Gating defects of a novel Na+ channel mutant causing hypokalemic periodic paralysis

Hypokalemic periodic paralysis type 2 (hypoPP2) is an inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the alpha subunit of the skeletal muscle Na+ channel (Nav1.4). All hypoPP2 mutations reported so far target an arginine residue of the voltage sensor S4 of domain II (R672/G/H/S). We identified a novel hypoPP2 mutation that neutralizes an arginine residue in DIII-S4 (R1132Q), and studied its functional consequences in HEK cells transfected with the human SCN4A cDNA. Whole-cell current recordings revealed an enhancement of both fast and slow inactivation, as well as a depolarizing shift of the activation curve. The unitary Na+ conductance remained normal in R1132Q and in R672S mutants, and cannot therefore account for the reduction of Na+ current presumed in hypoPP2. Altogether, our results provide a clear evidence for the role of R1132 in channel activation and inactivation, and confirm loss of function effects of hypoPP2 mutations leading to muscle hypoexcitability.     

Hyperkalemic periodic paralysis M1592V mutation modifies activation in human skeletal muscle Na+ channel

Mutations in thehuman skeletal muscle Na⁺ channelunderlie the autosomal dominant disease hyperkalemic periodic paralysis (HPP). Muscle fibers from affected individuals exhibit sustained Na⁺ currents thought to depolarizethe sarcolemma and thus inactivate normalNa⁺ channels. We expressed humanwild-type or M₁₅₉₂V mutant-subunits with the ₁-subunitin Xenopus laevis oocytes and recordedNa⁺ currents using two-electrodeand cut-open oocyte voltage-clamp techniques. The most prominentfunctional difference betweenM₁₅₉₂V mutant and wild-typechannels is a 5- to 10-mV shift in the hyperpolarized direction of thesteady-state activation curve. The shift in the activation curve forthe mutant results in a larger overlap with the inactivation curve thanthat observed for wild-type channels. Accordingly, the current throughM₁₅₉₂V channels displays a larger noninactivating component than does that through wild-type channels atmembrane potentials near 40 mV. The functional properties of theM₁₅₉₂V mutant resemble those ofthe previously characterized HPPT₇₀₄M mutant. Both clinicallysimilar phenotypes arise from mutations located at a distance from theputative voltage sensor of the channel.

     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Ion permeation and block of the gating pore in the voltage sensor of NaV1.4 channels with hypokalemic periodic paralysis mutations

Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge–carrying arginine residues in skeletal muscle Na_V1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only ∼1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- ± 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba²⁺ and Zn²⁺ in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba²⁺ and Zn²⁺ is increased at more negative holding potentials. The apparent dissociation constant (K_d) values for Zn²⁺ block for test pulses to −160 mV are 650 ± 150 µM, 360 ± 70 µM, and 95.6 ± 11 µM at holding potentials of 0 mV, −80 mV, and −120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent K_d for Gd³⁺ of 238 ± 14 µM at −80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal Na_V1.4 channel function. Together, our results demonstrate unique permeability of guanidine through Na_V1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.     

Structural effects of an LQT-3 mutation on heart Na+ channel gating

Computational methods that predict three-dimensional structures from amino acid sequences have become increasingly accurate and have provided insights into structure-function relationships for proteins in the absence of structural data. However, the accuracy of computational structural models requires experimental approaches for validation. Here we report direct testing of the predictions of a previously reported structural model of the C-terminus of the human heart Na(+) channel. We focused on understanding the structural basis for the unique effects of an inherited C-terminal mutation (Y1795C), associated with long QT syndrome variant 3 (LQT-3), that has pronounced effects on Na(+) channel inactivation. Here we provide evidence that this mutation, in which a cysteine replaces a tyrosine at position 1795 (Y1795C), enables the formation of disulfide bonds with a partner cysteine in the channel. Using the predictions of the model, we identify the cysteine and show that three-dimensional information contained in the sequence for the channel protein is necessary to understand the structural basis for some of the effects of the mutation. The experimental evidence supports the accuracy of the predicted structural model of the human heart Na(+) channel C-terminal domain and provides insight into a structural basis for some of the mutation-induced altered channel function underlying the disease phenotype.     

A mutation in the pore of the sodium channel alters gating.

Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure.     

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation

We examined theability of local anesthetics to correct altered inactivation propertiesof rat skeletal muscle Na⁺channels containing the equine hyperkalemic periodic paralysis (eqHPP)mutation when expressed in Xenopusoocytes. Increased time constants of current decay in eqHPP channelscompared with wild-type channels were restored by 1 mM benzocaine butwere not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to 10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward thatobserved for wild-type (WT) channels using selected concentrations ofbenzocaine, lidocaine, and mexiletine. Recovery from inactivation at80 mV (50-ms conditioning pulse) in eqHPP channels followed amonoexponential time course and was markedly accelerated compared withwild-type channels (_WT = 10.8 ± 0.9 ms; _eqHPP = 2.9 ± 0.4 ms). Benzocaine slowed the time course of recovery(_eqHPP,ben = 9.6 ± 0.4 msat 1 mM) in a concentration-dependent manner. In contrast, the recoveryfrom inactivation with lidocaine and mexiletine had a fast component(_fast,lid = 3.2 ± 0.2 ms;_fast,mex = 3.1 ± 0.2 ms),which was identical to the recovery in eqHPP channels without drug, anda slow component (_slow,lid = 1,688 ± 180 ms; _slow,mex = 2,323 ± 328 ms). The time constant of the slow component of therecovery from inactivation was independent of the drug concentration,whereas the fraction of current recovering slowly depended on drugconcentrations and conditioning pulse durations. Our results show thatlocal anesthetics are generally incapable of fully restoring normal WTbehavior in inactivation-deficient eqHPP channels.

     

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na⁺ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na⁺ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.     

Connexin phosphorylation as a regulatory event linked to channel gating

The main proteins required for functional gap junction channels are known as connexins and most of their isoforms indicate that they can become phosphorylated. Connexin phosphorylation has been reported to participate in modifying junctional communication and the mechanisms involved apparently depend on which kinase becomes involved. Although multiple reports have suggested a strong influence of phosphorylation on channel gating, not enough physiological studies have been performed to determine precisely the gating mechanisms implicated. Moreover, gap junction channels follow other various gating mechanisms, including voltage gating and chemical gating, where phosphorylation could act as a modulator. The quest for this chapter has been to discriminate those instances where phosphorylation acts directly as a gating trigger and where it acts indirectly or only as a modulator. Despite recent efforts, the mechanisms involved in all these cases are barely understood.     

Connexin phosphorylation as a regulatory event linked to channel gating

The main proteins required for functional gap junction channels are known as connexins and most of their isoforms indicate that they can become phosphorylated. Connexin phosphorylation has been reported to participate in modifying junctional communication and the mechanisms involved apparently depend on which kinase becomes involved. Although multiple reports have suggested a strong influence of phosphorylation on channel gating, not enough physiological studies have been performed to determine precisely the gating mechanisms implicated. Moreover, gap junction channels follow other various gating mechanisms, including voltage gating and chemical gating, where phosphorylation could act as a modulator. The quest for this chapter has been to discriminate those instances where phosphorylation acts directly as a gating trigger and where it acts indirectly or only as a modulator. Despite recent efforts, the mechanisms involved in all these cases are barely understood.     

Exploration of the pore structure of a peptide-gated Na+ channel

The FMRF-amide-activated sodium channel (FaNaC), a member of the ENaC/Degenerin family, is a homotetramer, each subunit containing two transmembrane segments. We changed independently every residue of the first transmembrane segment (TM1) into a cysteine and tested each position's accessibility to the cysteine covalent reagents MTSET and MTSES. Eleven mutants were accessible to the cationic MTSET, showing that TM1 faces the ion translocation pathway. This was confirmed by the accessibility of cysteines present in the acid-sensing ion channels and other mutations introduced in FaNaC TM1. Modification of accessibilities for positions 69, 71 and 72 in the open state shows that the gating mechanism consists of the opening of a constriction close to the intracellular side. The anionic MTSES did not penetrate into the channel, indicating the presence of a charge selectivity filter in the outer vestibule. Furthermore, amiloride inhibition resulted in the channel occlusion in the middle of the pore. Summarizing, the ionic pore of FaNaC includes a large aqueous cavity, with a charge selectivity filter in the outer vestibule and the gate close to the interior.     

More gating charges are needed to open a Shaker K+ channel than are needed to open an rBIIA Na+ channel

This study presents what is, to our knowledge, a novel technique by means of which the ratio of the single gating charges of voltage-gated rat brain IIA (rBIIA) sodium and Shaker potassium ion channels was estimated. In the experiment, multiple tandems of enhanced green fluorescent protein were constructed and inserted into the C-terminals of Na⁺ and K⁺ ion channels. cRNA of Na⁺ and K⁺ ion channels was injected and expressed in Xenopus laevis oocytes. The two electrode voltage-clamp technique allowed us to determine the total gating charge of sodium and potassium ion channels, while a relative measure of the amount of expressed channels could be established on the basis of the quantification of the fluorescence intensity of membrane-bound channels marked by enhanced green fluorescent proteins. As a result, gating charge and fluorescence intensity were found to be positively correlated. A relative comparison of the single gating charges of voltage-gated sodium and potassium ion channels could thus be established: the ratio of the single gating charges of the Shaker potassium channel and the rBIIA sodium channel was found to be 2.5 ± 0.4. Assuming the single channel gating charge of the Shaker K⁺ channel to be ∼13 elementary charges (well supported by other studies), this leads to approximately six elementary charges for the rBIIA sodium channel, which includes a fraction of gating charge that is missed during inactivation.     

A gating mutation in the internal pore of ASIC1a

Using a substituted cysteine accessibility scan, we have investigated the structures that form the internal pore of the acid-sensing ion channel 1a. We have identified the amino acid residues Ala-22, Ile-33, and Phe-34 in the amino terminus and Arg-43 in the first transmembrane helix, which when mutated into cysteine, were modified by intracellular application of MTSET, resulting in channel inhibition. The inhibition of the R43C mutant by internal MTSET requires opening of the channel. In addition, binding of Cd2+ ions to R43C slows the channel inactivation. This indicates that the first transmembrane helix undergoes conformational changes during channel inactivation. The effect of Cd2+ on R43C can be obtained with Cd2+ applied at either the extracellular or the intracellular side, indicating that R43C is located in the channel pore. The block of the A22C, I33C, and F34C mutants by MTSET suggests that these residues in the amino terminus of the channel also participate to the internal pore.     

Differential sialylation modulates voltage-gated Na+ channel gating throughout the developing myocardium

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an approximately 10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel alpha subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than alpha subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each alpha subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel alpha subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid-dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.     

Theoretical investigation of the neuronal Na+ channel SCN1A: abnormal gating and epilepsy

Epilepsy is a paroxysmal neurological disorder resulting from abnormal cellular excitability and is a common cause of disability. Recently, some forms of idiopathic epilepsy have been causally related to genetic mutations in neuronal ion channels. To understand disease mechanisms, it is crucial to understand how a gene defect can disrupt channel gating, which in turn can affect complex cellular dynamic processes. We develop a theoretical Markovian model of the neuronal Na+ channel NaV1.1 to explore and explain gating mechanisms underlying cellular excitability and physiological and pathophysiological mechanisms of abnormal neuronal excitability in the context of epilepsy. Genetic epilepsy has been shown to result from both mutations that give rise to a gain of channel function and from those that reduce the Na+ current. These data may suggest that abnormal excitation can result from both hyperexcitability and hypoexcitability, the mechanisms of which are presumably distinct, and as yet elusive. Revelation of the molecular origins will allow for translation into targeted pharmacological interventions that must be developed to treat syndromes resulting from divergent mechanisms. This work represents a first step in developing a comprehensive theoretical model to investigate the molecular mechanisms underlying runaway excitation that cause epilepsy.     

Contributions of charged residues in a cytoplasmic linking region to Na channel gating

Na channels inactivate quickly after opening, and the very highly positively charged cytoplasmic linking region between homologous domains III and IV of the channel molecule acts as the inactivation gate. To test the hypothesis that the charged residues in the domain III to domain IV linker have a role in channel function, we measured currents through wild-type and two mutant skeletal muscle Na channels expressed in Xenopus oocytes, each lacking two or three charged residues in the inactivation gate. Microscopic current measures showed that removing charges hastened activation and inactivation. Macroscopic current measures showed that removing charges altered the voltage dependence of inactivation, suggesting less coupling of the inactivation and activation processes. Reduced intracellular ionic strength shifted the midpoint of equilibrium activation gating to a greater extent, and shifted the midpoint of equilibrium inactivation gating to a lesser extent in the mutant channels. The results allow the possibility that an electrostatic mechanism contributes to the role of charged residues in Na channel inactivation gating.     

低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型的构建

目的：构建低钾型周期性麻痹相关的Cchl1a3基因R528H敲入小鼠模型。方法将 Cchl1a3-knock-in打靶载体电转染ES细胞,经过G418和Ganciclovoir筛选阳性ES细胞克隆并用PCR和DNA测序法鉴定。将阳性ES克隆注射到小鼠囊胚,获得嵌合体小鼠。通过杂交获得的杂合子小鼠与FLP小鼠交配繁育获得去neo杂合子小鼠,并用PCR和DNA测序进行鉴定。将去neo杂合子小鼠交配得到纯合子后代,进行生长发育等方面的观察。结果打靶载体成功转染ES细胞,PCR和DNA测序法证实9个ES细胞克隆发生正确的同源重组。通过显微注射获得7只嵌合体小鼠。将嵌合体小鼠交配繁育的杂合子小鼠和FLP小鼠交配获得9只去neo杂合子小鼠,最终得到15只去neo纯合子小鼠。该小鼠在发育至性成熟阶段,精神、饮食及活动状态良好,但是在4个月龄时逐渐出现脱毛,皮肤破溃甚至死亡。结论成功构建Cchl1a3基因 R528H 突变的纯合子小鼠,为研究人类CACNA1S基因功能和阐明低钾型周期性麻痹发生的分子机制奠定了基础。