When muscle Ca²⁺ channels carry monovalent cations through gating pores: insights into the pathophysiology of type 1 hypokalaemic periodic paralysis

Abstract

Patients suffering from type 1 hypokalaemic periodic paralysis (HypoPP1) experience attacks of muscle paralysis associated with hypokalaemia. The disease arises from missense mutations in the gene encoding the α1 subunit of the dihydropyridine receptor (DHPR), a protein complex anchored in the tubular membrane of skeletal muscle fibres which controls the release of Ca²⁺ from sarcoplasmic reticulum and also functions as a Ca²⁺ channel. The vast majority of mutations consist of the replacement of one of the outer arginines in S4 segments of the α1 subunit by neutral residues. Early studies have shown that muscle fibres from HypoPP1 patients are abnormally depolarized at rest in low K⁺ to the point of inducing muscle inexcitability. The relationship between HypoPP1 mutations and depolarization has long remained unknown. More recent investigations conducted in the closely structurally related voltage‐gated Na⁺ and K⁺ channels have shown that comparable S4 arginine substitutions gave rise to elevated inward currents at negative potentials called gating pore currents. Experiments performed in muscle fibres from different models revealed such an inward resting current through HypoPP1 mutated Ca²⁺ channels. In mouse fibres transfected with HypoPP1 mutated channels, the elevated resting current was found to carry H⁺ for the R1239H arginine‐to‐histidine mutation in a S4 segment and Na⁺ for the V876E HypoPP1 mutation, which has the peculiarity of not being located in S4 segments. Muscle paralysis probably results from the presence of a gating pore current associated with hypokalaemia for both mutations, possibly aggravated by external acidosis for the R1239H mutation.

Introduction

Contraction of the skeletal muscle fibre is elicited by the firing and propagation of action potentials along the plasma membrane. The main ion channels involved in muscle excitability are the voltage‐gated Na⁺ and K⁺ channels and the Cl⁻ channel. Mutations in the genes encoding the pore‐forming subunits of Na⁺ or Cl⁻ channels lead to muscle hyper‐excitability and stiffness or in contrast to muscle hypo‐excitability and weakness or even paralysis (Jurkat‐Rott & Lehmann‐Horn, 2005; Cannon, 2015). Mutations in the gene encoding the pore‐forming subunit of voltage‐gated Ca²⁺ channels, also present in skeletal muscle fibres, lead to another ion channelopathy called type 1 hypokalaemic periodic paralysis (HypoPP1). This muscle disorder is characterized by transient muscle inexcitability leading to muscle weakness or paralysis episodes. The causal relationship between the genetic mutations in the Ca²⁺ channel, which are for the vast majority located in the voltage‐sensing domain, and the loss of muscle excitability has remained elusive for more than 10 years. This is essentially because the non‐mutated Ca²⁺ channel was reputed to be not directly involved in muscle excitability. Nonetheless, structure‐function experiments in the closely structurally related voltage‐gated K⁺ channels yielded the unexpected finding that comparable mutations in the voltage‐sensing domain of the channel created an aberrant monovalent cation pathway called gating pore (Starace & Bezanilla, 2004). A number of strong lines of experimental evidence suggest that such a gating pore is also present in the HypoPP1 mutated Ca²⁺ channel and could reliably explain the pathophysiology of the disease.

The Ca²⁺ channel in skeletal muscle

In skeletal muscle, excitation‐contraction coupling refers to the sequence of events that occurs from the development of action potentials on the sarcolemma to the increase in intracellular Ca²⁺ that activates contraction. In this process, the Ca²⁺ channel anchored in the transverse tubular membrane, also called dihydropyridine receptor (DHPR), plays the critical role of voltage sensor. Propagation of action potentials on the surface and tubular membrane of the skeletal muscle fibre induces a change in the configuration of the DHPR which leads to opening of the ryanodine receptor (RyR1) anchored in the sarcoplasmic reticulum (SR) membrane, a Ca²⁺ channel controlling the release of Ca²⁺ ions into the cytosol. The control exerted by the DHPR on RyR1 operates in a voltage‐dependent manner through a direct physical interaction between the two proteins (Rios & Pizarro, 1991; Schneider, 1994; Melzer et al. 1995). The DHPR also behaves as an L‐type high‐threshold voltage‐gated Ca²⁺ channel which activates in response to long‐lasting depolarizations of the muscle membrane. The role of this Ca²⁺ influx is still a matter of debate, some groups claiming that it is irrelevant for muscle excitation‐contraction coupling and others arguing that it contributes in part to loading SR Ca²⁺ and maintaining release during long‐lasting activation of muscle fibres (Lee et al. 2015; Robin & Allard, 2015; Dayal et al. 2017; Flucher & Tuluc, 2017).

Type 1 hypokalaemic periodic paralysis

The absence of DHPR is lethal (Beam et al. 1986; Tanabe et al. 1988) and severe neuromuscular diseases arise from genetic defects in DHPR. One of these diseases is HypoPP1. This muscle disease is characterized by transient attacks of muscle weakness, of varying duration and severity with a matching falls in blood potassium. Factors that provoke attacks are carbohydrate‐rich meals, cold or rest after high levels of exertion, the latter being the most important. Attacks usually do not occur during exercise and mild exercise even appears to have a preventive effect. Interictally persisting weakness may progressively develop in parallel with formation of intramuscular vacuoles (Buruma & Schipperheyn, 1979). Acetazolamide, a carbonic anhydrase inhibitor, is the drug most commonly used for preventing paralysis attacks in HypoPP, but its mechanism of action remains elusive (Resnick et al. 1968; Wu et al. 2013).

The first DHPR mutations causing hypokalaemic periodic paralysis were identified in 1994 (Jurkat‐Rott et al. 1994; Ptacek et al. 1994). The DHPR is composed of the main subunit α1, also called Cav1.1, which carries the dual function of voltage sensor and voltage‐gated Ca²⁺ channel, and 4 auxiliary subunits β1, α2, δ and γ (Catterall, 2011). The α1 subunit, encoded by the CACNA1S gene, is composed of 4 homologous domains (I–IV), each of which contains 6 membrane‐spanning segments (S1–S6). The S1–S4 segments of each domain constitute the voltage‐sensing domain within which S4 segments enriched with positively charged amino acids detect the changes in membrane potential and translocate in the outward direction in response to depolarization (Fig. 1). The vast majority of HypoPP1 mutations affect the α1 subunit and are localized in one S4 segment of the protein (Cannon, 2010; Matthews & Hanna, 2010; Jurkat‐Rott et al. 2012; Moreau et al. 2014). However, two mutations, one localized in the S3 segment of domain III (V876E) and one in the S4–S5 loop of domain III (H916Q), were found to give rise to the same clinical features, suggesting that different pathophysiological mechanisms might lead to HypoPP1 (Ke et al. 2009; Li et al. 2012). Concerning the mutations affecting the S4 segments, they all consist of the replacement of the first or second outermost basic residue arginine by a much less charged amino acid, histidine, serine or glycine in domain II (R528H/G), domain III (R897S, R900S) or domain IV (R1239H/G) (Fig. 1).

Figure 1. Resting inward gating pore currents in the muscle DHPR.

A, the α1 subunit of DHPR is composed of 4 membrane domains (I–IV) that each contain 6 transmembrane segments. The 4 membrane domains are organized to form a pore that conducts Ca²⁺ ions and opens in response to depolarization. Domain I has been omitted for the sake of clarity. In each domain, segment 4 is enriched with positively charged residues (+) that confer voltage dependence to the channel. B, replacement of the second basic residue by a histidine in the S4 segment of domain IV for the R1239H HypoPP1 mutation and of valine by a glutamate in the S3 segment of domain III for the V876E HypoPP1 mutation generate an inward gating pore current at negative voltages through a pathway located within the voltage‐sensing domain and distinct from the main Ca²⁺ conducting pore. The gating pore current was found to be carried by H⁺ and Na⁺ for the R1239H and the V876E HypoPP1 mutations, respectively (Fuster et al. 2017a, b).

As a result of the genetic characterization of the disease, pathophysiological studies have obviously focused on the possible changes in the properties of the voltage‐gated Ca²⁺ channel and/or voltage control of SR Ca²⁺ release induced by the HypoPP1 mutations. In the late nineties the two most common mutations, R528H and R1239H, corresponding to the replacement of the first or second outermost arginine by a histidine in the S4 segment of domain II or IV, respectively, were the main mutations investigated either after expression in Xenopus oocytes, in a muscle cell line, or in cultured myotubes from patients’ muscle (Sipos et al. 1995; Lapie et al. 1996, Morrill et al. 1998; Jurkat‐Rott et al. 1998; Morrill & Cannon, 1999). Depending on the model systems used, and also certainly because of the difficulty of expressing the skeletal muscle Ca²⁺ channel in heterologous expression systems, voltage clamp experiments led to different outcomes. The R1239H mutation was found to induce a reduction in Ca²⁺ current density in all model systems but no change or a reduction in conductance was shown for the R528H mutation, even when it was investigated in equivalent model systems. By neutralizing one positive charge in the voltage sensor of the channel, mutations were also expected to disrupt the voltage dependence of the Ca²⁺ current. For the R1239H channel, the voltage dependence of activation was found to be either normal in myotubes or shifted toward positive potentials in Xenopus oocytes (Sipos et al. 1995; Morrill & Cannon, 1999), while, for the R528H channel, it was reported to be shifted toward negative potentials when expressed in Xenopus oocytes (Morrill & Cannon, 1999), but was unchanged when recorded in cultured myotubes from patients’ muscle or expressed in a muscle cell line (Jurkat‐Rott et al. 1998; Morrill et al. 1998). Even if such alterations in the biophysical properties of the Ca²⁺ channel and possibly of SR Ca²⁺ release explain muscle weakness, they do not readily explain the episodic nature of weakness and paralysis which characterizes the disorder. Until recently, the V876E mutation, which has the peculiarity of not being located in a S4 segment, had not been investigated.

Gating pores in voltage‐gated channels

During attacks of paralysis, muscle weakness is of the flaccid motor type with depression or abolition of tendon reflexes, suggesting that muscle fibres experience inexcitability (Buruma & Schipperheyn, 1979). In two studies performed in muscle fibres from human biopsies, upon reduction of extracellular K⁺, HypoPP fibres were found to exhibit a pathological depolarizing inward current prone to depolarize muscle cells and inactivate voltage‐gated Na⁺ channels (Rüdel et al. 1984; Ruff, 1999). The relationship between HypoPP1 mutations in the Ca²⁺ channel and the hypokalaemia‐induced transient depolarization of muscle fibres remained a mystery until the discovery of the existence of a permanent depolarizing current at resting potentials created by comparable mutations in the closely structurally related voltage‐gated Shaker K⁺ channel (Starace & Bezanilla, 2004). In studying the accessibility of charged residues in S4 segments using site‐directed histidine mutagenesis, it was indeed found that replacing the first S4 arginine by histidine in the Shaker K⁺ channel created a proton pore when the cell is hyperpolarized. Further experiments demonstrated that substituting smaller uncharged residues for arginine residues in S4 segments made the voltage‐sensing domain permeable to protons and/or cations, leading to a gating pore current called ω current, not only in voltage‐gated K⁺ channels but also in voltage‐gated Na⁺ channels (Sokolov et al. 2005; Tombola et al. 2005; Struyk & Cannon, 2007). These findings indicate that the arginines in S4 segments, through interactions with residues belonging to adjacent segments, form a very narrow septum that separates intra‐ and extracellular water crevices impermeable to ions. The replacement of arginine by neutral residues, by disrupting this interaction, bridges the internal and external solutions, generating in this way a gating pore open either in the resting state if substitution affects outer arginines or in the activated state if substitution affects inward gating charges.

Gating pores in HypoPP1 mutated Ca²⁺ channels

Two studies have postulated that the gating pore model depicted for K⁺ and Na⁺ channels also applies to HypoPP1 mutations, neutralizing the first or second arginine in a S4 segment of the Ca²⁺ channel. First, experiments in fibres from human muscle biopsies revealed a significant elevated leak current density at hyperpolarized potentials in low K⁺ in patients carrying the HypoPP1 mutations R528H or R1239H (Jurkat‐Rott et al. 2009). Second, a transgenic mouse model for HypoPP1 with a targeted Cav1.1 R528H mutation was generated and was shown to exhibit La³⁺‐sensitive inward currents active at negative voltages of significantly larger amplitude in R528H compared to control fibres (Wu et al. 2012). In order to complement these data and circumvent possible adaptive compensatory mechanisms and/or ultrastructural abnormalities that may develop in these model systems, our group acutely expressed in adult mouse hindlimb muscles wild‐type (WT), HypoPP1 R1239H and V876E mutant‐GFP‐tagged Cav1.1 α1 subunits (Fuster et al. 2017a, b). Striated patterns of expression of WT, R1239H and V876E subunits gave evidence for their proper localization in the t‐tubule membrane. As described in order model systems for the R1239H or for the R528H mutation, voltage clamp experiments revealed a mild reduction of L‐type voltage‐gated Ca²⁺ conductance and a negative shift of the voltage dependence in R1239H compared to WT fibres (Fuster et al. 2017a). The properties of the voltage‐gated Ca²⁺ channel were found not to be changed in fibres transfected with the HypoPP1 V876E mutant‐GFP‐tagged Cav1.1 α1 subunits, demonstrating and confirming that the HypoPP1 pathogenesis is not related to any change in the Ca²⁺ channel function of Cav1.1 (Fuster et al. 2017b).

More importantly, in the absence of external Na⁺ and K⁺ ions and low Cl⁻, R1239H fibres exhibited a significant larger leak conductance measured between −120 and −80 mV using descending voltage ramps from a holding potential of 0 mV. The calculated difference in the mean leak conductance between WT and R1239H fibres indicated that R1239H fibres exhibited an extra leak conductance of 19.6 S F⁻¹. This value was in reasonable agreement with the 28 S F⁻¹ value found for the R528H homozygous mutation in the transgenic mouse and 12 S F⁻¹ in fibres from patients who are heterozygous for this mutation (Jurkat‐Rott et al. 2009; Wu et al. 2012). In patients carrying the R1239H mutation, the leak conductance amounted to 19.5 μS cm⁻², which is twice the conductance of 19.6 S F⁻¹ we obtained for transfected mouse fibres, considering that human patients are heterozygous for this mutation as for the R528H mutation (Jurkat‐Rott et al. 2009). This discrepancy could arise from the fact that in our experimental conditions transfected channels stand together with endogenous channels. Finally, our value for conductance also agrees quite well with the newly identified R1242G mutant Cav1.1 expressed in myotubes (27.5 S F⁻¹), which also gives rise to a gating pore current but which is activated by depolarization that leads to normokalaemic periodic paralysis (Fan et al. 2013).

In transfected mouse muscle fibres, the leak conductance was found to be larger in R1239H fibres for voltages more negative than −40 mV, suggesting that the extra leak conductance in these fibres displayed an inward rectification and abrogated voltages less negative than −40 mV (Fuster et al. 2017a). Interestingly, inward rectification is an intrinsic property of gating pores induced by the neutralization of outer arginines in S4 segments of voltage‐gated K⁺ and Na⁺ channels. This inward rectification was shown to be imposed by a voltage‐dependent mechanism moving the S4 segments in the outward direction, disrupting the hydrophilic pathway created by the mutation and closing in this way the gating pore at positive potentials (Starace & Bezanilla, 2004; Sokolov et al. 2005; Tombola et al. 2005; Struyk & Cannon, 2007). In voltage‐clamped skeletal muscle fibres, S4 movements give rise to intramembrane charge movements that can be measured as small voltage‐activated outward currents (Collet et al. 2003). Interestingly, the voltage dependence of intramembrane charge movements correlates well with the voltage dependence of the extra leak current in R1239H fibres, since the leak current starts reducing around −50 mV when charge movements start activating with a reported half‐activation of −37 mV in mouse skeletal muscle (Collet et al. 2003). Therefore, the apparent inward rectification of the extra leak conductance in R1239H fibres is the first evidence of the creation of a gating pore in the HypoPP1 R1239H mutated Ca²⁺ channel. A second line of evidence is the potentiation of the R1239H extra leak current by external acidification. We indeed observed that a decrease in external pH from 7.2 to 6 (which should shift the electrochemical gradient for protons by 70 mV toward positive values) led to an increase in the leak conductance in WT and R1239H fibres, which was significantly larger in R1239H fibres, suggesting that the extra leak current in R1239H carries, at least in part, protons (Fig. 1). The leak current in R1239H fibres thus shares another comparable property with gating pore currents in voltage‐gated K⁺ and Na⁺ channels that were shown to be selective for protons when outer arginines are replaced by histidine, as is the case for the R1239H mutation (Starace & Bezanilla, 2004; Struyk & Cannon, 2007). Proton permeation through the extra leak conductance in R1239H fibres was confirmed using intracellular pH measurements, with the pH indicator BCECF showing that the influx of protons induced by external acidification, reflected by the rate of intracellular acidification, was larger in R1239H fibres (Fuster et al. 2017a; Fig. 1).

The generation of a gating pore seems to constitute a pathogenic mechanism common to all HypoPP1 disorders even when the mutation does not affect a S4 arginine residue. Indeed, mouse fibres expressing the HypoPP1 V876E mutant Cav1.1 α1 subunits were also found to exhibit an extra leak H⁺ conductance in the absence of external Na⁺ and K⁺ ions that displayed an inward rectification for potentials lower than −50 to −40 mV (Fuster et al. 2017b). However, in the presence of a physiological external solution, the proton influx at negative potentials reflected by the rate of intracellular acidification was not augmented in V876E fibres, indicating that the extra leak current carries monovalent cations other than protons. In contrast, the measurement of the rate of increase of intracellular Na⁺ influx provoked by readmission of Na⁺ in a Na⁺‐free external solution at −80 mV using the Na⁺ indicator SBFI showed a significant elevated rate of Na⁺ influx in V876E fibres. The relationship between the resting leak current and voltage revealed by voltage ramps in the presence of an external Tyrode solution confirmed the presence of an extra leak current in these mutant fibres, demonstrating that the elevated resting inward current was carried by Na⁺ ions. Although the V876E mutation does not directly affect an arginine residue in a S4 segment, the fact that it produced an extra leak inward current at rest probably suggests that a gating pore able to pass Na⁺ ions had been created (Fig. 1). Interestingly, Campos et al. (2007) have also shown in the Shaker K⁺ channel that mutations consisting of the replacement of an isoleucine by a histidine in S2 or S1 segments generated a gating pore by disrupting the hydrophobic barrier lined in part by isoleucine and the most extracellular arginine of S4 segment. It is thus likely that the valine at position 876 and one of the most extracellular charged residues in S4 segment constitute a narrow hydrophobic plug separating the water‐accessible crevices on each side of the membrane. This plug may be disrupted when valine is replaced by glutamate, bridging in this way the internal and external solutions through a gating pore, in the same manner as the gating pore created by arginine substitution in S4 segments. This local hydrophilic shortcut could be induced by a change in the electrostatic interactions with the positively charged residues of the S4 segment caused by the presence of the negatively charged residue glutamate or alternatively by widening the gating pore entrance through structural displacement of the S3 segment.

Gating pore current and pathophysiology of HypoPP1

It is thus very likely that the resting leak current flowing through gating pores induced by HypoPP1 mutations in the Ca²⁺ channel contributes to the sustained muscle depolarization that precipitates attacks of paralysis. Nonetheless, there is still a question about why paralysis and muscle weakness are transient while the extra leak current induced by the mutation flows continuously at rest. Because the resting K⁺ conductance, mainly through the inward rectifier K⁺ channels, is the most effective at maintaining the resting membrane potential in skeletal muscle, the fall in the external K⁺ concentration associated with paralysis attacks unambiguously plays a pivotal role in precipitating paralysis. Indeed, the membrane potential always settles at a value at which inward currents are balanced by outward K⁺ currents. Because of the presence of a large resting K⁺ conductance through inward rectifier K⁺ channels, this value is close to the K⁺ equilibrium potential (E _K = (RT/nF)ln([K⁺]_out/[K⁺]_int). At first sight, a decrease in the external K⁺ concentration ([K⁺]_out) is expected to give rise to hyperpolarization, since E _K becomes more negative. But, in parallel, very low concentrations of K⁺ induce a decrease in outward currents through inward rectifier K⁺ channels so that the inward currents dominate over the outward K⁺ currents. This provokes a paradoxical depolarization to voltages as low as −60 mV until activation of the voltage‐dependent outward rectifier K⁺ channels allows a new equilibrium between inward and outward currents to be reached (Struyk & Cannon, 2008; Jurkat‐Rott et al. 2009) (Fig. 2). While paradoxical depolarization is provoked by extremely low and non‐physiological K⁺ concentrations in control muscle fibres, addition of an inward current flowing through a gating pore, such as the one revealed in HypoPP1, is presumed to induce paradoxical depolarization and inexcitability in low K⁺ conditions to which normal muscle fibres adapt well and remain polarized and excitable (Struyk & Cannon, 2008; Jurkat‐Rott et al. 2009, 2012; Cannon, 2015).

Figure 2. Transmembrane ion movements involved in paralysis attacks in HypoPP1 disease.

A, in normal external K⁺ and in the presence of a neutral external pH, the outward current through inward rectifier K⁺ (Kir) channels largely dominates over inward leak currents including the H⁺ gating pore current through DHPR for the R1239H or the much larger Na⁺ gating pore current induced by the V876E mutation. In these conditions, the resting potential is close to the K⁺ equilibrium potential and negative enough to allow full activation of voltage‐gated Na⁺ channels (Nav) upon muscle excitation. B, the fall in external K⁺, possibly caused by over‐activity of the Na⁺/K⁺ pump, induces a decrease in the Kir conductance aggravated by external acidosis that further inhibits Kir channels and potentiates the inward H⁺ gating pore current for the R1239H mutation. For both mutations, the resting potential is then shifted toward less negative values for which outward currents through voltage‐gated K⁺ (Kv) channels plus Kir channels balance the inward H⁺ or Na⁺ depolarizing currents. This depolarized resting potential promotes inactivation of Nav, inexcitability and muscle paralysis. These conditions are possibly met during the period of rest following exercise (see text for details).

Identification of the cell mechanisms that lead to a decrease in extracellular K⁺ in HypoPP1 is thus of critical importance. Hyperactivity of the muscle Na⁺/K⁺‐ATPase seems to be one of the predominant factors causing low serum K⁺. The function of the muscle Na⁺/K⁺‐ATPase is indeed to maintain at rest or to restore during and after muscle activity the Na⁺ and K⁺ gradients by pumping K⁺ into and Na⁺ out of muscle fibres with high efficiency (Clausen, 2003). Because skeletal muscles represent close to half of the total body mass, any change in the activity of the pump in skeletal muscles has tremendous effects on extracellular K⁺. Firing of action potentials during muscle excitation is the primary condition leading to an acute increase in the transport activity of the pump. This hyperactivity helps to maintain extracellular K⁺ in the physiological range by taking up K⁺ ions released in particular through voltage‐dependent outward rectifier K⁺ channels that contribute to the repolarization phase of action potentials. However, during the resting period following exercise, a sustained post‐exercise hypokalaemia has been reported to occur, suggesting that over‐activity of the pump is maintained long after the end of exercise when muscle K⁺ loss has ceased (Mebdo & Sejersted, 1990; Lindinger, 1995; Clausen, 2013). This post‐exercise hypokalaemia probably represents an important triggering factor for paralysis in HypoPP1 patients during the resting period following intense muscle exercise, a condition routinely reported to trigger episodes of paralysis in HypoPP (Fig. 2). In parallel, it is worth noting that other well‐known activators of muscle pump‐mediated K⁺ uptake, such as catecholamines and insulin, also promote hypokalaemia. Such hypokalaemia promoted by pump activation could explain why attacks can be also triggered by stress or rich carbohydrate meals, considering in the latter case that insulin has an additional inhibitory effect on Kir currents (Ruff, 1999).

Proton permeation through the gating pore generated by the R1239H mutation may also have pathophysiological relevance. Muscle cells indeed undergo marked changes in intra‐ but also extracellular pH during and after physical exertion, which should affect the magnitude of the leak inward proton current by changing the transmembrane electrochemical gradient for H⁺. During intense muscle exercise, protons are produced intracellularly and are also released in the blood stream. Acidification is, however, much larger in the cytosol than in the extracellular medium, providing conditions that should attenuate the inward proton current and could even have preventive effects. In contrast, at the end of exercise, while intracellular pH rapidly returns to pre‐exercise values, arterial blood pH was shown to remain acidic much longer and had not recovered even 30 min after the end of the exercise (Stringer et al. 1992; Juel et al. 2004). It is therefore during the recovery period that the inward driving force for protons should be the highest and consequently the leak proton inward current through the gating pore maximal. Potentiation of the inward proton current through the gating pore should then favour the occurrence of paralytic attacks at rest after intense muscle exercise. Indeed, we found that the Kir conductance was reduced in response to a decrease in external pH in mouse muscle fibres (Fuster et al. 2017a). Therefore, external acidosis may further worsen the conditions that lead to loss of excitability in HypoPP1 muscle, not only through potentiation of the inward gating pore current but also through a decrease in the inward rectifier K⁺ conductance (Fig. 2).

Interestingly, in a study investigating the predictive role of genotype in the efficacy of acetazolamide in HypoPP, it was reported that there was a greater chance of benefit in patients with mutations in the gene encoding Cav1.1 and especially with mutations that resulted in amino acids being replaced by histidine (Matthews et al. 2011). Acetazolamide is a membrane‐permeant carbonic anhydrase inhibitor that reduces the intracellular availability of H⁺ and HCO₃ ⁻ ions. Although the actual mechanism of action of the drug remains to be elucidated, the beneficial effect of acetazolamide might be explained by its regulating action on intracellular H⁺ handling that the proton leak through gating pores might disrupt. In addition, the H⁺ selectivity of the R1239H‐induced gating pore could explain the intramuscular Na⁺ overload that has been reported in R1239H patients and that was shown to be correlated with muscle weakness (Jurkat‐Rott et al. 2009). The elevated resting proton influx in R1239H fibres may indeed over‐activate the Na⁺/H⁺ exchanger, the main mechanism of H⁺ extrusion in muscle which is coupled to Na⁺ influx (Aickin & Thomas, 1977), leading eventually to chronic Na⁺ overload. For other HypoPP1 mutations, Na⁺ overload may directly result from the elevated inward Na⁺ leak when the HypoPP1 mutation leads to Na⁺ selectivity through gating pores, as is the case for the V876E mutation.

It is very likely that the severity of HypoPP1 clinical symptoms is related to the magnitude of the resting leak gating pore current. Indeed, the leak gating pore conductance generated by the V876E mutation was estimated at 68 S F⁻¹, i.e. at least 3 times larger than that measured for the R1239H mutation (Fuster et al. 2017b). In agreement with this very large leak, the V876E mutation has been characterized by an uncommon early age of onset of the disease, high penetrance and a very severe prognosis (Ke et al. 2009). In this case, the gating pore current was found to carry Na⁺ and not H⁺ (Fuster et al. 2017b) so that external pH should have little influence on membrane excitability. It can thus be suggested that for the V876E mutation hypokalaemia is the sole factor precipitating paralytic attacks, the large resting leak inward current making the fibres hypersensitive to K⁺ changes.

Concluding remarks

It has now become quite clear that the development of an inward gating pore current in the voltage‐sensing domain of HypoPP1 mutated voltage‐gated Ca²⁺ channels plays a critical role in the pathophysiology of the HypoPP1 disease. Moreover, gating pore currents have also been found in voltage‐gated Na⁺ channels carrying comparable mutations in S4 segments and leading to another type of HypoPP, HypoPP2, with the same clinical symptoms as HypoPP1 (Sokolov et al. 2007; Struyk & Cannon, 2007). Proton permeation through gating pores generated by the R1239H mutation, which has also been demonstrated in the Na⁺ channel for histidine‐to‐arginine substitutions in S4 segments, implies that acidification of the intra‐ or extracellular medium will be protective or detrimental by decreasing or increasing, respectively, the inward driving force for protons. However, it remains to be demonstrated whether the magnitude of the changes in extracellular pH is large enough to substantially affect the resting leak current. Furthermore, it is very unlikely that external acidosis can trigger paralysis on its own in the absence of a drop in extracellular K⁺. The decrease in serum K⁺ could even be the unique factor that triggers the attacks of paralysis for the R1239H mutation. This is certainly the case for the V876E mutation since Cav1.1 carries Na⁺ and not H⁺ in physiological saline. The fact that this mutation does not directly affect an arginine residue in a S4 segment, but yet generates a gating pore current, confirms that an elevated resting leak inward current is critical in the HypoPP1 pathogenesis. Further investigations are required to find pharmacological compounds that suppress or at least attenuate the leak inward current through closed muscle Ca²⁺ channels.

Additional information

Competing interests

The authors declare that they have no competing interests.

Funding

Research of the authors is supported by the University Lyon 1, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (Inserm) and the Association Française contre les Myopathies (AFM‐Téléthon).

Biographies

Bruno Allard is Professor of Physiology at the University Claude Bernard Lyon 1. He works in the NeuroMyoGene Institute, where his research is focused on excitability and Ca²⁺ signalling in normal and diseased muscle using electrophysiological approaches, Ca²⁺ imaging and gene transfer.

Clarisse Fuster is a third year PhD student working under the supervision of Professor Allard in the NeuroMyoGene Institute. Her PhD work, which is partly reported in the present review, aimed at measuring the changes in transmembrane ion movements in mouse muscle fibres acutely expressing mutant HypoPP1 Ca²⁺ channels.