Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac

Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1–R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.     

Isoform-specific lidocaine block of sodium channels explained by differences in gating

When depolarized from typical resting membrane potentials (V(rest) approximately -90 mV), cardiac sodium (Na) currents are more sensitive to local anesthetics than brain or skeletal muscle Na currents. When expressed in Xenopus oocytes, lidocaine block of hH1 (human cardiac) Na current greatly exceeded that of mu1 (rat skeletal muscle) at membrane potentials near V(rest), whereas hyperpolarization to -140 mV equalized block of the two isoforms. Because the isoform-specific tonic block roughly parallels the drug-free voltage dependence of channel availability, isoform differences in the voltage dependence of fast inactivation could underlie the differences in block. However, after a brief (50 ms) depolarizing pulse, recovery from lidocaine block is similar for the two isoforms despite marked kinetic differences in drug-free recovery, suggesting that differences in fast inactivation cannot entirely explain the isoform difference in lidocaine action. Given the strong coupling between fast inactivation and other gating processes linked to depolarization (activation, slow inactivation), we considered the possibility that isoform differences in lidocaine block are explained by differences in these other gating processes. In whole-cell recordings from HEK-293 cells, the voltage dependence of hH1 current activation was approximately 20 mV more negative than that of mu1. Because activation and closed-state inactivation are positively coupled, these differences in activation were sufficient to shift hH1 availability to more negative membrane potentials. A mutant channel with enhanced closed-state inactivation gating (mu1-R1441C) exhibited increased lidocaine sensitivity, emphasizing the importance of closed-state inactivation in lidocaine action. Moreover, when the depolarization was prolonged to 1 s, recovery from a "slow" inactivated state with intermediate kinetics (I(M)) was fourfold longer in hH1 than in mu1, and recovery from lidocaine block in hH1 was similarly delayed relative to mu1. We propose that gating processes coupled to fast inactivation (activation and slow inactivation) are the key determinants of isoform-specific local anesthetic action.     

Altered sodium and gating current kinetics in frog skeletal muscle caused by low external pH

The effect of low pH on the kinetics of Na channel ionic and gating currents was studied in frog skeletal muscle fibers. Lowering external pH from 7.4 to 5.0 slows the time course of Na current consistent with about a +25-mV shift in the voltage dependence of activation and inactivation time constants. Similar shifts in voltage dependence adequately describe the effects of low pH on the tail current time constant (+23.3 mV) and the gating charge vs. voltage relationship (+22.1 mV). A significantly smaller shift of +13.3 mV described the effect of pH 5.0 solution on the voltage dependence of steady state inactivation. Changes in the time course of gating current at low pH were complex and could not be described as a shift in voltage dependence. tau g, the time constant that describes the time course of the major component of gating charge movement, was slowed in pH 5.0 solution by a factor of approximately 3.5 for potentials from -60 to +45 mV. We conclude that the effects of low pH on Na channel gating cannot be attributed simply to a change in surface potential. Therefore, although it may be appropriate to describe the effect of low pH on some Na channel kinetic properties as a "shift" in voltage dependence, it is not appropriate to interpret such shifts as a measure of changes in surface potential. The maximum gating charge elicited from a holding potential of -150 mV was little affected by low pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH modification of human T-type calcium channel gating

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.     

Use-dependent potentiation of the Nav1.6 sodium channel

Nav1.2 and Nav1.6 are two voltage-gated sodium channel isoforms that are abundant in the adult central nervous system. These channels are expressed in different cells and localized in different neuronal regions, which may reflect functional specialization. To examine this possibility, we compared the properties of Nav1.2 and Nav1.6 in response to a rapid series of repetitive depolarizations. Currents through Nav1.6 coexpressed with beta1 demonstrated use-dependent potentiation during a rapid train of depolarizations. This potentiation was in contrast to the use-dependent decrease in current for Nav1.2 with beta1. The voltage dependence of potentiation correlated with the voltage dependence of activation, and it still occurred when fast inactivation was removed by mutation. Rapid stimulation accelerated a slow phase of activation in the Nav1.6 channel that had fast inactivation removed, resulting in faster channel activation. Although the Nav1.2 channel with fast inactivation removed also demonstrated slightly faster activation, that channel showed very pronounced slow inactivation compared to Nav1.6. These results indicate that potentiation of Nav1.6 sodium currents results from faster channel activation, and that this effect is masked by slow inactivation in Nav1.2. The data suggest that Nav1.6 might be more resistant to inactivation, which might be helpful for high-frequency firing at nodes of Ranvier compared to Nav1.2.     

Role of auxiliary beta1-, beta2-, and beta3-subunits and their interaction with Na(v)1.8 voltage-gated sodium channel

The nociceptive C-fibers of the dorsal root ganglion express several sodium channel isoforms that associate with one or more regulatory beta-subunits (beta1-beta4). To determine the effects of individual and combinations of the beta-subunit isoforms, we co-expressed Nav1.8 in combination with these beta-subunits in Xenopus oocytes. Whole-cell inward sodium currents were recorded using the two-microelectrode voltage clamp method. Our studies revealed that the co-expression beta1 alone or in combination with other beta-subunits enhanced current amplitudes, accelerated current decay kinetics, and negatively shifted the steady-state curves. In contrast, beta2 alone and in combination with beta1 altered steady-state inactivation of Nav1.8 to more depolarized potentials. Co-expression of beta3 shifted steady-state inactivation to more depolarized potentials; however, combined beta1beta3 expression caused no shift in channel availability. The results in this study suggest that the functional behavior of Nav1.8 will vary depending on the type of beta-subunit that expressed under normal and disease states.     

Inactivation viewed through single sodium channels

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Hysteresis in the voltage dependence of HCN channels: conversion between two modes affects pacemaker properties

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) ion channels are important for rhythmic activity in the brain and in the heart. In this study, using ionic and gating current measurements, we show that cloned spHCN channels undergo a hysteresis in their voltage dependence during normal gating. For example, both the gating charge versus voltage curve, Q(V), and the conductance versus voltage curve, G(V), are shifted by about +60 mV when measured from a hyperpolarized holding potential compared with a depolarized holding potential. In addition, the kinetics of the tail current and the activation current change in parallel to the voltage shifts of the Q(V) and G(V) curves. Mammalian HCN1 channels display similar effects in their ionic currents, suggesting that the mammalian HCN channels also undergo voltage hysteresis. We propose a model in which HCN channels transit between two modes. The voltage dependence in the two modes is shifted relative to each other, and the occupancy of the two modes depends on the previous activation of the channel. The shifts in the voltage dependence are fast (tau approximately 100 ms) and are not accompanied by any apparent inactivation. In HCN1 channels, the shift in voltage dependence is slower in a 100 mM K extracellular solution compared with a 1 mM K solution. Based on these findings, we suggest that molecular conformations similar to slow (C-type) inactivation of K channels underlie voltage hysteresis in HCN channels. The voltage hysteresis results in HCN channels displaying different voltage dependences during different phases in the pacemaker cycle. Computer simulations suggest that voltage hysteresis in HCN channels decreases the risk of arrhythmia in pacemaker cells.     

Open- and closed-state fast inactivation in sodium channels: differential effects of a site-3 anemone toxin

The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.     

Block of sodium conductance and gating current in squid giant axons poisoned with quaternary strychnine.

Quaternary strychnine blocks sodium channels from the axoplasmic side, probably by insertion into the inner channel mouth. Block is strongly voltage dependent, being more pronounced in depolarized than in resting axons. Using potential steps as a means to modulate the level of block, we investigate strychnine effects on sodium and gating currents at +50 and -50 mV. We analyze our data in terms of the simplest possible model, wherein only an open channel may receive and retain a strychnine molecule. Our main findings are (a) block by strychnine and inactivation resemble each other and (b) block of sodium and gating currents by strychnine happen with closely similar time-courses. Our data support the hypothesis of Armstrong and Bezanilla (1977) wherein an endogenous blocking particle causes inactivation by inserting itself into the inner mouth of the sodium channel. Quaternary strychnine may act as an artificial substitute for the hypothetical endogenous blocking particle. Further, we suggest that at least 90% of the rapid asymmetrical displacement current in squid axons is sodium channel gating current, inasmuch as quaternary strychnine can block 90% of the displacement current simultaneously with sodium current.     

The ladder-shaped polyether toxin gambierol anchors the gating machinery of Kv3.1 channels in the resting state

Voltage-gated potassium (Kv) and sodium (Nav) channels are key determinants of cellular excitability and serve as targets of neurotoxins. Most marine ciguatoxins potentiate Nav channels and cause ciguatera seafood poisoning. Several ciguatoxins have also been shown to affect Kv channels, and we showed previously that the ladder-shaped polyether toxin gambierol is a potent Kv channel inhibitor. Most likely, gambierol acts via a lipid-exposed binding site, located outside the K⁺ permeation pathway. However, the mechanism by which gambierol inhibits Kv channels remained unknown. Using gating and ionic current analysis to investigate how gambierol affected S6 gate opening and voltage-sensing domain (VSD) movements, we show that the resting (closed) channel conformation forms the high-affinity state for gambierol. The voltage dependence of activation was shifted by >120 mV in the depolarizing direction, precluding channel opening in the physiological voltage range. The (early) transitions between the resting and the open state were monitored with gating currents, and provided evidence that strong depolarizations allowed VSD movement up to the activated-not-open state. However, for transition to the fully open (ion-conducting) state, the toxin first needed to dissociate. These dissociation kinetics were markedly accelerated in the activated-not-open state, presumably because this state displayed a much lower affinity for gambierol. A tetrameric concatemer with only one high-affinity binding site still displayed high toxin sensitivity, suggesting that interaction with a single binding site prevented the concerted step required for channel opening. We propose a mechanism whereby gambierol anchors the channel’s gating machinery in the resting state, requiring more work from the VSD to open the channel. This mechanism is quite different from the action of classical gating modifier peptides (e.g., hanatoxin). Therefore, polyether toxins open new opportunities in structure–function relationship studies in Kv channels and in drug design to modulate channel function.     

Modulation of aminopyridine block of potassium currents in squid axon.

Aminopyridines are known to block potassium (K) currents in excitable membranes in a manner dependent upon membrane potential, such that the block is relieved by depolarization and restored upon repolarization. In the present study, the effects of aminopyridines on voltage-dependent potassium (K) channels were examined in internally perfused, voltage-clamped squid giant axons. The time course of block restoration after conditioning depolarization was found to be modulated by membrane electric field, K-channel gating, and external cations. Depolarized holding potentials accelerated block restoration without altering steady-state block levels, suggesting that the voltage dependence of block restoration may be related to K channel gating rather than drug binding per se. In support of this notion, low external calcium concentration, which shifts the voltage dependence of K-channel gating to more negative potentials, also accelerated block restoration. Conversely, the relationship between the rate of block restoration and membrane holding potential was shifted in the depolarizing direction by phloretin, an agent that shifts the dependence of K-channel opening on membrane potential in a similar manner. Modification of K-channel gating also was found to alter the rate of block restoration. Addition of internal zinc or internal treatment with glutaraldehyde slowed the time course of both K-channel activation and aminopyridine block restoration. Aminopyridines also were found to interact in the K channel with external Cs+, NH4+, and Rb+, each of which slowed aminopyridine block restoration. Our results suggest that aminopyridines enter and occlude K channels, and that the availability of the binding site may be modulated by channel gating such that access is limited by the probability of the channel reaching an intermediate closed state at the resting potential.     

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.     

A naturally occurring amino acid substitution in the voltage-dependent sodium channel selectivity filter affects channel gating

The pore of sodium channels contains a selectivity filter made of 4 amino acids, D/E/K/A. In voltage sensitive sodium channel (Nav) channels from jellyfish to human the fourth amino acid is Ala. This Ala, when mutated to Asp, promotes slow inactivation. In some Nav channels of pufferfishes, the Ala is replaced with Gly. We studied the biophysical properties of an Ala-to-Gly substitution (A1529G) in rat Nav1.4 channel expressed in Xenopus oocytes alone or with a β1 subunit. The Ala-to-Gly substitution does not affect monovalent cation selectivity and positively shifts the voltage-dependent inactivation curve, although co-expression with a β1 subunit eliminates the difference between A1529G and WT. There is almost no difference in channel fast inactivation, but the β1 subunit accelerates WT current inactivation significantly more than it does the A1529G channels. The Ala-to-Gly substitution mainly influences the rate of recovery from slow inactivation. Again, the β1 subunit is less effective on speeding recovery of A1529G than the WT. We searched Nav channels in numerous databases and noted at least four other independent Ala-to-Gly substitutions in Nav channels in teleost fishes. Thus, the Ala-to-Gly substitution occurs more frequently than previously realized, possibly under selection for alterations of channel gating.     

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.     

Modifications of human cardiac sodium channel gating by UVA light

Voltage-gated Na(+) channels are membrane proteins responsible for the generation of action potentials. In this report we demonstrate that UVA light elicits gating changes of human cardiac Na+ channels. First, UVA irradiation hampers the fast inactivation of cardiac Nav1.5 Na(+) channels expressed in HEK293t cells. A maintained current becomes conspicuous during depolarization and reaches its maximal quasi steady-state level within 5-7 min. Second, the activation time course is slowed by UVA light; modification of the activation gating by UVA irradiation continues for 20 min without reaching steady state. Third, along with the slowed activation time course, the peak current is reduced progressively. Most Na(+) currents are eliminated during 20 min of UVA irradiation. Fourth, UVA light increases the holding current nonlinearly; this phenomenon is slow at first but abruptly fast after 20 min. Other skeletal muscle Nav1.4 isoforms and native neuronal Na(+) channels in rat GH(3) cells are likewise sensitive to UVA irradiation. Interestingly, a reactive oxygen metabolite (hydrogen peroxide at 1.5%) and an oxidant (chloramine-T at 0.5 mM) affect Na(+) channel gating similarly, but not identically, to UVA. These results together suggest that UVA modification of Na(+) channel gating is likely mediated via multiple reactive oxygen metabolites. The potential link between oxidative stress and the impaired Na(+) channel gating may provide valuable clues for ischemia/reperfusion injury in heart and in CNS.     

A gating charge interaction required for late slow inactivation of the bacterial sodium channel NavAb

Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V_1/2 of approximately −98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ∼75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.     

Rate of opening of a population of Na channels in frog skeletal muscle fibers

Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.