Voltage-sensitive and solvent-sensitive processes in ion channel gating. Kinetic effects of hyperosmolar media on activation and deactivation of sodium channels.

Kinetic effects of osmotic stress on sodium ionic and gating currents have been studied in crayfish giant axons after removal of fast inactivation with chloramine-T. Internal perfusion with media made hyperosmolar by addition of formamide or sucrose, reduces peak sodium current (before and after removal of fast inactivation with chloramine-T), increases the half-time for activation, but has no effect on tail current deactivation rate(s). Kinetics of ON and OFF gating currents are not affected by osmotic stress. These results confirm (and extend to sodium channels) the separation of channel gating mechanisms into voltage-sensitive and solvent-sensitive processes recently proposed by Zimmerberg J., F. Bezanilla, and V. A. Parsegian. (1990. Biophys. J. 57:1049-1064) for potassium delayed rectifier channels. Additionally, the kinetic effects produced by hyperosmolar media seem qualitatively similar to the kinetic effects of heavy water substitution in crayfish axons (Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J. 57:745-758). However, our observations are incompatible with models in which voltage-sensitive and solvent-sensitive gating processes are presumed to be either (a) strictly sequential or, (b) parallel and independent. We introduce a variant of the parallel model which includes explicit coupling between voltage-sensitive and solvent-sensitive processes. Simulations of this model, in which the total coupling energy is as small as 1/10th of kT, demonstrate the characteristic kinetic changes noted in our data.     

Gating current harmonics. III. Dynamic transients and steady states with intact sodium inactivation gating.

Internally perfused squid giant axons with intact sodium inactivation gating were prepared for gating current experiments. Gating current records were obtained in sinusoidally driven dynamic steady states and as dynamic transients as functions of the mean membrane potential and the frequency of the command sinusoid. Controls were obtained after internal protease treatment of the axons that fully removed inactivation. The nonlinear analysis consisted of determining and interpreting the harmonic content in the current records. The results indicate the presence of three kinetic processes, two of which are associated with activation gating (the so-called primary and secondary processes), and the third with inactivation gating. The dynamic steady state data show that inactivation gating does not contribute a component to the gating current, and has no direct voltage-dependence of its own. Rather, the inactivation kinetics appear to be coupled to the primary activation kinetics, and the coupling mechanism appears to be one of reciprocal steric hindrance between two molecular components. The mechanism allows the channel to become inactivated without first entering the conducting state, and will do so in about 40 percent of depolarizing voltage-clamp steps to 0 mV. The derived model kinetics further indicate that the conducting state may flicker between open and closed with the lifetime of either state being 10 microseconds. Dynamic transients generated by the model kinetics (i.e., the behavior of the harmonic components as a function of time after an instantaneous change in the mean membrane potential from a holding potential of -80 mV) match the experimental dynamic transients in all details. These transients have a duration of 7-10 ms (depending on the level of depolarization), and are the result of the developing inactivation following the discontinuous voltage change. A detailed hypothetical molecular model of the channel and gating machinery is presented.     

Gating current kinetics in Myxicola giant axons. Order of the back transition rate constants.

Gating current, Ig, was recorded in Myxicola axons with series resistance compensation and higher time resolution than in previous studies. Ig at ON decays as two exponentials with time constants, tau ON-F and tau ON-S, very similar to squid values. No indication of an additional very fast relaxation was detected, but could be still unresolved. Ig at OFF also displays two exponentials, neither reflecting recovery from charge immobilization. Deactivation of the two I(ON) components may proceed with well-separated exponentials at -100 mV. INa tail currents at OFF also display two exponentials plus a third very slow relaxation of 5-9% of the total tail current. The very slow component is probably deactivation of a very small subpopulation of TTX sensitive channels. A -100 mV, means for INa tail component time constants (four axons) are 76 microseconds (range: 53-89 microseconds) and 344 microseconds (range: 312-387 microseconds), and for IOFF (six axons) 62 microseconds (range: 34-87 microseconds) and 291 microseconds (range: 204-456 microseconds) in reasonable agreement. INa ON activation time constant, tau A, is clearly slower than tau ON-F at all potentials. Except for the interval -30 to -15 mV, tau A is clearly faster than tau ON-S, and has a different dependency on potential. tau ON-S is several fold smaller than tau h. Computations with a closed2----closed1----open activation model indicated Na tail currents are consistent with a closed1----open rate constant greater than the closed2----closed1.     

Shaker potassium channel gating. II: Transitions in the activation pathway

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single- channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.     

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.     

Modifications of sodium channel gating in Myxicola giant axons by deuterium oxide, temperature, and internal cations.

In dialyzed Myxicola axons substitution of heavy water (D2O) externally and internally slows both sodium and potassium kinetics and decreases the maximum conductances. Furthermore, this effect is strongly temperature dependent, the magnitude of the slowing produced by D2O substitution decreasing with increasing temperature over the range 3-14 degrees C with a Q10 of approximately 0.71. The relatively small magnitude of the D2O effect, combined with its strong temperature dependence, suggests that the rate limiting process producing a conducting channel involves appreciable local changes in solvent structure. Maximum conductances in the presence of D2O were decreased by approximately 30%, while the voltage dependences of both gNa and gK were not appreciably changed. In contrast to the effects of heavy water substitution on the ionic currents, membrane asymmetry currents were not altered by D2O, suggesting that gating charge movement may preceed by several steps the final transformation of the Na+ channel to a conducting state. In Myxicola axons the effect of temperature alone on asymmetry current kinetics can be well described via a simple temporal expansion equivalent to a Q10 of 2.2, which is somewhat less than the Q10 of GNa activation. The integral of membrane asymmetry current, representing maximum charge movement, is however not appreciably altered by temperature.     

Gating current "fractionation" in crayfish giant axons.

Effects of changes in initial conditions on the magnitude and kinetics of gating current and sodium current were studied in voltage-clamped, internally-perfused, crayfish giant axons. We examined the effects of changes in holding potential, inactivating prepulses, and recovery from inactivation in axons with intact fast inactivation. We also studied the effects of brief interpulse intervals in axons pretreated with chloramine-T for removal of fast inactivation. We find marked effects of gating current kinetics induced by both prepulse inactivation and brief interpulse intervals. The apparent changes in gating current relaxation rates cannot be explained simply by changes in gating charge magnitude (charge immobilization) combined with "Cole-Moore-type" time shifts. Rather they appear to indicate selective suppression of kinetically-identifiable components within the control gating currents. Our results provide additional support for a model involving parallel, nonidentical, gating particles.     

FPL 64176 modification of Ca(V)1.2 L-type calcium channels: dissociation of effects on ionic current and gating current

FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit Ca(V)1.2 (alpha1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with beta1a and alpha2delta1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced approximately 5-fold for a voltage step from -90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (< -250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions.     

Voltage-gated transient currents in bovine adrenal fasciculata cells. I. T-type Ca2+ current

The whole cell version of the patch clamp technique was used to identify and characterize voltage-gated Ca2+ channels in enzymatically dissociated bovine adrenal zona fasciculata (AZF) cells. The great majority of cells (84 of 86) expressed only low voltage-activated, rapidly inactivating Ca2+ current with properties of T-type Ca2+ current described in other cells. Voltage-dependent activation of this current was fit by a Boltzmann function raised to an integer power of 4 with a midpoint at -17 mV. Independent estimates of the single channel gating charge obtained from the activation curve and using the "limiting logarithmic potential sensitivity" were 8.1 and 6.8 elementary charges, respectively. Inactivation was a steep function of voltage with a v1/2 of -49.9 mV and a slope factor K of 3.73 mV. The expression of a single Ca2+ channel subtype by AZF cells allowed the voltage-dependent gating and kinetic properties of T current to be studied over a wide range of potentials. Analysis of the gating kinetics of this Ca2+ current indicate that T channel activation, inactivation, deactivation (closing), and reactivation (recovery from inactivation) each include voltage-independent transitions that become rate limiting at extreme voltages. Ca2+ current activated with voltage- dependent sigmoidal kinetics that were described by an m4 model. The activation time constant varied exponentially at test potentials between -30 and +10 mV, approaching a voltage-independent minimum of 1.6 ms. The inactivation time constant (tau i) also decreased exponentially to a minimum of 18.3 ms at potentials positive to 0 mV. T channel closing (deactivation) was faster at more negative voltages; the deactivation time constant (tau d) decreased from 8.14 +/- 0.7 to 0.48 +/- 0.1 ms at potentials between -40 and -150 mV. T channels inactivated by depolarization returned to the closed state along pathways that included two voltage-dependent time constants. tau rec-s ranged from 8.11 to 4.80 s when the recovery potential was varied from - 50 to -90 mV, while tau rec-f decreased from 1.01 to 0.372 s. At potentials negative to -70 mV, both time constants approached minimum values. The low voltage-activated Ca2+ current in AZF cells was blocked by the T channel selective antagonist Ni2+ with an IC50 of 20 microM. At similar concentrations, Ni2+ also blocked cortisol secretion stimulated by adrenocorticotropic hormone. Our results indicate that bovine AZF cells are distinctive among secretory cells in expressing primarily or exclusively T-type Ca2+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2Δ2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2Δ2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.     

Fluctuations in ion channel gating currents. Analysis of nonstationary shot noise.

Conti and Stühmer (1989. Eur. Biophys. J. 17:53-59) have measured the nonstationary shot noise in the gating current of a population of sodium channels. Here we present expressions for the autocovariance and variance of such noise from general Markov models of channel kinetics, based on the theoretical work of E. Frehland. We compare the predictions of the independent, two-state gating model used by Conti and Stühmer with a six-state model of sodium channel activation based on the work of Armstrong and Gilly (1979. J. Gen. Physiol. 74:691-711). We find that Conti and Stühmer's experiment would not be able to distinguish between these schemes. We describe experimental conditions under which better model discrimination would be possible.     

Slowing of sodium channel opening kinetics in squid axon by extracellular zinc

The interaction of Zn ion on Na channels was studied in squid giant axons. At a concentration of 30 mM Zn2+ slows opening kinetics of Na channels with almost no alteration of closing kinetics. The effects of Zn2+ can be expressed as a "shift" of the gating parameters along the voltage axis, i.e., the amount of additional depolarization required to overcome the Zn2+ effect. In these terms the mean shifts caused by 30 mM Zn2+ were +29.5 mV for Na channel opening (on) kinetics (t1/2 on), +2 mV for closing (off) kinetics (tau off), and +8.4 mV for the gNa-V curve. Zn2+ does not change the shape of the instantaneous I-V curve for inward current, but reduces it in amplitude by a factor of or approximately 0.67. Outward current is unaffected. Effects of Zn2+ on gating current (measured in the absence of TTX) closely parallel its actions on gNa. On gating current kinetics are shifted by +27.5 mV, off kinetics by +6 mV, and the Q-V distribution by +6.5 mV. Kinetic modeling shows that Zn2+ slows the forward rate constants in activation without affecting backward rate constants. More than one of the several steps in activation must be affected. The results are not compatible with the usual simple theory of uniform fixed surface charge. They suggest instead that Zn2+ is attracted by a negatively charged element of the gating apparatus that is present at the outer membrane surface at rest, and migrates inward on activation.     

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

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

Proton and zinc effects on HERG currents.

The proton and Zn2+ effects on the human ether-a-go-go related gene (HERG) channels were studied after expression in Xenopus oocytes and stable transfection in the mammalian L929 cell line. Experiments were carried out using the two-electrode voltage clamp at room temperature (oocytes) or the whole-cell patch clamp technique at 35 degrees C (L929 cells). In oocytes, during moderate extracellular acidification (pHo = 6.4), current activation was not shifted on the voltage axis, the time course of current activation was unchanged, but tail current deactivation was dramatically accelerated. At pHo < 6.4, in addition to accelerating deactivation, the time course of activation was slower and the midpoint voltage of current activation was shifted to more positive values. Protons and Zn2+ accelerated the kinetics of deactivation with apparent Kd values about one order of magnitude lower than for tail current inhibition. For protons, the Kd values for the effect on tail current amplitude versus kinetics were, respectively, 1.8 microM (pKa = 5.8) and 0.1 microM (pKa = 7.0). In the presence of Zn2+, the corresponding Kd values were, respectively, 1.2 mM and 169 microM. In L929 cells, acidification to pHo = 6.4 did not shift the midpoint voltage of current activation and had no effect on the time course of current activation. Furthermore, the onset and recovery of inactivation were not affected. However, the acidification significantly accelerated tail current deactivation. We conclude that protons and Zn2+ directly interact with HERG channels and that the interaction results, preferentially, in the regulation of channel deactivation mechanism.     

Gating current harmonics. IV. Dynamic properties of secondary activation kinetics in sodium channel gating.

The kinetics for sodium channel gating appear to involve three coupled processes: (a) "primary" activation, (b) "secondary" activation, and (c) inactivation. Gating current data obtained in dynamic steady states with sinusoidal voltage-clamp were analyzed to give further details about the secondary activation process in sodium channel gating. Unlike primary activation and inactivation, the secondary activation kinetics involve physical processes that become defined when the data are analyzed as a function of the sinusoid frequency in addition to mean membrane potential. The effects of these processes are described, and a physical interpretation is presented.     

Sodium and gating current time shifts resulting from changes in initial conditions

The sodium and gating currents of the squid giant axon elicited by a depolarizing pulse are delayed, with little change in shape, as a result of a hyperpolarizing prepulse. The delays are almost completely saturated, at approximately 45 microseconds, for prepulses to -140 mV. At 8 degrees C they develop with time constants of between 60 and 180 microseconds for prepulses in the -130- to -150-mV range. There is a correlation between the extra charge moved during the gating current and the increase in the time delay of the sodium current as the magnitude of the hyperpolarizing prepulse is increased. These results strengthen the conclusion that the gating current is indeed closely associated with the process of sodium channel opening and provide information concerning the kinetics of the early steps, which are hidden in ionic current measurements. The main features of the gating and sodium current time shifts and the correlation between charge movement and time shifts are duplicated by a sequential six-state model for sodium activation.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

Some kinetic and steady-state properties of sodium channels after removal of inactivation

To study the kinetic and steady-state properties of voltage-dependent sodium conductance activation, squid giant axons were perfused internally with either pronase or N-bromoacetamide and voltage clamped. Parameters of activation, tau m and gNa(V), and deactivation, tau Na, were measured and compared with those obtained from control axons under the assumption that gNa oc m3h of the Hodgkin-Huxley scheme. tau m(V) values obtained from the turn-on of INa agree well with control axons and previous determinations by others. tau Na(V) values derived from Na tail currents were also unchanged by pronase treatment and matched fairly well previously published values. tau m(V) obtained from 3 x tau Na(V) were much larger than tau m(V) obtained from INa turn-on at the same potentials, resulting in a discontinuous distribution. Steady-state In (gNa/gNa max - gNa) vs. voltage was not linear and had a limiting logarithmic slope of 5.3 mV/e-fold gNa. Voltage step procedures that induce a second turn-on of INa during various stages of the deactivation (Na tail current) process reveal quasiexponential activation at early stages that becomes increasingly sigmoid as deactivation progresses. For moderate depolarizations, primary and secondary activation kinetics are superimposable. These data suggest that, although m3 can describe the shape of INa turn-on, it cannot quantitatively account for the kinetics of gNa after repolarization. Kinetic schemes for gNa in which substantial deactivation occurs by a unique pathway between conducting and resting states are shown to be unlikely. It appears that the rate-limiting step in linear kinetic models of activation may be between a terminal conducting state and the adjacent nonconducting intermediate.     

High intracellular pH reversibly prevents gating-charge immobilization in squid axons

Squid giant axons were used to study the reversible effects of high intracellular pH (pHi) on gating currents. Under depolarization, when Na channels are activated, internal solutions buffered at high pHi (10.2) affect considerably the time course of gating charge associated with channel closing, QOFF, with almost no alteration of QON records. In particular, at pHi 10.2 the charge corresponding to the fast phase of IgOFF, measured after long depolarizing pulses (7.7 ms), was consistently larger than that recorded at physiological pHi (7.2). This suggests that high pH prevents immobilization of gating charges induced by Na inactivation. In this respect, the present data agree reasonably well with previous observations, which show that pHi greater than 7.2 reversibly removes the fast Na inactivation with little effects on activation kinetics (Carbone, E., P. L. Testa, and E. Wanke, 1981, Biophys. J., 35:393-413; Brodwick, M.S., and D. C. Eaton, 1978, Science [Wash. DC], 200:1494-1496). Unexpectedly, high pH increases the amount of charge associated with the slow phase of IgOFF. In our opinion, this might be the result of either an increment of the net charge produced by the exposure to high pHi or that gating charges that return to the closed state might experience a larger fraction of the potential drop across the membrane (Neumcke, B., W. Schwarz, and R. Stampfli, 1980, Biophys. J., 31:325-332).