Voltage-dependent Gating of Single Wild-Type and S4 Mutant KAT1 Inward Rectifier Potassium Channels

The voltage-dependent gating mechanism of KAT1 inward rectifier potassium channels was studied using single channel current recordings from Xenopus oocytes injected with KAT1 mRNA. The inward rectification properties of KAT1 result from an intrinsic gating mechanism in the KAT1 channel protein, not from pore block by an extrinsic cation species. KAT1 channels activate with hyperpolarizing potentials from −110 through −190 mV with a slow voltage-dependent time course. Transitions before first opening are voltage dependent and account for much of the voltage dependence of activation, while transitions after first opening are only slightly voltage dependent. Using burst analysis, transitions near the open state were analyzed in detail. A kinetic model with multiple closed states before first opening, a single open state, a single closed state after first opening, and a closed-state inactivation pathway accurately describes the single channel and macroscopic data. Two mutations neutralizing charged residues in the S4 region (R177Q and R176L) were introduced, and their effects on single channel gating properties were examined. Both mutations resulted in depolarizing shifts in the steady state conductance–voltage relationship, shortened first latencies to opening, decreased probability of terminating bursts, and increased burst durations. These effects on gating were well described by changes in the rate constants in the kinetic model describing KAT1 channel gating. All transitions before the open state were affected by the mutations, while the transitions after the open state were unaffected, implying that the S4 region contributes to the early steps in gating for KAT1 channels.     

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.     

Inter-Subunit Interactions across the Upper Voltage Sensing-Pore Domain Interface Contribute to the Concerted Pore Opening Transition of Kv Channels

The tight electro-mechanical coupling between the voltage-sensing and pore domains of Kv channels lies at the heart of their fundamental roles in electrical signaling. Structural data have identified two voltage sensor pore inter-domain interaction surfaces, thus providing a framework to explain the molecular basis for the tight coupling of these domains. While the contribution of the intra-subunit lower domain interface to the electro-mechanical coupling that underlies channel opening is relatively well understood, the contribution of the inter-subunit upper interface to channel gating is not yet clear. Relying on energy perturbation and thermodynamic coupling analyses of tandem-dimeric Shaker Kv channels, we show that mutation of upper interface residues from both sides of the voltage sensor-pore domain interface stabilizes the closed channel state. These mutations, however, do not affect slow inactivation gating. We, moreover, find that upper interface residues form a network of state-dependent interactions that stabilize the open channel state. Finally, we note that the observed residue interaction network does not change during slow inactivation gating. The upper voltage sensing-pore interaction surface thus only undergoes conformational rearrangements during channel activation gating. We suggest that inter-subunit interactions across the upper domain interface mediate allosteric communication between channel subunits that contributes to the concerted nature of the late pore opening transition of Kv channels.     

A sodium channel gating model based on single channel, macroscopic ionic, and gating currents in the squid giant axon.

Sodium channel gating behavior was modeled with Markovian models fitted to currents from the cut-open squid giant axon in the absence of divalent cations. Optimum models were selected with maximum likelihood criteria using single-channel data, then models were refined and extended by simultaneous fitting of macroscopic ionic currents, ON and OFF gating currents, and single-channel first latency densities over a wide voltage range. Best models have five closed states before channel opening, with inactivation from at least one closed state as well as the open state. Forward activation rate constants increase with depolarization, and deactivation rate constants increase with hyperpolarization. Rates of inactivation from the open or closed states are generally slower than activation or deactivation rates and show little or no voltage dependence. Channels tend to reopen several times before inactivating. Macroscopic rates of activation and inactivation result from a combination of closed, open and inactivated state transitions. At negative potentials the time to first opening dominates the macroscopic current due to slow activation rates compared with deactivation rates: channels tend to reopen rarely, and often inactivate from closed states before they reopen. At more positive potentials, the time to first opening and burst duration together produce the macroscopic current.     

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.     

Y1767C, a novel SCN5A mutation, induces a persistent Na+ current and potentiates ranolazine inhibition of Nav1.5 channels

Long QT syndrome type 3 (LQT3) has been traced to mutations of the cardiac Na(+) channel (Na(v)1.5) that produce persistent Na(+) currents leading to delayed ventricular repolarization and torsades de pointes. We performed mutational analyses of patients suffering from LQTS and characterized the biophysical properties of the mutations that we uncovered. One LQT3 patient carried a mutation in the SCN5A gene in which the cysteine was substituted for a highly conserved tyrosine (Y1767C) located near the cytoplasmic entrance of the Na(v)1.5 channel pore. The wild-type and mutant channels were transiently expressed in tsA201 cells, and Na(+) currents were recorded using the patch-clamp technique. The Y1767C channel produced a persistent Na(+) current, more rapid inactivation, faster recovery from inactivation, and an increased window current. The persistent Na(+) current of the Y1767C channel was blocked by ranolazine but not by many class I antiarrhythmic drugs. The incomplete inactivation, along with the persistent activation of Na(+) channels caused by an overlap of voltage-dependent activation and inactivation, known as window currents, appeared to contribute to the LQTS phenotype in this patient. The blocking effect of ranolazine on the persistent Na(+) current suggested that ranolazine may be an effective therapeutic treatment for patients with this mutation. Our data also revealed the unique role for the Y1767 residue in inactivating and forming the intracellular pore of the Na(v)1.5 channel.     

A negatively charged residue in the outer mouth of rat sodium channel determines the gating kinetics of the channel

Previous studies using combined techniques of site-directed mutagenesis and electrophysiology of voltage-gated Na(+) channels have demonstrated that there are significant overlaps in the regions that are important for the two fundamental properties of the channels, namely gating and permeation. We have previously shown that a pore-lining residue, W402 in S5-S6 region (P loop) in domain I of the micro1 skeletal muscle Na(+) channel, was important in the gating of the channel. Here, we determined the role of an adjacent pore-lining negatively charged residue (E403) in channel gating. Charge neutralization or substitution with positively charged side chain at this position resulted in a marked delay in the rate of recovery from slow inactivation. Indeed, the fast inactivation process appeared intact. Restoration of the negatively charged side chain with a sulfhydryl modifier, MTS-ethylsulfonate, resulted in a reactivation profile from a slow-inactivated state, which was indistinguishable from that of the wild-type channels. We propose an additional functional role for the negatively charged residue. Assuming no major changes in the pore structure induced by the mutations, the negatively charged residue E403 may work in concert with other pore regions during recovery from slow inactivation of the channel. Our data represent the first report indicating the role of negative charge in the slow inactivation of the voltage-gated Na(+) channel.     

Removal of the outermost arginine in IVS4 segment of the Ca(V)3.1 channel affects amplitude but not voltage dependence of gating current

Positively charged amino acids in S4 segments of voltage-dependent Ca(V)3.1 channel form putative voltage sensor. Previously we have shown that exchange of uppermost positively charged arginine in IVS4 segment for cysteine (mutation R1717C) affected deactivation and inactivation, but not activation of macroscopic current. Now we compared gating currents from both channels. Maximal amplitude of charge movement in R1717C channel decreased but voltage-dependent characteristics of charge movement were not significantly altered. We concluded that mutation of R1717C affects the coupling between S4 activation and pore opening, but not the S4 activation itself.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

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.     

Activating cystic fibrosis transmembrane conductance regulator channels with pore blocker analogs

Cystic fibrosis (CF) is caused by mutations that disrupt the surface localization and/or gating of the CF transmembrane conductance regulator (CFTR) chloride channel. The most common CF mutant is deltaF508-CFTR, which inefficiently traffics to the surfaces of most cells. The deltaF508 mutation may also disrupt the opening of CFTR channels once they reach the cell surface, but the extent of this gating defect is unclear. Here, we describe potent activators of wild-type and deltaF508-CFTR channels that are structurally related to 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a negatively charged pore blocker that we show to have mixed agonistic activity (channel activation plus voltage-dependent pore block). These CFTR agonists include 1) an uncharged NPPB analog that stimulates channel opening at submicromolar concentrations without blocking the pore and 2) curcumin, a dietary compound recently reported to augment deltaF508-CFTR function in mice by an unknown mechanism. The uncharged NPPB analog enhanced the activities of wild-type and deltaF508-CFTR channels both in excised membrane patches and in intact epithelial monolayers. This compound increased the open probabilities of deltaF508-CFTR channels in excised membrane patches by 10-15-fold under conditions in which wild-type channels were already maximally active. Our results support the emerging view that CFTR channel activity is substantially reduced by the deltaF508 mutation and that effective CF therapies may require the use of channel openers to activate mutant CFTR channels at the cell surface.     

Tuning of EAG K+ channel inactivation: Molecular determinants of amplification by mutations and a small molecule

Ether-à-go-go (EAG) and EAG-related gene (ERG) K(+) channels are close homologues but differ markedly in their gating properties. ERG1 channels are characterized by rapid and extensive C-type inactivation, whereas mammalian EAG1 channels were previously considered noninactivating. Here, we show that human EAG1 channels exhibit an intrinsic voltage-dependent slow inactivation that is markedly enhanced in rate and extent by 1-10 μM 3-nitro-N-(4-phenoxyphenyl) benzamide, or ICA105574 (ICA). This compound was previously reported to have the opposite effect on ERG1 channels, causing an increase in current magnitude by inhibition of C-type inactivation. The voltage dependence of 2 μM ICA-induced inhibition of EAG1 current was half-maximal at -73 mV, 62 mV negative to the half-point for channel activation. This finding suggests that current inhibition by the drug is mediated by enhanced inactivation and not open-channel block, where the voltage half-points for current inhibition and channel activation are predicted to overlap, as we demonstrate for clofilium and astemizole. The mutation Y464A in the S6 segment also induced inactivation of EAG1, with a time course and voltage dependence similar to that caused by 2 μM ICA. Several Markov models were investigated to describe gating effects induced by multiple concentrations of the drug and the Y464A mutation. Models with the smallest fit error required both closed- and open-state inactivation. Unlike typical C-type inactivation, the rate of Y464A- and ICA-induced inactivation was not decreased by external tetraethylammonium or elevated [K(+)](e). EAG1 channel inactivation introduced by Y464A was prevented by additional mutation of a nearby residue located in the S5 segment (F359A) or pore helix (L434A), suggesting a tripartite molecular model where interactions between single residues in S5, S6, and the pore helix modulate inactivation of EAG1 channels.     

A mutation causing pseudohypoaldosteronism type 1 identifies a conserved glycine that is involved in the gating of the epithelial sodium channel.

Pseudohypoaldosteronism type 1 (PHA-1) is an inherited disease characterized by severe neonatal salt-wasting and caused by mutations in subunits of the amiloride-sensitive epithelial sodium channel (ENaC). A missense mutation (G37S) of the human ENaC beta subunit that causes loss of ENaC function and PHA-1 replaces a glycine that is conserved in the N-terminus of all members of the ENaC gene family. We now report an investigation of the mechanism of channel inactivation by this mutation. Homologous mutations, introduced into alpha, beta or gamma subunits, all significantly reduce macroscopic sodium channel currents recorded in Xenopus laevis oocytes. Quantitative determination of the number of channel molecules present at the cell surface showed no significant differences in surface expression of mutant compared with wild-type channels. Single channel conductances and ion selectivities of the mutant channels were identical to that of wild-type. These results suggest that the decrease in macroscopic Na currents is due to a decrease in channel open probability (P(o)), suggesting that mutations of a conserved glycine in the N-terminus of ENaC subunits change ENaC channel gating, which would explain the disease pathophysiology. Single channel recordings of channels containing the mutant alpha subunit (alphaG95S) directly demonstrate a striking reduction in P(o). We propose that this mutation favors a gating mode characterized by short-open and long-closed times. We suggest that determination of the gating mode of ENaC is a key regulator of channel activity.     

Cross talk between activation and slow inactivation gates of Shaker potassium channels

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.     

The selectivity filter of the voltage-gated sodium channel is involved in channel activation

Amino acids located in the outer vestibule of the voltage-gated Na+ channel determine the permeation properties of the channel. Recently, residues lining the outer pore have also been implicated in channel gating. The domain (D) IV P-loop residue alanine 1529 forms a part of the putative selectivity filter of the adult rat skeletal muscle (mu1) Na+ channel. Here we report that replacement of alanine 1529 by aspartic acid enhances entry to an ultra-slow inactivated state. Ultra-slow inactivation is characterized by recovery time constants on the order of approximately 100 s from prolonged depolarizations and by the fact that entry to this state can be reduced by binding to the pore of a mutant mu-conotoxin GIIIA, suggesting that ultra-slow inactivation may reflect a structural rearrangement of the outer vestibule. The voltage dependence of ultra-slow inactivation in DIV-A1529D is U-shaped, with a local maximum near -60 mV, whereas activation is maximal only above -20 mV. Furthermore, a train of brief depolarizations produces more ultra-slow inactivation than a single maintained depolarization of the same duration. These data suggest that ultra-slow inactivation emanates from "partially activated" closed states and that the P-loop in DIV may undergo a conformational change during channel activation, which is accentuated by DIV-A1529D.     

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (Na_V) and K (K_V) channels. Na_V channels activate much faster and allow the initial Na⁺ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in K_V channels but an equally highly conserved threonine in Na_V channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker K_V channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of Na_V channels comes from a stronger dielectric polarization by threonine (Na_V channel) produced as the first gating charge enters the gating pore compared with isoleucine (K_V channel).     

Determinants of voltage-dependent gating and open-state stability in the S5 segment of Shaker potassium channels.

The best-known Shaker allele of Drosophila with a novel gating phenotype, Sh(5), differs from the wild-type potassium channel by a point mutation in the fifth membrane-spanning segment (S5) (Gautam, M., and M.A. Tanouye. 1990. Neuron. 5:67-73; Lichtinghagen, R., M. Stocker, R. Wittka, G. Boheim, W. Stühmer, A. Ferrus, and O. Pongs. 1990. EMBO [Eur. Mol. Biol. Organ.] J. 9:4399-4407) and causes a decrease in the apparent voltage dependence of opening. A kinetic study of Sh(5) revealed that changes in the deactivation rate could account for the altered gating behavior (Zagotta, W.N., and R.W. Aldrich. 1990. J. Neurosci. 10:1799-1810), but the presence of intact fast inactivation precluded observation of the closing kinetics and steady state activation. We studied the Sh(5) mutation (F401I) in ShB channels in which fast N-type inactivation was removed, directly confirming this conclusion. Replacement of other phenylalanines in S5 did not result in substantial alterations in voltage-dependent gating. At position 401, valine and alanine substitutions, like F401I, produce currents with decreased apparent voltage dependence of the open probability and of the deactivation rates, as well as accelerated kinetics of opening and closing. A leucine residue is the exception among aliphatic mutants, with the F401L channels having a steep voltage dependence of opening and slow closing kinetics. The analysis of sigmoidal delay in channel opening, and of gating current kinetics, indicates that wild-type and F401L mutant channels possess a form of cooperativity in the gating mechanism that the F401A channels lack. The wild-type and F401L channels' entering the open state gives rise to slow decay of the OFF gating current. In F401A, rapid gating charge return persists after channels open, confirming that this mutation disrupts stabilization of the open state. We present a kinetic model that can account for these properties by postulating that the four subunits independently undergo two sequential voltage-sensitive transitions each, followed by a final concerted opening step. These channels differ primarily in the final concerted transition, which is biased in favor of the open state in F401L and the wild type, and in the opposite direction in F401A. These results are consistent with an activation scheme whereby bulky aromatic or aliphatic side chains at position 401 in S5 cooperatively stabilize the open state, possibly by interacting with residues in other helices.     

Simulation of Na channel inactivation by thiazine dyes

Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.     

Pore structure influences gating properties of the T-type Ca2+ channel alpha1G

The selectivity filter of all known T-type Ca2+ channels is built by an arrangement of two glutamate and two aspartate residues, each one located in the P-loops of domains I-IV of the alpha1 subunit (EEDD locus). The mutations of the aspartate residues to glutamate induce changes in the conduction properties, enhance Cd2+ and proton affinities, and modify the activation curve of the channel. Here we further analyze the role of the selectivity filter in the gating mechanisms of T-type channels by comparing the kinetic properties of the alpha1G subunit (CaV3.1) to those of pore mutants containing aspartate-to-glutamate substitution in domains III (EEED) or IV (EEDE). The change of the extracellular pH induced similar effects on the activation properties of alpha1G and both pore mutants, indicating that the larger affinity of the mutant channels for protons is not the cause of the gating modifications. Both mutants showed alterations in several gating properties with respect to alpha1G, i.e., faster macroscopic inactivation in the voltage range from -10 to 50 mV, positive voltage shift and decrease in the voltage sensitivity of the time constants of activation and deactivation, decrease of the voltage sensitivity of the steady-state inactivation, and faster recovery from inactivation for long repolarization periods. Kinetic modeling suggests that aspartate-to-glutamate mutations in the EEDD locus of alpha1G modify the movement of the gating charges and alter the rate of several gating transitions. These changes are independent of the alterations of the selectivity properties and channel protonation.     

Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin

The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage- dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O- ->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.