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Voltage gated sodium channels (Nav) are transmembrane proteins responsible for action potential initiation. Mutations mainly located in the voltage sensor domain (VSD) of Nav1.5, the cardiac sodium channel, have been associated with the development of arrhythmias combined with dilated cardiomyopathy. Gating pore currents have been observed with three unrelated mutations associated with similar clinical phenotypes. However, gating pores have never been associated with mutations outside the first domain of Nav1.5. The aim of this study was to explore the possibility that gating pore currents might be caused by the Nav1.5 R225P and R814W mutations (R3, S4 in DI and DII, respectively), which are associated with rhythm disturbances and dilated cardiomyopathy. Nav1.5 WT and mutant channels were transiently expressed in tsA201 cells. The biophysical properties of the alpha pore currents and the presence of gating pore currents were investigated using the patch-clamp technique. We confirmed the previously reported gain of function of the alpha pores of the mutant channels, which mainly consisted of increased window currents mostly caused by shifts in the voltage dependence of activation. We also observed gating pore currents associated with the R225P and R814W mutations. This novel permeation pathway was open under depolarized conditions and remained temporarily open at hyperpolarized potentials after depolarization periods. Gating pore currents could represent a molecular basis for the development of uncommon electrical abnormalities and changes in cardiac morphology. We propose that this biophysical defect be routinely evaluated in the case of Nav1.5 mutations on the VSD. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels are known to play a pivotal role in perception and transmission of pain sensations. Gain-of-function mutations in the genes encoding the peripheral neuronal sodium channels, hNav1.7-1.9, cause human painful diseases. Thus while treatment of chronic pain remains an unmet clinical need, sodium channel blockers are considered as promising druggable targets. In a previous study, we evaluated the analgesic activity of sumatriptan, an agonist of serotonin 5HT1B/D receptors, and some new chiral bioisosteres, using the hot plate test in the mouse. Interestingly, we observed that the analgesic effectiveness was not necessarily correlated to serotonin agonism. In this study, we evaluated whether sumatriptan and its congeners may inhibit heterologously expressed hNav1.7 sodium channels using the patch-clamp method. We show that sumatriptan blocks hNav1.7 channels only at very high, supratherapeutic concentrations. In contrast, its three analogs, namely 20b, (R)-31b, and (S)-22b, exert a dose and use-dependent sodium channel block. At 0.1 and 10 Hz stimulation frequencies, the most potent compound, (S)-22b, was 4.4 and 1.7 fold more potent than the well-known sodium channel blocker mexiletine. The compound induces a negative shift of voltage dependence of fast inactivation, suggesting higher affinity to the inactivated channel. Accordingly, we show that (S)- 22b likely binds the conserved local anesthetic receptor within voltage-gated sodium channels. Combining these results with the previous ones, we hypothesize that usedependent sodium channel blockade contributes to the analgesic activity of (R)-31b and (S)-22b. These later compounds represent promising lead compounds for the development of efficient analgesics, the mechanism of action of which may include a dual action on sodium channels and 5HT1D receptors. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium channels are intrinsic plasma membrane proteins that initiate the action potential in electrically excitable cells. They are composed of a pore-forming α-subunit and associated β-subunits. The β1-subunit was the first accessory subunit to be cloned. It can be important for controlling cell excitability and modulating multiple aspects of sodium channel physiology. Mutations of β1 are implicated in a wide variety of inherited pathologies, including epilepsy and cardiac conduction diseases. This review summarizes β1-subunit related channelopathies pointing out the current knowledge concerning their genetic background and their underlying molecular mechanisms. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE APETx2, a toxin from the sea anemone Anthropleura elegantissima, inhibits acid-sensing ion channel 3 (ASIC3)-containing homo- and heterotrimeric channels with IC50 values < 100 nM and 0.1-2 µM respectively. ASIC3 channels mediate acute acid-induced and inflammatory pain response and APETx2 has been used as a selective pharmacological tool in animal studies. Toxins from sea anemones also modulate voltage-gated Na+ channel (Nav) function. Here we tested the effects of APETx2 on Nav function in sensory neurones. EXPERIMENTAL APPROACH Effects of APETx2 on Nav function were studied in rat dorsal root ganglion (DRG) neurones by whole-cell patch clamp. KEY RESULTS APETx2 inhibited the tetrodotoxin (TTX)-resistant Nav 1.8 currents of DRG neurones (IC50, 2.6 µM). TTX-sensitive currents were less inhibited. The inhibition of Nav 1.8 currents was due to a rightward shift in the voltage dependence of activation and a reduction of the maximal macroscopic conductance. The inhibition of Nav 1.8 currents by APETx2 was confirmed with cloned channels expressed in Xenopus oocytes. In current-clamp experiments in DRG neurones, the number of action potentials induced by injection of a current ramp was reduced by APETx2. CONCLUSIONS AND IMPLICATIONS APETx2 inhibited Nav 1.8 channels, in addition to ASIC3 channels, at concentrations used in in vivo studies. The limited specificity of this toxin should be taken into account when using APETx2 as a pharmacological tool. Its dual action will be an advantage for the use of APETx2 or its derivatives as analgesic drugs. [ABSTRACT FROM AUTHOR]

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of β- and γ-epithelial Na[sup +] channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in β- and γ-ENaC subunits (βY618A, β-P616L, β-R564stop, and γ-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCD[sub c14]), and transepithelial Na[sup +] transport was assessed by measurements of the benzamil-sensitive short-circuit current (I[sub sc]). Cells that express ENaC mutants of the PY motif showed a five- to six-fold higher basal I[sub sc] compared with control cells and responded to stimulation by aldosterone (10[sup -6] M) or vasopressin (10[sup -9] M) with a further increase in I[sub sc]. The rates of the initial increases in I[sub sc] after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na[sup +] absorption in the distal nephron of patients with Liddle's syndrome. [ABSTRACT FROM AUTHOR]

Deals with a study which identified a conserved Ser residue at the N-terminal end of the second transmembrane domain of the epithelial sodium channel (ENaC) alpha subunit which allows potassium ions to permeate the channel. Methods of the study; Analysis of the sing-channel conductance and conductance ratios; Results and discussion.

Acid-sensing ion channels (ASICs) are neuronal Na+-conducting channels activated by extracellular acidification. ASICs are involved in pain sensation, expression of fear, and neurodegeneration after ischemic stroke. Functional ASICs are composed of three identical or homologous subunits, whose extracellular part has a handlike structure. Currently, it is unclear how protonation of residues in extracellular domains controls ASIC activity. Knowledge of these mechanisms would allow a rational development of drugs acting on ASICs. Protonation may induce conformational changes that control the position of the channel gate. We used voltage-clamp fluorometry with fluorophores attached to residues in different domains of ASIC1a to detect conformational changes. Comparison of the timing of fluorescence and current signals identified residues involved in movements that preceded desensitization and may therefore be associated with channel opening or early steps leading to desensitization. Other residues participated in movements intimately linked to desensitization and recovery from desensitization. Fluorescence signals of all mutants were detected at more alkaline pH than ionic currents. Their midpoint of pH dependence was close to that of steady-state desensitization, whereas the steepness of the pH fluorescence relationship was closer to that of current activation. A sequence of movements was observed upon acidification, and its backward movements during recovery from desensitization occurred in the reverse order, indicating that the individual steps are interdependent. Furthermore, the fluorescence signal of some labeled residues in the finger domain was strongly quenched by a Trp residue in the neighboring β-ball domain. Upon channel activation, their fluorescence intensity increased, indicating that the finger moved away from the β ball. This extensive analysis of activity-dependent conformational changes in ASICs sheds new light on the mechanisms by which protonation controls ASIC activity. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) are neuronal Na + -permeable ion channels activated by extracellular acidification. ASICs are involved in learning, fear sensing, pain sensation and neurodegeneration. Increasing the extracellular Ca 2+ concentration decreases the H + sensitivity of ASIC1a, suggesting a competition for binding sites between H + and Ca 2+ ions. Here, we predicted candidate residues for Ca 2+ binding on ASIC1a, based on available structural information and our molecular dynamics simulations. With functional measurements, we identified several residues in cavities previously associated with pH-dependent gating, whose mutation reduced the modulation by extracellular Ca 2+ of the ASIC1a pH dependence of activation and desensitization. This occurred likely owing to a disruption of Ca 2+ binding. Our results link one of the two predicted Ca 2+ -binding sites in each ASIC1a acidic pocket to the modulation of channel activation. Mg 2+ regulates ASICs in a similar way as does Ca 2+ . We show that Mg 2+ shares some of the binding sites with Ca 2+ . Finally, we provide evidence that some of the ASIC1a Ca 2+ -binding sites are functionally conserved in the splice variant ASIC1b. Our identification of divalent cation-binding sites in ASIC1a shows how Ca 2+ affects ASIC1a gating, elucidating a regulatory mechanism present in many ion channels.

Acid-sensing ion channels (ASICs) are activated by extracellular acidification. Because ASIC currents are transient, these channels appear to be ideal sensors for detecting the onset of rapid pH changes. ASICs are involved in neuronal death after ischemic stroke, and in the sensation of inflammatory pain. Ischemia and inflammation are associated with a slowly developing, long-lasting acidification. Recent studies indicate however that ASICs are unable to induce an electrical signaling activity under standard experimental conditions if pH changes are slow. In situations associated with slow and sustained pH drops such as high neuronal signaling activity and ischemia, the extracellular K + concentration increases, and the Ca 2+ concentration decreases. We hypothesized that the concomitant changes in H + , K + , and Ca 2+ concentrations may allow a long-lasting ASIC-dependent induction of action potential (AP) signaling. We show that for acidification from pH7.4 to pH7.0 or 6.8 on cultured cortical neurons, the number of action potentials and the firing time increased strongly if the acidification was accompanied by a change to higher K + and lower Ca 2+ concentrations. Under these conditions, APs were also induced in neurons from ASIC1a -/- mice, in which a pH of ≤ 5.0 would be required to activate ASICs, indicating that ASIC activation was not required for the AP induction. Comparison between neurons of different ASIC genotypes indicated that the ASICs modulate the AP induction under such changed ionic conditions. Voltage-clamp measurements of the Na + and K + currents in cultured cortical neurons showed that the lowering of the pH inhibited Na + and K + currents. In contrast, the lowering of the Ca 2+ together with the increase in the K + concentration led to a hyperpolarizing shift of the activation voltage dependence of voltage-gated Na + channels. We conclude that the ionic changes observed during high neuronal activity mediate a sustained AP induction caused by the potentiation of Na + currents, a membrane depolarization due to the changed K + reversal potential, the activation of ASICs, and possibly effects on other ion channels. Our study describes therefore conditions under which slow pH changes induce neuronal signaling by a mechanism involving ASICs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, LR, declared a shared committee with one of the authors, SK, at the time of review., (Copyright © 2021 Alijevic, Peng and Kellenberger.)

Members of the ENaC/degenerin family of ion channels include the epithelial sodium channel (ENaC), acid-sensing ion channels (ASICs) and the nematode Caenorhabditis elegans degenerins. These channels are activated by a variety of stimuli such as ligands (ASICs) and mechanical forces (degenerins), or otherwise are constitutively active (ENaC). Despite their functional heterogeneity, these channels might share common basic mechanisms for gating. Mutations of a conserved residue in the extracellular loop, namely the 'degenerin site' activate all members of the ENaC/degenerin family. Chemical modification of a cysteine introduced in the degenerin site of rat ENaC (betaS518C) by the sulfhydryl reagents MTSET or MTSEA, results in a approximately 3-fold increase in the open probability. This effect is due to an 8-fold shortening of channel closed times and an increase in the number of long openings. In contrast to the intracellular gating domain in the N-terminus which is critical for channel opening, the intact extracellular degenerin site is necessary for normal channel closing, as illustrated by our observation that modification of betaS518C destabilises the channel closed state. The modification by the sulfhydryl reagents is state- and size-dependent consistent with a conformational change of the degenerin site during channel opening and closing. We propose that the intracellular and extracellular modulatory sites act on a common channel gate and control the activity of ENaC at the cell surface.