Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I)

In skeletal muscle, slow inactivation (SI) of Na_V1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na_V1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na_V1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1–160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na_V1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (∼10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.     

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na_Vs, are responsible for the rapid rise of action potentials in excitable tissues. Na_V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na_V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na_V1.4 (skeletal muscle) and Na_V1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na_V1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na_V isoform has been challenging because they usually show undesired crossreactivity for different Na_V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa_V1.4 or CTNa_V1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa_V1.4 and CTNa_V1.5 with high affinity (K_D ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na_V1.4 and Na_V1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na_V1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na_V reagents to capture Na_Vs from cell lysates and as molecular visualization agents for Na_Vs.     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

Slow inactivation in voltage-gated sodium channels

Slow inactivation in voltage-gated sodium channels is a biophysical process that governs the availability of sodium channels over extended periods of time. Slow inactivation, therefore, plays an important role in controlling membrane excitability, firing properties, and spike frequency adaptation. Defective slow inactivation is associated with several diseases of cell excitability, such as hyperkalemic periodic paralysis, myotonia, idiopathic ventricular fibrillation and long-QT syndrome. These associations underscore the physiological importance of this phenomenon. Nevertheless, our understanding of the molecular substrates for slow inactivation is still fragmentary. This review covers the current state of knowledge concerning the molecular underpinnings of slow inactivation, and its relationship with other biophysical processes of voltage-gated sodium channels.     

Effects of lactate on the voltage-gated sodium channels of rat skeletal muscle: modulating current opinion

During muscle contraction, lactate production and translocation across the membrane increase. While it has recently been shown that lactate anion acts on chloride channel, less is known regarding a potential effect on the voltage-gated sodium channel (Na(v)) of skeletal muscle. The electrophysiological properties of muscle Na(v) were studied in the absence and presence of lactate (10 mM) by using the macropatch-clamp method in dissociated fibers from rat peroneus longus (PL). Lactate in the external medium (petri dish + pipette) increases the maximal sodium current, while the voltage dependence of activation and fast inactivation are shifted toward the hyperpolarized potentials. Lactate induces a leftward shift in the relationship between the kinetic parameters and the imposed potentials, resulting in an earlier recruitment of muscle Na(v). In addition, lactate significantly decreases the time constant of activation at voltages more negative than -10 mV, corresponding to an acceleration of Na(v) activation. The slow inactivation process is decreased by lactate, corresponding to an enhancement in the number of excitable Na(v). In an additional series of experiments, lactate (10 mM) was only added to the petri dish, while the pipette remained sealed on the membrane area. With this approach, the electrophysiological properties of Na(v) were unaffected by lactate compared with the control condition. Altogether, these data indicate that lactate modulates muscle Na(v) properties by an extracellular pathway. These effects are consistent with an enhancement in excitability, providing new insights into the role of lactate in muscle physiology.     

Impaired slow inactivation in mutant sodium channels.

Hyperkalemic periodic paralysis (HyperPP) is a disorder in which current through Na+ channels causes a prolonged depolarization of skeletal muscle fibers, resulting in membrane inexcitability and muscle paralysis. Although HyperPP mutations can enhance persistent sodium currents, unaltered slow inactivation would effectively eliminate any sustained currents through the mutant channels. We now report that rat skeletal muscle channels containing the mutation T698M, which corresponds to the human T704M HyperPP mutation, recover very quickly from prolonged depolarizations. Even after holding at -20 mV for 20 min, approximately 25% of the maximal sodium current is available subsequent to a 10-ms hyperpolarization (-100 mV). Under the same conditions, recovery is less than 3% in wild-type channels and in the F1304Q mutant, which has impaired fast inactivation. This effect of the T698M mutation on slow inactivation, in combination with its effects on activation, is expected to result in persistent currents such as that seen in HyperPP muscle.     

Recombinant human voltage-gated skeletal muscle sodium channels are pharmacologically functional in planar lipid bilayers

Human voltage-gated sodium ion channels are major sites of action for drugs and toxins that modulate cellular excitability, and are therefore key molecular targets for ion channel research, high throughput screening for new drugs, and toxin detection. Protein suitable for these applications must be produced in a functionally active form. We report the successful use of ion metal affinity chromatography (IMAC) to purify C-terminal polyhistidine tagged human skeletal muscle voltage-gated sodium (hSkM1-HT) channels from Sf9 insect cells; hSkM1 channels were pharmacologically functional when reconstituted into liposomes and incorporated into planar bilayer lipid membranes. hSkM1-HT single channel currents activated by veratridine had a conductance of 21 pS and those activated by brevetoxin, 16 pS. Channel activity was inhibited by tetrodotoxin and saxitoxin. This protein is suitable for the development of biosensor and high throughput screening technologies.     

Effects of temperature on slow and fast inactivation of rat skeletal muscle Na+ channels

Patch-clampstudies of mammalian skeletal muscleNa⁺ channels are commonly done atsubphysiological temperatures, usually room temperature. However, atsubphysiological temperatures, mostNa⁺ channels are inactivated atthe cell resting potential. This study examined the effects oftemperature on fast and slow inactivation ofNa⁺ channels to determine iftemperature changed the fraction of Na⁺ channels that were excitableat resting potential. The loose patch voltage clamp recordedNa⁺ currents(I_Na) in vitroat 19, 25, 31, and 37°C from the sarcolemma of rat type IIbfast-twitch omohyoid skeletal muscle fibers. Temperature affected thefraction of Na⁺ channels that wereexcitable at the resting potential. At 19°C, only 30% of channelswere excitable at the resting potential. In contrast, at 37°C, 93%of Na⁺ channels were excitable atthe resting potential. Temperature did not alter the resting potentialor the voltage dependencies of activation or fast inactivation.I_Na available atthe resting potential increased with temperature because thesteady-state voltage dependence of slow inactivation shifted in adepolarizing direction with increasing temperature. The membranepotential at which half of the Na⁺channels were in the slow inactivated state was shifted by +16 mV at37°C compared with 19°C. Consequently, the low availability ofexcitable Na⁺ channels atsubphysiological temperatures resulted from channels being in the slow,inactivated state at the resting potential.

     

Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells

Voltage-gated Na(+) channel currents (I(Na)) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na(+) with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type Ca(2+) channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia.     

Floxed allele for conditional inactivation of the voltage-gated sodium channel Scn8a (NaV1.6)

The sodium channel gene Scn8a encodes the channel NaV1.6, which is widely distributed in the central and peripheral nervous system. NaV1.6 is the major channel at the nodes of Ranvier in myelinated axons. Mutant alleles of mouse Scn8a result in neurological disorders including ataxia, tremor, paralysis, and dystonia. We generated a floxed allele of Scn8a by inserting loxP sites around the first coding exon. The initial targeted allele containing the neo-cassette was a severe hypomorph. In vivo deletion of the neo-cassette by Flp recombinase produced a floxed allele that generates normal expression of NaV1.6 protein. Ubiquitous deletion of the floxed exon by Cre recombinase in ZP3-Cre transgenic mice produced the Scn8a(del) allele. The null phenotype of Scn8a(del) homozygotes confirms the in vivo inactivation of Scn8a. Conditional inactivation of the floxed allele will make it possible to circumvent the lethality that results from complete loss of Scn8a in order to investigate the physiologic role of NaV1.6 in subpopulations of neurons.     

Residue-specific effects on slow inactivation at V787 in D2-S6 of Na(v)1.4 sodium channels

Slow inactivation in voltage-gated sodium channels (NaChs) occurs in response to depolarizations of seconds to minutes and is thought to play an important role in regulating membrane excitability and action potential firing patterns. However, the molecular mechanisms of slow inactivation are not well understood. To test the hypothesis that transmembrane segment 6 of domain 2 (D2-S6) plays a role in NaCh slow inactivation, we substituted different amino acids at position V787 (valine) in D2-S6 of rat skeletal muscle NaCh mu(1) (Na(v)1.4). Whole-cell recordings from transiently expressed NaChs in HEK cells were used to study and compare slow inactivation phenotypes between mutants and wild type. V787K (lysine substitution) showed a marked enhancement of slow inactivation. V787K enters the slow-inactivated state approximately 100x faster than wild type (tau(1) approximately 30 ms vs. approximately 3 s), and occurs at much more hyperpolarized potentials than wild type (V(1/2) of s(infinity) curve approximately -130 mV vs. approximately -75 mV). V787C (cysteine substitution) showed a resistance to slow inactivation, i.e., opposite to that of V787K. Entry into the slow inactivation state in V787C was slower (tau(1) approximately 5 s), less complete, and less voltage-dependent (V(1/2) of s(infinity) curve approximately -50 mV) than in wild type. Application of the cysteine modification agent methanethiosulfonate ethylammonium (MTSEA) to V787C demonstrated that the 787 position undergoes a relative change in molecular conformation that is associated with the slow inactivation state. Our results suggest that the V787 position in Na(v)1.4 plays an important role in slow inactivation gating and that molecular rearrangement occurs at or near residue V787 in D2-S6 during NaCh slow inactivation.     

Modeling of benzocaine analog interactions with the D4S6 segment of NaV4.1 voltage-gated sodium channels

Local anesthetics (LAs) are compounds that inhibit the propagation of action potentials in excitable tissues by blocking voltage-gated Na+ channels. Mutagenesis studies have demonstrated that several amino acid residues are important sites of LA interaction with the channel, but these studies provide little information regarding the molecular forces that govern drug-binding interactions, including the binding orientation of drugs. We used computational methods to construct a simple model of benzocaine analog binding with the D4S6 segment of rat skeletal muscle (NaV4.1) sodium channels. The model revealed that four hydrophobic residues form a binding cavity for neutral LAs, and docking studies indicated that increasing hydrophobicity among the benzocaine analogs allowed a better fit within the binding cavity. The similarities between our simple model and published experimental data suggested that modeling of LA interactions with sodium channels, along with experimental approaches, could further enhance our understanding of LA interactions with sodium channels.     

New insights from modelling studies and molecular dynamics simulations of the DIS5-S6 extracellular linker of the skeletal muscle sodium channel NaV1.4

In the CryoEM-structure of the hSkMNaV1.4 ion channel (PDB:6AGF), the 59-residue DI_S5-S6 linker peptide was omitted due to absence of electron density. This peptide is intriguing – comprised of unique sequence and found only in mammalian skeletal muscle sodium ion channels. To probe potential physiological and evolutionary significance, we constructed an homology model of the complete hSkMNaV1.4 channel. Rather than a flexible random coil potentiating drift across the channel, the linker folds into a compact configuration through self-assembling secondary structural elements. Analogous sequences from 48 mammalian organisms show hypervariability with between 40% and 100% sequence similarity. To investigate structural implications, sequences from 14 representative organisms were additionally modelled. All showed highly conserved N-and C-terminal residues closely superimposed, suggesting a critical functional role. An optimally located asparagine residue within the conserved region was investigated for N-linked glycosylation and MD simulations carried out. Results suggest a complex glycan added at this site in the linker may form electrostatic interactions with the DIV voltage sensing domain and be mechanistically involved in channel gating. The relationship of unique sequence, compact configuration, potential glycosylation and MD simulations are discussed relative to SkMNaV1.4 structure and function.     

Conotoxin modulation of voltage-gated sodium channels

The rising phase of the action potential in excitable cells is mediated by voltage-gated sodium channels (VGSCs), of which there are nine mammalian subtypes with distinct tissue distribution and biophysical properties. The involvement of certain VGSC subtypes in disease states such as pain and epilepsy highlights the need for agents that modulate VGSCs in a subtype-specific manner. Conotoxins from marine snails of the Conus genus constitute a promising source of such modulators, since these peptide toxins have evolved to become selective for various membrane receptors, ion channels and transporters in excitable cells. This review covers the structure and function of three classes of conopeptides that modulate VGSCs: the pore-blocking mu-conotoxins, the delta-conotoxins which delay or inhibit VGSC inactivation, and the muO-conotoxins which inhibit VGSC Na(+) conductance independent of the tetrodotoxin binding site. Some of these toxins have potential therapeutic and research applications, in particular the muO-conotoxins, which may develop into potential drug leads for the treatment of pain states.     

Gamma 1 subunit interactions within the skeletal muscle L-type voltage-gated calcium channels

Voltage-gated calcium channels mediate excitationcontraction coupling in the skeletal muscle. Their molecular composition, similar to neuronal channels, includes the pore-forming alpha(1) and auxiliary alpha(2)delta, beta, and gamma subunits. The gamma subunits are the least characterized, and their subunit interactions are unclear. The physiological importance of the neuronal gamma is emphasized by epileptic stargazer mice that lack gamma(2). In this study, we examined the molecular basis of interaction between skeletal gamma(1) and the calcium channel. Our data show that the alpha(1)1.1, beta(1a), and alpha(2)delta subunits are still associated in gamma(1) null mice. Reexpression of gamma(1) and gamma(2) showed that gamma(1), but not gamma(2), incorporates into gamma(1) null channels. By using chimeric constructs, we demonstrate that the first half of the gamma(1) subunit, including the first two transmembrane domains, is important for subunit interaction. Interestingly, this chimera also restores calcium conductance in gamma(1) null myotubes, indicating that the domain mediates both subunit interaction and current modulation. To determine the subunit of the channel that interacts with gamma(1), we examined the channel in muscular dysgenesis mice. Cosedimentation experiments showed that gamma(1) and alpha(2)delta are not associated. Moreover, alpha(1)1.1 and gamma(1) subunits form a complex in transiently transfected cells, indicating direct interaction between the gamma(1) and alpha(1)1.1 subunits. Our data demonstrate that the first half of gamma(1) subunit is required for association with the channel through alpha(1)1.1. Because subunit interactions are conserved, these studies have broad implications for gamma heterogeneity, function and subunit association with voltage-gated calcium channels.     

Interaction between fast and slow inactivation in Skm1 sodium channels.

Rat skeletal muscle (Skm1) sodium channel alpha and beta 1 subunits were coexpressed in Xenopus oocytes, and resulting sodium currents were recorded from on-cell macropatches. First, the kinetics and steady-state probability of both fast and slow inactivation in Skm1 wild type (WT) sodium channels were characterized. Next, we confirmed that mutation of IFM to QQQ (IFM1303QQQ) in the DIII-IV 'inactivation loop' completely removed fast inactivation at all voltages. This mutation was then used to characterize Skm1 slow inactivation without the presence of fast inactivation. The major findings of this paper are as follows: 1) Even with complete removal of fast inactivation by the IFM1303QQQ mutation, slow inactivation remains intact. 2) In WT channels, approximately 20% of channels fail to slow-inactivate after fast-inactivating, even at very positive potentials. 3) Selective removal of fast inactivation by IFM1303QQQ allows slow inactivation to occur more quickly and completely than in WT. We conclude that fast inactivation reduces the probability of subsequent slow inactivation.