Modification of sodium and gating currents by amino group specific cross-linking and monofunctional reagents.

To test the possible role of lysine residues in Na channel function the effects of several imidoesters on Na and gating currents were studied in voltage-clamped single frog nerve fibers. Mono- and bisimidoesters were used. These reagents modify amino groups exclusively and do not change the net charge. The three bisimidoesters used easily introduce cross-links between neighboring amino groups. Their structure is almost identical; only the length of the spacers between the two amino-reactive groups is different. An irreversible reduction of Na currents and gating currents was observed with the longest (dimethyl suberimidate [DMS]) and the shortest (dimethyl adipimidate [DMA]) of the cross-linkers used. Of the three cross-linking reagents only the shortest made Na current inactivation slow and incomplete. The steady-state inactivation curve, h infinity (E), was shifted by greater than 25 mV in the hyperpolarizing direction by each of the reagents. The voltage dependence of activation, however, remained unchanged. Furthermore, the effects of two different monoimidoesters (ethyl acetimidate [EAI] and isethionyl acetimidate [IAI]) on gating currents were tested. EAI can penetrate a membrane, whereas IAI is membrane impermeant. IAI was almost without effect, whereas EAI caused a considerable reduction of the gating currents. EAI and DMS reduced the Qoff/Qon ratio without affecting the decay of the Na currents. The results show that lysine residues are critically involved in Na channel gating.     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Destruction of Sodium Conductance Inactivation in Squid Axons Perfused with Pronase

We have studied the effects of the proteolytic enzyme Pronase on the membrane currents of voltage-clamped squid axons. Internal perfusion of the axons with Pronase rather selectively destroys inactivation of the Na conductance (g_Na). At the level of a single channel, Pronase probably acts in an all-or-none manner: each channel inactivates normally until its inactivation gate is destroyed, and then it no longer inactivates. Pronase reduces _Na, possibly by destroying some of the channels, but after removal of its inactivation gate a Na channel seems no longer vulnerable to Pronase. The turn-off kinetics and the voltage dependence of the Na channel activation gates are not affected by Pronase, and it is probable that the enzyme does not affect these gates in any way. Neither the K channels nor their activation gates are affected in a specific way by Pronase. Tetrodotoxin does not protect the inactivation gates from Pronase, nor does maintained inactivation of the Na channels during exposure to Pronase. Our results suggest that the inactivation gate is a readily accessible protein attached to the inner end of each Na channel. It is shown clearly that activation and inactivation of Na channels are separable processes, and that Na channels are distinct from K channels.     

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.     

Site-directed sulfhydryl labeling of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae: helix VIII comprises a portion of the sodium ion channel

Helix VIII of the beta-subunit of the oxaloacetate decarboxylase of Klebsiella pneumoniae contains the functionally important residues betaN373, betaG377, betaS382, and betaR389. Using a functional oxaloacetate decarboxylase mutant devoid of Cys residues in the beta-subunit, each amino acid residue in helix VIII was replaced individually with Cys. Structural and dynamic features of this region were studied by using site-directed sulfhydryl modification of 20 single-Cys replacement mutants with methanethiosulfonate (MTS) reagents in the absence or presence of Na(+) ions. The pattern of accessibility of the MTS reagents from the periplasmic side of helix VIII shows a periodicity which suggests that this region is alpha-helical. In particular, a water-accessible face comprising betaN373, betaG377, betaS382, betaM386, and betaV390 may be part of a Na(+) channel. Cys residues introduced in the cytoplasmically oriented part of helix VIII were accessible to three different water-soluble MTS compounds and therefore believed to be exposed to water on this side of the membrane. Most residues located in the upper part of helix VIII (residues betaN373-betaV381C) were protected by Na(+) ions for inactivation by the MTS reagents. The distinct results on accessibility toward the different MTS reagents obtained in the presence or absence of Na(+) ions may suggest a conformational change upon binding of Na(+) in this region. The betaR389C mutant had a reduced activity and a pH optimum at pH 9, which could be restored to a wild-type pH optimum of 6.5 and to a 400% gain in activity upon chemical modification with 2-aminoethyl methanethiosulfonate.     

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na(+) channels

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na(+) channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na(+) current (I(Na)). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I(Na). Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4min), there is little effect on I(Na). However, with longer exposures (4-8min), there is a large decrease in I(Na), but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) - MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na(+) channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

Phosphorylation of S1505 in the cardiac Na+ channel inactivation gate is required for modulation by protein kinase C

Inactivation of both brain and cardiac Na+ channels is modulated by activation of protein kinase C (PKC) but in different ways. Previous experiments had shown that phosphorylation of serine 1506 in the highly conserved loop connecting homologous domains III and IV (LIII/IV) of the brain Na+ channel alpha subunit is necessary for all effects of PKC. Here we examine the importance of the analogous serine for the different modulation of the rH1 cardiac Na+ channel. Serine 1505 of rH1 was mutated to alanine to prevent its phosphorylation, and the resulting mutant channel was expressed in 1610 cells. Electrophysiological properties of these mutant channels were indistinguishable from those of wild-type (WT) rH1 channels. Activation of PKC with 1-oleoyl-2-acetyl-sn-glycerol (OAG) reduced WT Na+ current by 49.3 +/- 4.2% (P < 0.01) but S1505A mutant current was reduced by only 8.5 +/- 5.4% (P = 0.29) when the holding potential was -94 mV. PKC activation also caused a -17-mV shift in the voltage dependence of steady-state inactivation of the WT channel which was abolished in the mutant. Thus, phosphorylation of serine 1505 is required for both the negative shift in the inactivation curve and the reduction in Na+ current by PKC. Phosphorylation of S1505/1506 has common and divergent effects in brain and cardiac Na+ channels. In both brain and cardiac Na+ channels, phosphorylation of this site by PKC is required for reduction of peak Na+ current. However, phosphorylation of S1506 in brain Na+ channels slows and destabilizes inactivation of the open channel. Phosphorylation of S1505 in cardiac, but not S1506 in brain, Na+ channels causes a negative shift in the inactivation curve, indicating that it stabilizes inactivation from closed states. Since LIII/IV containing S1505/S1506 is completely conserved, interaction of the phosphorylated serine with other regions of the channel must differ in the two channel types.     

Interactions of monovalent cations with sodium channels in squid axon. I. Modification of physiological inactivation gating

Inactivation of Na channels has been studied in voltage-clamped, internally perfused squid giant axons during changes in the ionic composition of the intracellular solution. Peak Na currents are reduced when tetramethylammonium ions (TMA+) are substituted for Cs ions internally. The reduction reflects a rapid, voltage-dependent block of a site in the channel by TMA+. The estimated fractional electrical distance for the site is 10% of the channel length from the internal surface. Na tail currents are slowed by TMA+ and exhibit kinetics similar to those seen during certain drug treatments. Steady state INa is simultaneously increased by TMA+, resulting in a "cross-over" of current traces with those in Cs+ and in greatly diminished inactivation at positive membrane potentials. Despite the effect on steady state inactivation, the time constants for entry into and exit from the inactivated state are not significantly different in TMA+ and Cs+. Increasing intracellular Na also reduces steady state inactivation in a dose-dependent manner. Ratios of steady state INa to peak INa vary from approximately 0.14 in Cs+- or K+-perfused axons to approximately 0.4 in TMA+- or Na+-perfused axons. These results are consistent with a scheme in which TMA+ or Na+ can interact with a binding site near the inner channel surface that may also be a binding or coordinating site for a natural inactivation particle. A simple competition between the ions and an inactivation particle is, however, not sufficient to account for the increase in steady state INa, and changes in the inactivation process itself must accompany the interaction of TMA+ and Na+ with the channel.     

N-type inactivation and the S4-S5 region of the Shaker K+ channel

The intracellular segment of the Shaker K+ channel between transmembrane domains S4 and S5 has been proposed to form at least part of the receptor for the tethered N-type inactivation "ball." We used the approach of cysteine substitution mutagenesis and chemical modification to test the importance of this region in N-type inactivation. We studied N-type inactivation or the block by a soluble inactivation peptide ("ball peptide") before and after chemical modification by methanethiosulfonate reagents. Particularly at position 391, chemical modification altered specifically the kinetics of ball peptide binding without altering other biophysical properties of the channel. Results with reagents that attach different charged groups at 391 C suggested that there are both electrostatic and steric interactions between this site and the ball peptide. These findings identify this site to be in or near the receptor site for the inactivation ball. At many of the other positions studied, modification noticeably inhibited channel current. The accessible cysteines varied in the state-dependence of their modification, with five- to tenfold changes in reactions rate depending on the gating state of the channel.     

Functional role of the N-terminus of Na(+),K(+)-ATPase alpha-subunit as an inactivation gate of palytoxin-induced pump channel

The N-terminus of the Na(+),K(+)-ATPase alpha-subunit shows some homology to that of Shaker-B K(+) channels; the latter has been shown to mediate the N-type channel inactivation in a ball-and-chain mechanism. When the Torpedo Na(+),K(+)-ATPase is expressed in Xenopus oocytes and the pump is transformed into an ion channel with palytoxin (PTX), the channel exhibits a time-dependent inactivation gating at positive potentials. The inactivation gating is eliminated when the N-terminus is truncated by deleting the first 35 amino acids after the initial methionine. The inactivation gating is restored when a synthetic N-terminal peptide is applied to the truncated pumps at the intracellular surface. Truncated pumps generate no electrogenic current and exhibit an altered stoichiometry for active transport. Thus, the N-terminus of the alpha-subunit appears to act like an inactivation gate and performs a critical step in the Na(+),K(+)-ATPase pumping function.     

The extracellular domain of the beta1 subunit is both necessary and sufficient for beta1-like modulation of sodium channel gating.

The type IIA voltage-gated sodium Na(+) channel from rat brain is composed of a large, pore-forming alpha subunit and the auxiliary subunits beta1 and beta2. When expressed in Xenopus oocytes, the beta1 subunit modulates the gating properties of the type IIA alpha subunit, resulting in acceleration of both inactivation and recovery from inactivation and in a negative shift in the voltage dependence of fast inactivation. The beta1 subunit is composed of an extracellular domain with a single immunoglobulin-like fold, a single transmembrane segment, and a small intracellular domain. A series of chimeras with exchanges of domains between the Na(+) channel beta1 and beta2 subunits and between beta1 and the structurally related protein myelin P0 were constructed and analyzed by two-microelectrode voltage clamp in Xenopus oocytes. Only chimeras containing the beta1 extracellular domain were capable of beta1-like modulation of Na(+) channel gating. Neither the transmembrane segment nor the intracellular domain was required for modulation, although mutation of Glu(158) within the transmembrane domain altered the voltage dependence of steady-state inactivation. A truncated beta1 subunit was engineered in which the beta1 extracellular domain was fused to a recognition sequence for attachment of a glycosylphosphatidylinositol membrane anchor. The beta1(ec)-glycosylphosphatidylinositol protein fully reproduced modulation of Na(+) channel inactivation and recovery from inactivation by wild-type beta1. Our findings demonstrate that extracellular domain of the beta1 subunit is both necessary and sufficient for the modulation of Na(+) channel gating.     

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.     

The non-steroidal anti-inflammatory drug, diclofenac, inhibits Na(+) current in rat myoblasts

The inhibitory effect of diclofenac, a non-steroidal anti-inflammatory drug (NSAID), on the voltage-gated inward Na+ current (I(Na)) in cultured rat myoblasts was investigated using the whole-cell voltage-clamp technique. At concentrations of 10 nM-100 microM, diclofenac produced a dose-dependent and reversible inhibition of I(Na) with an IC50 of 8.51 microM, without modulating the fast activation and inactivation process. The inhibitory effect of diclofenac took place at resting channels and increased with more depolarizing holding potential. In addition to inhibiting the Na+ current amplitude, diclofenac significantly modulated the steady-state inactivation properties of the Na+ channels, but did not alter the steady-state activation. The steady-state inactivation curve was significantly shifted towards the hyperpolarizing potential in the presence of diclofenac. Furthermore, diclofenac treatment resulted in a fairly slow recovery from inactivation of the Na+ channel. The inhibitory effect of diclofenac was enhanced by repetitive pulses and was inflected by changing frequency; the blocking effect at higher frequency was significantly greater than at lower frequency. Both intracellular and extracellular application of diclofenac could inhibit I(Na), indicating that diclofenac may exert its channel inhibitory action both inside and outside the channel sites. Our data directly demonstrate that diclofenac can inhibit the inward Na+ channels in rat myoblasts. Some different inhibitory mechanisms from that in neuronal Na+ channels are discussed.     

A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.     

Interaction of sulfhydryl reagents with A-type channels of Lymnaea neurons.

The effect of sulfhydryl reagents on macroscopic inactivation of A-current in internally perfused Lymnaea neurons under voltage-clamp conditions was investigated. It was found that the binding of Hg2+ rather than PHMB with channel proteins resulted in a strong decrease of the peak current and the inactivation rate. Hg2+ markedly influenced the steady-state inactivation but did not change the rate of recovery from inactivation. It was found that both reagents reacted with the same groups of the channel protein and that those are most likely sulfhydryl groups. These groups seemed not to be involved in the gating charge movement. Hg2+ ions can immobilize some part of the gating charge thereby resulting in strong changes of the steady-state inactivation.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Slow inactivation differs among mutant Na channels associated with myotonia and periodic paralysis.

Several heritable forms of myotonia and hyperkalemic periodic paralysis (HyperPP) are caused by missense mutations in the alpha subunit of the skeletal muscle Na channel (SkM1). These mutations impair fast inactivation or shift activation toward hyperpolarized potentials, inducing persistent Na currents that may cause muscle depolarization, myotonia, and onset of weakness. It has been proposed that the aberrant Na current and resulting weakness will be sustained only if Na channel slow inactivation is also impaired. We therefore measured slow inactivation for wild-type and five mutant Na channels constructed in the rat skeletal muscle isoform (rSkM1) and expressed in HEK cells. Two common HyperPP mutations (T698M in domain II-S5 and M1585V in IV-S6) had defective slow inactivation. This defect reduced use-dependent inhibition of Na currents elicited during 50-Hz stimulation. A rare HyperPP mutation (M1353V in IV-S1) and mutations within the domain III-IV linker that cause myotonia (G1299E) or myotonia plus weakness (T1306M) did not impair slow inactivation. We also observed that slow inactivation of wild-type rSkM1 was incomplete; therefore it is possible that stable membrane depolarization and subsequent muscle weakness may be caused solely by defects in fast inactivation or activation. Model simulations showed that abnormal slow inactivation, although not required for expression of a paralytic phenotype, may accentuate muscle membrane depolarization, paralysis, and sensitivity to hyperkalemia.     

Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line.

Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters. Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded. The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons. The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd. Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function.     

NH2-terminal inactivation peptide binding to C-type-inactivated Kv channels

In many voltage-gated K(+) channels, N-type inactivation significantly accelerates the onset of C-type inactivation, but effects on recovery from inactivation are small or absent. We have exploited the Na(+) permeability of C-type-inactivated K(+) channels to characterize a strong interaction between the inactivation peptide of Kv1.4 and the C-type-inactivated state of Kv1.4 and Kv1.5. The presence of the Kv1.4 inactivation peptide results in a slower decay of the Na(+) tail currents normally observed through C-type-inactivated channels, an effective blockade of the peak Na(+) tail current, and also a delay of the peak tail current. These effects are mimicked by addition of quaternary ammonium ions to the pipette-filling solution. These observations support a common mechanism of action of the inactivation peptide and intracellular quaternary ammonium ions, and also demonstrate that the Kv channel inner vestibule is cytosolically exposed before and after the onset of C-type inactivation. We have also examined the process of N-type inactivation under conditions where C-type inactivation is removed, to compare the interaction of the inactivation peptide with open and C-type-inactivated channels. In C-type-deficient forms of Kv1.4 or Kv1.5 channels, the Kv1.4 inactivation ball behaves like an open channel blocker, and the resultant slowing of deactivation tail currents is considerably weaker than observed in C-type-inactivated channels. We present a kinetic model that duplicates the effects of the inactivation peptide on the slow Na(+) tail of C-type-inactivated channels. Stable binding between the inactivation peptide and the C-type-inactivated state results in slower current decay, and a reduction of the Na(+) tail current magnitude, due to slower transition of channels through the Na(+)-permeable states traversed during recovery from inactivation.