving mavacamten, but also lay the groundwork for a potential methodology to investigate the sarcomeric basis of chronotropic incompetence in disease states to motivate new therapeutic interventions.