©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Critical Role for Transmembrane Segment IVS6 of the Sodium Channel Subunit in Fast Inactivation (*)

Jancy C. McPhee , David S. Ragsdale , Todd Scheuer , William A. Catterall

From the (1) Department of Pharmacology SJ-30, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Fast Na channel inactivation is thought to occur by the binding of an intracellular inactivation gate to regions around or within the Na channel pore through hydrophobic interactions. Previous studies indicate that the intracellular loop between domains III and IV of the Na channel subunit (L) forms the inactivation gate. A three-residue hydrophobic motif (IFM) is an essential structural feature of the gate and may serve as an inactivation particle that binds within the pore. In this study, we used alanine-scanning mutagenesis to examine the functional role of amino acid residues in transmembrane segment IVS6 of the Na channel subunit in fast inactivation. Mutant F1764A, in the center of IVS6, and mutant V1774A, near its intracellular end, exhibited substantial sustained Na currents at the end of 30-ms depolarizations. The double mutation F1764A/V1774A almost completely abolished fast inactivation, demonstrating a critical role for these amino acid residues in the process of inactivation. Single channel analysis of these three mutants revealed continued reopenings late in 40-ms depolarizing pulses, indicating that the stability of the inactivated state was substantially impaired compared with wild type. In addition, the cumulative first latency distribution for the V1774A mutation contained a new component arising from opening transitions from the destabilized inactivated state. Substitution of multiple amino acid residues showed that the disruption of inactivation was not correlated with the hydrophobicity of the substitution at position 1774, in contrast to the expectation if this residue interacts directly with the IFM motif. Thermodynamic cycle analysis of simultaneous mutations in the IFM motif and in IVS6 suggested that mutations in these two regions independently disrupt inactivation, consistent with the conclusion that they do not interact directly. Furthermore, a peptide containing the IFM motif (acetyl-KIFMK-amide) restored inactivation to the F1764A/V1774A IVS6 mutant, indicating that the binding site for the IFM motif remains intact in these mutants. These results suggest that the amino acid residues 1764 and 1774 in IVS6 do not directly interact with the IFM motif of the inactivation gate but instead play a novel role in fast inactivation of the Na channel.


INTRODUCTION

Ionic current through voltage-dependent Na channels initiates the action potential in neurons and other excitable cells. Upon membrane depolarization, Na channels undergo conformational changes through a series of nonconducting closed states to a conducting open state and then to a nonconducting, inactivated state within 1 or 2 ms. Upon membrane repolarization, Na channels recover from fast inactivation within a few milliseconds and are again available for activation. Understanding the molecular basis of fast inactivation gating is an important step in defining the mechanisms of Na channel function.

The rat brain Na channel is a heterotrimeric protein consisting of (260-kDa), 1 (36-kDa), and 2 (33-kDa) subunits (reviewed in Catterall(1992)). The subunit, a 2005-amino acid glycoprotein, contains four homologous domains, each with six probable -helical transmembrane segments (S1-S6; Noda et al., 1986b; Auld et al., 1988). The 1 subunit, a 218-amino acid glycoprotein, has a single transmembrane segment (Isom et al., 1992). Only the cloned subunit is required for the expression of functional Na channels in Xenopus laevis oocytes (Noda et al., 1986a; Goldin et al., 1986). Co-expression of the and 1 subunits, however, is required for the expression of Na channels displaying normal time course and voltage dependence of inactivation (Isom et al., 1992).

Treatment of the intracellular surface of Na and K channels with proteolytic enzymes specifically disrupts fast inactivation, implicating peptide segment(s) on the cytoplasmic surface of these channels in the inactivation process (Armstrong et al., 1973; Armstrong, 1981; Hoshi et al., 1991). One current hypothesis for the mechanism of fast inactivation is that a tethered inactivation gate, composed of these cytoplasmic peptide segments, binds to a region within the pore during fast inactivation, blocking ion conductance and immobilizing gating charge (Armstrong and Bezanilla, 1977; Armstrong, 1981; Hoshi et al., 1991; Zagotta et al., 1990). Studies using site-directed antibodies and site-directed mutagenesis indicate that the intracellular loop between homologous domains III and IV of the subunit (L) is involved in inactivation (Vassilev et al., 1988; 1989; Stühmer et al., 1989). Mutation of three hydrophobic amino acids, Ile, Phe, and Met (IFM), within this loop to glutamine disables fast inactivation (West et al., 1992a). In contrast, mutations of charged amino acid residues elsewhere in this loop do not disrupt inactivation (Moorman et al., 1990; Patton et al., 1992). Small peptides containing the IFM sequence are sufficient to restore fast inactivation to Na channels with mutations in L, demonstrating that the IFM motif has affinity for a region within the Na channel pore (Eaholtz et al., 1994). These results have led to the proposal that Na channel inactivation occurs when L, serving as an inactivation gate, closes over the intracellular mouth of the pore and binds to other regions of the Na channel. The IFM residues are thought to act as a hydrophobic molecular latch that holds the gate shut by forming a hydrophobic interaction with residues within or near the intracellular mouth of the pore (West et al., 1992a; Scheuer et al., 1993; Eaholtz et al., 1994).

The actions of local anesthetics and functionally related drugs on Na channels are similar to the actions of the fast inactivation gate in that they act from the cytoplasmic side of the Na channel to inhibit ionic current and immobilize gating charge (Cahalan and Almers, 1979; Hille, 1992). Some local anesthetics, like lidocaine, stabilize the inactivated state (Hille, 1977; Bean et al., 1983), whereas other compounds, like N-methylstrychnine, prevent inactivation (Cahalan and Almers, 1979). These observations suggest that local anesthetics and related drugs interact with regions of the Na channel that are important for inactivation. We have recently identified amino acid residues in the S6 transmembrane segment of domain IV of the Na channel subunit (segment IVS6) that are critical determinants of local anesthetic action (Ragsdale et al., 1994), and we have shown that mutation of a cluster of three residues near the cytoplasmic end of IVS6 strongly disrupts fast inactivation (McPhee et al., 1994). These results indicate that segment IVS6 is important for both drug action and fast Na channel inactivation. In the present work, we have undertaken a systematic analysis of the role of segment IVS6 in inactivation by alanine-scanning site-directed mutagenesis and identified individual amino acid residues that are important for inactivation.


EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis

Mutants were produced in a 1719-base pair DNA fragment excised from the rat brain type IIA Na channel subunit cDNA (Auld et al., 1988; Auld et al., 1990) and inserted into M13 mp18. A single-stranded, uridine-containing form of the template was isolated from dut ungE.coli cells. Oligonucleotides containing the mutation to be introduced were annealed to the template and used to direct the transcription of the complementary strand containing thymidine in vitro. After transforming normal bacteria with this heterodimeric, double-stranded template, only the mutant, thymidine-containing strand was replicated, whereas the wild type (WT),() uridine-containing strand was not. The double-stranded mutant DNA was isolated and sequenced to confirm the identity of the mutation. The full-length, mutant Na channel was produced by subcloning the mutant cassette into either vector ZEMRVSP6-2580 (West et al., 1992b) or vector pVA2580 (Auld et al., 1988) containing the remainder of the Na channel and a bacteriophage RNA polymerase promoter (Kunkel, 1985; West et al., 1991; West et al., 1992b).

NaChannel Expression

RNA encoding WT and mutant sodium channel subunits and WT 1 subunits was synthesized in vitro using the Ambion mMessage mMachine kit containing either SP6 or T7 bacteriophage RNA polymerase for the ZemRVSP6 or pVA2580 cDNAs, respectively. In preparation for expression of WT and mutant Na channels, pieces of ovary were surgically removed from anesthetized X. laevis frogs. Oocytes were separated and defolliculated by shaking gently for 2 h in 1.5 mg/ml collagenase in OR-2 (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl, and 5 mM HEPES, pH 7.5). After an overnight incubation at 18 °C in Barth's medium (88 mM NaCl, 1 mM KCl, 0.82 mM MgSO, 0.33 mM Ca(NO), 0.41 mM CaCl, 2.4 mM NaHCO, and 10 mM HEPES, pH 7.4) supplemented with 5% fetal bovine serum and 40 µg/ml gentamicin, healthy stage V and VI oocytes were pressure-injected with 50 nl of a 10:1 mixture of rat brain Na channel 1 and subunit mRNA with concentrations ranging from 5 to 50 ng/µl of injected solution. For injection, RNA was diluted in a solution of 1 mM Tris-HCl, pH 7.5, and 0.01 mM EDTA. Injected oocytes were maintained for 2-3 days as described above before electrophysiological recording.

Electrophysiological Recording

Two-microelectrode voltage clamp recordings were obtained from injected oocytes using a Dagan CA-1 voltage clamp (Dagan Corp., Minneapolis, MN). The amplitude of expressed Na currents was typically 1-5 µA. The bath was continuously perfused with Frog Ringer (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl, 10 mM HEPES, pH 7.2). Recording electrodes contained 3 M KCl and had resistances of <0.5 megaohms. Pulses were applied, and data were acquired using a personal computer-based data acquisition system (Basic-Fastlab, Indec Systems, Sunnyvale, CA). Maximum possible series resistance compensation was used to avoid errors due to relatively large Na currents and to maximize resolution of their rapid kinetics. Capacity transients were partially canceled using the internal clamp circuitry. The remaining transients and leak were subtracted using the P/4 procedure (Armstrong and Bezanilla, 1974).

The cell-attached configuration of the patch clamp technique (Hamill et al., 1981) was used to obtain both macropatch and single channel data. Oocytes were incubated in 200 mM potassium aspartate, 20 mM KCl, 1 mM MgCl, 10 mM EGTA, and 10 mM HEPES for 5-10 min prior to removing the vitelline layer with fine forceps (Methfessel et al., 1986). Oocytes were then transferred to a solution containing 110 mM KCl, 10 mM NaCl, 10 mM EGTA, 1 mM MgCl, 10 mM HEPES, pH 7.2, to depolarize the membrane potential. Microelectrode measurement of oocyte membrane potential after incubation in this solution gave a value of -3.25 ± 0.25 mV (n = 4). For experiments with the peptide blocker, macropatches were excised and placed beneath a stream of bath solution containing 50 µM KIFMK peptide. Pipettes were pulled from Corning 7052 (Garner Glass Co., Claremont, CA), coated with Sylgard, and fire-polished before filling with Frog Ringer. Single channel currents from the patch clamp amplifier (List EPC-7) were sampled at 50 kHz and filtered at 5 kHz. Macropatch records were filtered at 7 kHz.

Data Analysis

Normalized conductance-voltage curves and inactivation curves were fit with the expression A/{1 + exp[(V - V)/k]} + (1 - A), where V is the test pulse voltage (for activation) or prepulse voltage (for inactivation), V is the midpoint of the curve, k is a slope factor, and A is the amplitude. Least squares fitting was done with the Sigma-Plot program (Jandel Scientific, San Rafael, CA).

Single channel openings were detected using standard half-amplitude threshold analysis after filtering the data at 2 kHz (Colquhoun and Sigworth, 1993). Patches contained from one to four channels. Channel number was estimated from the maximum number of overlapping openings at potentials where the probability of opening was high. Open times, closed times, and first latencies were analyzed using PSTAT software (Axon Instruments, Foster City, CA). Overlapping events were omitted from analysis.

RESULTS

Point Mutations in Segment IVS6 Affect Fast Inactivation

Fig. 1A illustrates the amino acid sequence of segment IVS6, which contains both hydrophobic and neutral polar amino acids. In order to identify functionally important residues, we substituted alanine at each position by oligonucleotide-directed mutagenesis. Alanine is small and minimally hydrophobic, has a small effect on protein secondary structure, and is frequently located in -helical transmembrane regions (Richardson, 1981; Zhang et al., 1992; Eriksson et al., 1992; Blaber et al., 1993). Accordingly, alanine substitutions should change the size and hydrophobicity of each residue in the helix without disrupting overall protein structure. We also mutated methionine 1770 to valine to recreate a naturally occurring mutation associated with hyperkalemic periodic paralysis in the skeletal muscle Na channel (Rojas et al., 1991).


Figure 1: Effects of point mutations in transmembrane segment IVS6 on Na channel macroscopic inactivation. A, single letter code for the amino acid sequence of transmembrane segment IVS6 of the WT rat brain type IIA Na channel subunit. Sequence numbers of the amino and carboxyl termini are given (Auld et al., 1988). Each native residue was individually mutated to alanine. Methionine 1770 was also mutated to valine. B, two-microelectrode voltage clamp recordings of currents due to expression of WT, F1764A, V1774A, and F1764A/V1774A Na channel subunits in combination with 1 subunits in Xenopus oocytes. Currents were evoked by 30-ms pulses to -10 mV from a holding potential of -90 mV. Normalized currents are shown. Oocytes were injected with RNA encoding the appropriate subunit construct as well as a 10-fold excess of RNA encoding the rat brain 1 subunit (Isom et al., 1992). C, fraction of current that fails to inactivate for each mutant construct. Currents were elicited by pulses to -5 mV as described above. The fraction of non-inactivating current was determined as the current 15 ms after the beginning of the pulse divided by the peak inward current. Mean ± S.E. is plotted for each IVS6 mutant. The asterisks indicate significant differences from WT as determined by t test (p < 0.01). Data are from 3-10 experiments for each mutant. The histogram data shown at position 1770 is for mutant M1770A. The data for the mutant M1770V (not shown) were also not significantly different from WT.



We co-expressed WT and mutant Na channel type IIA subunit RNAs with 1 subunit RNA in X. laevis oocytes and analyzed Na currents due to expression of WT and mutant Na channels by two-microelectrode voltage clamp recording. Coexpression of WT and 1 subunits yielded Na currents that closely resemble native rat brain Na currents in that they inactivated rapidly and almost completely (Fig. 1B) (Isom et al., 1992). A number of mutations in segment IVS6 caused incomplete inactivation, resulting in sustained currents at the end of a 30-ms-long depolarization (Fig. 1, B and C). The mutants F1764A and V1774A exhibited the largest sustained Na currents (Fig. 1C), whereas mutants S1763A, Y1771A, I1775A, and L1776A had significantly increased but smaller sustained Na currents. Thirteen of the remaining mutants (including M1770V) did not significantly affect inactivation, while inactivation of Y1759A and V1768A was significantly more complete than inactivation of WT.

The large sustained currents caused by the mutations F1764A and V1774A suggested that these two residues might play a particularly important role in Na channel inactivation. This suggestion is further supported by the finding that simultaneous mutation of both residues (mutant F1764A/V1774A) almost completely eliminated fast inactivation (Fig. 1, B and C). These data indicate that segment IVS6 plays an essential role in Na channel inactivation and that residues Phe and Val within this segment may be particularly important in the inactivation process.

In addition to producing sustained currents, IVS6 mutations also altered the voltage dependence of steady state activation and inactivation (, Fig. 2). For example, the voltage for half-maximal activation of mutants V1774A and F1764A/V1774A was shifted by -8 to -10 mV compared with WT (, Fig. 2A). In contrast, the voltage for half-maximal activation of F1764A and several other mutants was unaffected (, Fig. 2A). Apparent shifts in activation toward more negative membrane potentials are expected for mutations that strongly inhibit inactivation because of the kinetic overlap of the activation and inactivation gating processes (Gonoi et al., 1984; Gonoi and Hille, 1987). The lack of a strict quantitative correlation between the extent of disruption of inactivation and shifts in the voltage dependence of activation among our different mutants suggests that IVS6 mutations may also have small direct effects on the voltage dependence of activation.


Figure 2: Effects of point mutations on Na current activation and inactivation. A, activation curves. Currents were elicited by 30-ms-long two-microelectrode voltage clamp pulses to a range of test potentials from a holding potential of -90 mV. Peak inward current (I) was measured. Conductance was determined as I/(55 mV - V), where V was the test pulse voltage and +55 mV was the approximate reversal potential in our recording conditions. Normalized conductance was fit with a Boltzmann relationship (see ``Experimental Procedures''), and mean V and k values were determined for each mutant (Table I). The curves shown are plots of the Boltzmann relationship using these mean values. B, steady state inactivation curves. Currents were elicited by test pulses to 0 mV following 100-ms conditioning pulses to various potentials from a holding potential of -90 mV. Peak test pulse current was plotted as a function of prepulse potential, normalized and fit with the Boltzmann equation. Mean V and k were determined for each mutant construct (Table I). Curves shown are plots of the Boltzmann equation using these mean values. C, recovery from inactivation. Na channels were inactivated by 15-ms-long pulses to 0 mV. The membrane was then repolarized to a recovery potential for an interval of variable duration that was then followed by a test pulse to 0 mV. Peak current during the test pulse was divided by peak current during the inactivating pulse and plotted as a function of time between the pulses. Such plots were fit with single exponential functions to determine the recovery time constant at each recovery potential. The time constant of the fit is plotted as a function of recovery potential.



Steady state inactivation was determined using a test pulse applied after a 100-ms voltage step to various prepulse potentials. Mutations F1764A and F1764A/V1774A shifted the voltage dependence of steady state inactivation by +6 mV and +15 mV, respectively, whereas V1774A was similar to WT (, Fig. 2B). Prepulses more depolarized than -30 mV resulted in almost complete inactivation of WT channels. In contrast, the steady state inactivation curves for F1764A, V1774A, and F1764A/V1774A approached nonzero asymptotes with strong depolarization (Fig. 2B). This residual, non-inactivating component of the Na current was 31 ± 1% for V1774A, 12 ± 1% for F1764A, and 79 ± 4% for F1764A/V1774A but only 3 ± 0.3% for WT. Thus, the non-inactivating currents of mutant Na channels, detected during 30-ms-long test depolarizations (Fig. 1), showed little additional decay at 100 ms.

Mutations F1764A and V1774A also accelerated the rate of recovery from inactivation at hyperpolarized potentials. We examined the time course of recovery from inactivation by stepping the voltage to 0 mV for 15 ms to inactivate channels and then repolarizing to negative potentials for recovery intervals of varying duration. The time constants for recovery derived from an exponential fit to the data are plotted as a function of recovery voltage in Fig. 2C. For F1764A and V1774A, the time constant for recovery was 2-3-fold faster than WT at all potentials. This result gives further evidence that the inactivated state is destabilized by these mutations.

Inactivation of Mutant NaChannels Is Less Complete at Positive Voltages

To examine the inactivation properties of WT and the F1764A, V1774A, and F1764A/V1774A mutants in more detail, we recorded macroscopic currents from cell-attached patches (macropatches), which gives better time resolution and voltage control than two-microelectrode voltage clamp. Fig. 3A shows typical records obtained by depolarization to +20 mV. The overall characteristics of the records in the macropatches were similar to those obtained in two-microelectrode recordings although the fraction of sustained Na current was smaller. This difference between the two recording techniques was probably due to incomplete resolution of the peak current in two-microelectrode recording. With strong depolarizations as depicted in Fig. 3A, activation is fast compared with inactivation (Aldrich et al., 1983; Aldrich and Stevens, 1987), and the time constant () of current decay reflects mainly the rate of channel inactivation. The of WT current decay was 0.21 ± 0.008 ms. For F1764A, the of the current decay was similar to WT, 0.27 ± 0.003 ms, indicating that this mutation did not substantially disrupt the rate of channel entry into the inactivated state. In contrast, the time constant for V1774A (0.49 ± 0.03 ms) was slower than WT, suggesting that for these channels, entry into the inactivated state was slowed. Thus, F1764A and V1774A mutations greatly destabilized the inactivated state, but entry into the inactivated state was unaffected or slightly slowed.


Figure 3: Voltage dependence of macroscopic inactivation. A, normalized currents evoked by depolarizations to +20 mV from a holding potential of -140 mV, in cell-attached macropatches on X. laevis oocytes expressing WT, F1764A, V1774A, and F1764A/V1774A mutants. Each trace is an average of 10-15 sweeps. B, normalized currents from a cell-attached macropatch on an oocyte expressing mutant V1774A elicited by 15-ms pulses to -20 and +20 mV. C, the fraction of non-inactivating current in cell-attached macropatches on oocytes expressing WT (), F1764A (), and V1774A () was determined at each potential and plotted as a function of test pulse voltage. This fraction was defined as the ratio of sustained current at 10 ms after the beginning of the depolarizing pulse to peak current. The data are from three, three, and five experiments, respectively. D, normalized tail current amplitudes in cell-attached macropatches expressing F1764A, V1774A, and F1764A/V1774A () mutants as a function of depolarizing pulse voltage. Tail currents were measured at -140 mV following 11-ms depolarizing pulses to the indicated potentials. Peak amplitudes were normalized to the tail obtained after the pulse to +130 mV for each mutant. Data are from representative experiments displayed as a semilog plot.



The inactivation of mutant F1764A and V1774A becomes progressively impaired with increasingly positive depolarizing pulses. For example, the records illustrated in Fig. 3B show that mutant V1774A inactivated more completely at -20 mV than at +20 mV. The progressively larger fraction of sustained current observed at more positive voltages reveals this voltage-dependent impairment of inactivation (Fig. 3, B and C). In contrast, the fraction of sustained WT current was not voltage-dependent.

To examine this point more carefully, we measured tail currents recorded after repolarization to -140 mV following these 11-ms-long depolarizing pulses (Fig. 3D). These tail currents are inward Na currents flowing through Na channels that were open at the end of the depolarizing pulse and then closed progressively following repolarization. Their amplitudes are proportional to the fraction of Na channels that were open at the end of the depolarizing pulse. They are large and easily measured because of the greatly increased driving force for Na influx at -140 mV.

Tail current amplitudes for both F1764A and V1774A increased progressively and did not saturate at positive potentials. Instead, the amplitudes of these tail currents continued to increase at strongly positive membrane potentials (Fig. 3D) where activation was essentially complete (see Fig. 2A). This increase in tail current amplitude at positive potentials reflects the progressive impairment of inactivation at these potentials and the increased fraction of sustained Na current at the end of more positive depolarizing pulses (Fig. 3, B and C). In contrast, for mutant F1764A/V1774A, the amplitude of tail currents saturates at positive voltages. Inactivation of this mutant is minimal at all potentials, and the probability of channel opening is already high (see Fig. 4 ); therefore, further destabilization of inactivation at positive potentials can have little additional effect. The destabilization of inactivation with increased depolarization may be a general property of Na channels. Similar behavior is seen in naturally occurring Na channels with incomplete inactivation such as those of the squid giant axon (Chandler and Meves, 1970; Correa and Bezanilla, 1994a). This voltage-dependent impairment of inactivation is normally masked in WT Na channels with virtually irreversible inactivation, such as those studied here, but is revealed by our mutations which destabilize the inactivated state.


Figure 4: Single channel records and ensemble averages. Examples of single channel traces (uppertrace) and ensemble averages (lowertrace) from cell-attached patches for each of the indicated constructs. All depolarizations were to -20 mV. Arrows indicate the beginning of 40-ms depolarizations from a holding potential of -140 mV. The number of channels in the patches was two, two, two, and one for WT, F1764A, V1774A, and F1764A/V1774A, respectively. Single channel current magnitudes for each mutant at -20 mV were similar to WT (WT, i = 1.26 ± 0.02 pA (n = 5 patches); F1764A, i = 1.35 ± 0.07 (n = 2); V1774A, i = 1.32 ± 0.06 (n = 4); F1764A/V1774A, i = 1.36 ± 0.06 (n = 3)). Sweeps of hyperactive moding behavior (Patlak and Ortiz, 1986; Nilius, 1988; Zhou et al., 1991) or series of null sweeps representing slow inactivation were omitted from ensemble averages (Horn et al., 1984).



Gating of Single Mutant NaChannels Is Altered

To determine the mechanisms underlying the different inactivation gating of the WT and mutant Na channels observed in measurements of macroscopic Na currents, we examined the behavior of single Na channels (Fig. 4). Gating of single sodium channels is described in terms of the general kinetic model shown in Fig. S1(Armstrong and Bezanilla, 1977; Aldrich et al., 1983; Horn and Vandenberg, 1984; Scanley et al., 1990).


Figure S1: Scheme 1.



Upon depolarization, sodium channels undergo voltage-dependent transitions through multiple closed states (states C through C), open (state O), and then inactivate (state I). The inactivated state can also be reached from one or more closed states as illustrated in Fig. S1. At depolarized potentials, inactivation of WT sodium channels is irreversible on the millisecond time scale.

In response to a 40-ms depolarization to -20 mV, WT Na channels opened once or twice at the beginning of the depolarization but then inactivated and did not reopen until the membrane patch was repolarized (Fig. 4). Ensemble averages of WT single channel current records had a time course like WT currents in whole cell and macropatch recordings. F1764A, V1774A, and F1764A/V1774A, on the other hand, all exhibited repeated openings during 40-ms depolarizations (Fig. 4), suggesting that inactivation was rapidly reversible for these mutations. Ensemble averages showed sustained Na currents for V1774A and F1764A/V1774A similar to those observed in macroscopic current recordings. For F1764A, no clear sustained current was detected in ensemble averages despite the frequent channel openings observed late in the pulse. This finding is consistent with the relatively small sustained currents seen with this mutant at the macroscopic level (Fig. 3, A and C). These results indicate that the behavior of single WT and mutant channels observed in patch recordings accounts for the properties of Na currents observed at the macroscopic level.

With strong depolarizations, the single channel open time gives an estimate of the rate of channel entry into the inactivated state from the open state, because the channel leaves the open state primarily by entry into the inactivated state rather than by return to a closed state at depolarized voltages (Armstrong, 1981; Aldrich et al., 1983; Armstrong and Bezanilla, 1977). Mutations that slow the entry into the inactivated state from the open state are expected to increase single channel mean open time. Distributions of open times at -20 mV for typical WT, F1764A, V1774A, and F1764A/V1774A single channel recording experiments are illustrated in Fig. 5as histograms. WT, F1764A, and V1774A single channel data were well fit with single exponentials, suggesting that each of these channels had a single open state with a short lifetime. The time constants () of these fits correspond to the mean open time. The values for WT, F1764A, and V1774A were 0.24 ± 0.03 ms (n = 5), 0.21 ± 0.04 ms (n = 2), and 0.33 ± 0.01 ms (n = 3), respectively. The values for WT and F1764A were similar, indicating that the F1764A mutation did not alter the rate of entry into the inactivated state. The V1774A mean open time was slightly longer than that for WT, suggesting that the rate of entry into the inactivated state is slightly slower for this mutant. The mean open times obtained from single channel recordings were similar to the values for inactivation determined from macropatches. The open times were not obviously voltage-dependent, consistent with the idea that the rate of entry into the inactivated state from the open state is voltage-independent (Armstrong, 1981; Aldrich et al., 1983; Armstrong and Bezanilla, 1977; Zagotta and Aldrich, 1990).


Figure 5: Properties of single channel open times. Open time histograms are displayed for WT, and the F1764A, V1774A, and F1764A/V1774A mutants. Data were collected during pulses to -20 mV. For WT, F1764A, and V1774A, the solidlines are fits of single exponentials to the binned data at times 100 µs. Time constants are 0.23, 0.18, and 0.33 ms, respectively. For F1764/V1774, the data were fit with the sum of two exponentials of approximately equal weight with time constants of 0.46 and 1.20 ms. The dottedlines in each mutant panel are single exponentials with the WT time constant of 0.23 ms. These histograms are representative plots from five, two, three, and three experiments for WT, F1764A, V1774A, and F1764A/V1774A, respectively.



Fits of the open times of F1764A/V1774A required two exponentials of nearly equal weight, suggesting that the extensive disruption of inactivation seen with this mutant reveals a second open state (Fig. 5). The mean open times from three experiments were 0.37 ± 0.06 ms and 1.01 ± 0.16 ms. Two open states have been previously suggested for Na channels from squid giant axon and mouse neuroblastoma cells but are not normally observed for the WT brain channel (Nagy et al., 1983; Nagy, 1987; Chandler and Meves, 1970; Correa and Bezanilla, 1994b).

The first latency is the time before the first opening of a single Na channel following depolarization and indicates the rate of transition of Na channels through the multiple closed states in the activation pathway and subsequent channel opening (Fig. S1) (Armstrong and Bezanilla, 1977; Armstrong, 1981; Aldrich et al., 1983; Zagotta and Aldrich, 1990). Fig. 6A shows first latency distributions for WT, V1774A, and F1764A/V1774A, corrected for the number of channels in the patch (Patlak and Horn, 1982) and plotted as a cumulative probability density function versus time. These plots illustrate the rate and final extent of opening of single Na channels as a function of time after depolarization. For WT at -20 mV, single channels opened rapidly, but the cumulative first latency curve reached a maximum probability of only 0.65 because 35% of the depolarizations failed to elicit channel openings and therefore resulted in null sweeps. At the holding potential of -140 mV, it is unlikely that any of the channels were inactivated. Thus, null sweeps represent the irreversible transitions of WT Na channels directly from closed states in the activation pathway to the inactivated state during depolarization (Fig. S1) (Aldrich et al., 1983; Aldrich and Stevens, 1987).


Figure 6: Properties of single channel closed times. A, cumulative first latency distributions for WT, V1774A, and F1764A/V1774A. Distributions at -20 mV are compared after correcting for channel number (Patlak and Horn, 1982). For V1774A, two distributions are shown, one obtained from a single channel patch and the other obtained from a two-channel patch. The similarity of the two distributions is an empirical verification of the correction procedure. B, representative 40-ms-long traces of single V1774A channel activity during depolarizations to the indicated voltages are shown. This patch contained one channel. C, mean closed times for V1774A. Data are from a single channel patch. Mean closed times were determined with first latencies excluded and are displayed as a semilog plot. The solidline is a least squares fit with a slope corresponding to an e-fold change in closed time per 35 mV. Qualitatively similar behavior was seen in three other patches containing from two to five channels.



The cumulative first latency distribution for V1774A also had a rapid component with a maximum open probability of about 0.6, but virtually no null sweeps were observed for this mutant. Instead, the first latency distribution for V1774A had an additional slow component, and the total distribution increased to a probability of opening of 1.0 by 20 ms (Fig. 6A). The fast component of this distribution reflects channels that opened normally, whereas the slow component likely represents channels that inactivated from closed states and then opened. This component appears as delayed openings in the V1774A mutant because the inactivated state is rapidly reversible, but it appears as nulls in WT recordings because WT inactivation is irreversible at -20 mV on the time scale of our recordings.

The first latency distribution for F1764A/V1774A approached an open probability of 1.0 as for mutant V1774A; however, there was no slow component to the distribution. Either this mutation blocked transitions from the closed states to the inactivated state or these channels leave the inactivated state so rapidly that the overall first latency distribution is unaffected. The similar time course for the rapid component of the cumulative first latency distribution for WT and both mutant channels indicates that the activation process is not strongly slowed by these mutations.

For the mutant V1774A, we consistently observed an increase in single channel activity throughout the duration of a stimulus pulse with increasingly strong depolarizations (Fig. 6B). This behavior suggests that the degree of destabilization of inactivation in this mutant was voltage-dependent, as observed in recordings of sustained macroscopic Na currents and tail currents (Fig. 3). We examined this phenomenon in more detail by measuring closed times in a V1774A patch that contained only a single active channel. The voltage dependence of these closed times is illustrated in Fig. 6C, where mean closed times are plotted as a function of membrane potential. The shortest mean closed time was greater than 2 ms. This long duration is much longer than the mean first latency for channel activation from closed states (Fig. 6A), indicating that the mean closed times are dominated by openings of single sodium channels from the destabilized inactivated state. The mean closed times were shorter with stronger depolarizations. This voltage dependence of the closed times in V1774A indicates that the rate for exiting the inactivated state is increased with depolarization. This increased frequency of reopening at more positive membrane potentials may underlie the increase in sustained macroscopic currents and tail currents observed at more positive depolarizing potentials with this mutant (Fig. 3).

Mutational Tests for Hydrophobic Interactions of Residues Phe and Val in Segment IVS6 with the IFM Motif

Studies using site-directed antibodies (Vassilev et al., 1988, 1989) and mutagenesis (Stühmer et al., 1989) indicate that the intracellular loop between domains III and IV of the subunit forms the Na channel inactivation gate. Three hydrophobic residues, Ile, Phe, and Met (IFM), are crucial components of this inactivation gate, and mutations of residue Phe to hydrophilic residues are sufficient to nearly completely prevent fast inactivation (West et al., 1992a). The IFM motif is thought to bind to a hydrophobic site in or near the inner mouth of the channel pore (Scheuer et al., 1993; Eaholtz et al., 1994). One possible interpretation of the results of mutations F1764A and/or V1774A is that the native residues at these positions form the binding site for the IFM motif and interact directly with it. We investigated this possibility by analyzing two additional types of mutants.

First, we investigated the role of hydrophobicity at the 1774 position. If this residue forms part of a hydrophobic binding site for IFM, then substituting serine or asparagine, two polar residues, for the native valine should disrupt inactivation even more than substituting alanine. When analyzed as described in Fig. 1, the fraction of sustained Na current for mutant V1774S was 0.28 ± 0.02 at 15 ms after the beginning of a pulse to -5 mV, which is smaller than that found for the mutant V1774A (0.38 ± 0.01). The fraction of sustained current for mutant V1774N was 0.07 ± 0.003, which is only slightly larger than WT (0.04 ± 0.005) (see Fig. 1B for V1774A and WT). These data suggest that the effects of mutations at position 1774 are not closely correlated with the hydrophobicity of the substituted amino acid. In contrast, a series of substitutions for Phe in the IFM motif did show a close correlation of inactivation with hydrophobicity (Scheuer et al., 1993). Thus, it is unlikely that Phe in the IFM motif forms a required hydrophobic interaction with residue Val during the inactivation process.

As a second strategy to investigate whether the residues Phe and Val of transmembrane segment IVS6 and residue Phe of the IFM motif interact, we examined whether the mutation F1489W, which increases the size and hydrophobicity of the critical residue of the IFM motif, could compensate for the reduced size and hydrophobicity of the F1764A and V1774A mutants and restore inactivation when combined with these IVS6 mutations. This approach is analogous to the analysis of suppressor mutants isolated by selection in genetic screens. Fig. 7compares Na currents elicited with depolarizing pulses to +20 mV in macropatches expressing WT, the single mutants F1489W, F1764A, and V1774A, or the double mutants F1489W/F1764A and F1489W/V1774A. F1489W caused a small sustained current, similar in size to that observed for F1764A. Combining F1489W with F1764A or V1774A resulted in substantially greater disruption of inactivation than observed for either of the single mutants. Thus, Trp is not as effective as Phe in mediating inactivation of Na channels having WT or mutant IVS6 segments, and mutation F1489W does not compensate for the effects of F1764A or V1774A.


Figure 7: Analysis of single and double mutants of the III-IV loop residue F1489W and the IVS6 residues F1764A or V1774A. Normalized currents from cell-attached macropatches on X. laevis oocytes expressing WT, F1489W, F1764A, V1774A, F1489W/F1764A, and F1489W/V1774A mutants at +20 mV are shown. Each trace is an average of 10-15 sweeps. Data for the thermodynamic analysis described in the text is from four, three, two, four, four, and two patches, respectively. The mean values for the fraction of sustained current for WT, F1489W, F1764A, V1774A, F1489W/F1764A, and F1489W/V1774A were 0.01 ± 0.005, 0.04 ± 0.003, 0.08 ± 0.03, 0.28 ± 0.02, 0.15 ± 0.02, and 0.80 ± 0.002.



The results of these double mutation experiments argue against a direct hydrophobic interaction between the critical Phe residue of the IFM motif and either the Phe or Val residues. To test this hypothesis more quantitatively, we examined the independence of the changes in the free energy (G) of the inactivation process caused by these mutations using a thermodynamic cycle analysis originally developed by Carter et al.(1984) to examine structural changes in the active site of tyrosyl-tRNA synthetase. According to this analysis, if two residues do not interact, then the changes in the free energy of the inactivation process caused by their mutation should be independent of each other and therefore additive if the two mutations are combined. Conversely, if the residues interact, then the changes in free energy of the inactivation process caused by their mutation may be interdependent and therefore substantially greater than or less than additive.

For macroscopic Na currents recorded during strong depolarizations, the fraction of sustained current (S) is approximately equal to k/(k + k), where k is the rate constant governing the transition from the open to inactivated state, and k is the rate constant governing the reverse transition from the inactivated state to the open state (Fig. S1). This equation can be rearranged to give the equilibrium constant K = k/k = (1/S) - 1. The free energy of inactivation is G = -RTlnK, where R and T are the universal gas constant and temperature, respectively. The values for G of inactivation at +20 mV and their standard errors (Taylor, 1982) for WT and each mutant were calculated from the mean values for the fraction of sustained Na current for each channel given in the legend to Fig. 7. The changes in these values, (G) that were caused by each mutation are shown in Fig. S2 .


Figure S2: Scheme 2.



In these thermodynamic cycles, the sum of (G) values in the transitions from WT to each of the individual mutations (e.g. WT F1764A + WT F1489W) should equal the predicted (G) value for the double mutant (e.g. WT F1764A + F1764A F1489W/F1764A) if the two individual mutations act independently. The sum of the free energy changes from WT to the double mutant in either direction around the cycle gives an identical value for (G), as required for a thermodynamic cycle. For the double mutant F1489W/F1764A, (G)was 1.60 ± 0.29 kcal/mol, a value indistinguishable from the value of 1.92 ± 0.29 kcal/mol expected for independent effects. For the double mutant F1489W/V1774A, (G) was 3.41 ± 0.27 kcal/mol, a value similar to the value of 2.84 ± 0.39 kcal/mol expected for independent effects. This thermodynamic analysis, therefore, supports the conclusion that the mutations at positions 1764 and 1774 act independently of the mutation at position 1489 to disrupt inactivation (Carter et al., 1984).

Effects of Mutations in Segment IVS6 on Interaction with a Free Peptide Containing the IFM Motif

Intracellular application of the peptide acetyl-KIFMK-amide (KIFMK) containing the IFM motif restores fast inactivation to Na channels with mutations in their intrinsic IFM motif (Eaholtz et al., 1994). Application of KIFMK to the intracellular surface of Na channels in excised membrane patches also restores fast inactivation and binds to open Na channels with a Kof 21 µM (Eaholtz et al., 1995). As an additional test of the hypothesis that Phe and Val interact with the IFM motif during inactivation, we examined the effect of application of the peptide KIFMK to the intracellular surface of excised membrane patches from Xenopus oocytes expressing these mutant Na channels. Application of 50 µM KIFMK to excised macropatches containing Na channels with the F1489Q mutation, which disables the intrinsic inactivation gate, caused strong, time-dependent block of the current elicited by a depolarizing pulse to -20 mV (Fig. 8A). These results are consistent with restoration of fast inactivation by the peptide (Eaholtz et al., 1994). The same concentration of peptide similarly blocked the Na current conducted by the F1764A/V1774A mutant (Fig. 8B). The restoration of inactivation of the IVS6 double mutant by KIFMK was comparable with that observed with mutant F1489Q. In contrast, the inactive peptide acetyl-KIQMK-amide (Eaholtz et al., 1994) at 300 µM did not restore inactivation of F1764A/V1774A (data not shown). These results indicate that the mutations at positions 1764 and 1774 do not alter the binding site for the free peptide containing the IFM motif.


Figure 8: Effect of KIFMK peptide on excised, inside-out macropatches from oocytes expressing the F1489Q (A) and F1764A/V1774A (B) mutants. Currents elicited by depolarizing pulses to -20 mV from a holding potential of -140 mV in the absence and presence of 50 µM KIFMK peptide are shown. The traces shown were obtained without averaging and are representative of three patch experiments for each mutant construct.



DISCUSSION

We have identified amino acid residues in transmembrane segment IVS6 of the Na channel subunit that are critical for fast inactivation. Point mutations near the middle (F1764A) and toward the cytoplasmic end (V1774A) of this transmembrane segment caused sustained Na currents, indicating that these mutations destabilized the inactivated state. The double mutation F1764A/V1774A prevented fast inactivation nearly completely. Single channel analysis revealed that the sustained currents caused by mutation of these residues were due to a large increase in the frequency of reopenings from the inactivated state without a major alteration in the rate of activation or the rate of entry into the inactivated state. These reopenings account for the sustained currents seen at the macroscopic level. Apparently, the Phe and Val residues play a particularly important role in fast inactivation.

Inactivation of Na channels is hypothesized to involve a cytoplasmic inactivation gate folding into and occluding the ion conducting pore. Experimental evidence with site-directed antibodies (Vassilev et al., 1988, 1989) and mutations (Stühmer et al., 1989) indicates that the intracellular loop connecting domains III and IV acts as the inactivation gate. The hydrophobic motif IFM is a critical component of the inactivation gate, and these residues are thought to interact with a receptor site that becomes available in the activated Na channel (West et al., 1992a; Eaholtz et al., 1994). This interaction is due primarily to hydrophobic forces (Scheuer et al., 1993). Although mutations of amino acid residues in segment IVS6 disrupt the stability of the inactivated state, our results indicate that these residues probably do not form this hydrophobic receptor for the IFM motif. Thus, the destabilization of inactivation caused by substitution at position 1774 was not correlated with amino acid hydrophobicity; thermodynamic analysis of double mutations at positions 1764 and 1774 in IVS6 and at position 1489 in the IFM motif suggest that the critical residues in these two regions act independently in the process of inactivation; and the KIFMK peptide restored inactivation to the F1764A/V1774A double mutant as effectively as to a mutation in the IFM motif itself. The results of each of these tests contradict the predictions of models in which these IVS6 residues interact directly with the IFM motif. Therefore, we have identified amino acid residues in segment IVS6 of the Na channel that are critical for fast inactivation and for the stability of the inactivated state but do not appear to interact directly with the IFM motif in the fast inactivation gate.

Additional support for a role of segment IVS6 in Na channel inactivation comes from studies of mutations in the skeletal muscle Na channel that cause hyperkalemic periodic paralysis. One of these mutations is analogous to M1770V within segment IVS6 (Rojas et al., 1991). Expression of skeletal muscle Na channels containing this mutation in mammalian cells causes small sustained Na currents (Cannon and Strittmatter, 1993). In our work, expression of rat brain Na channels with this mutation in Xenopus oocytes did not produce major effects on inactivation, indicating that the role of this amino acid residue may be less critical than others identified in this study. Mutations causing substantial inhibition of Na channel inactivation as observed in our experiments would probably be lethal and therefore are not expected to be found in human genetic studies.

Segment IVS6 is considered to be an helix (Noda et al., 1986b; Durell and Guy, 1992). Helical wheel analysis (Schiffer and Edmundson, 1967) indicates that Phe and Val fall on the same face of the helix separated by an angle of approximately 80° and are 3 helical turns (15 Å) distant from each other. Interestingly, two other mutations that disrupted inactivation, Y1771A and I1775A, also fall on the same helix face. The mutations F1764A and Y1771A also reduce the affinity of Na channels for local anesthetic drugs by up to 2 orders of magnitude (Ragsdale et al., 1994). These drugs are thought to bind to a receptor site within the pore of the Na channel and to interact with the closed inactivation gate (reviewed in Hille(1992)). Thus, an intriguing possibility is that these residues are oriented toward the inner lumen of the ion conducting pore, where they play a role in stabilizing both drug binding and channel inactivation.

Our experimental results place limitations on the possible mechanisms by which these amino acid residues participate in fast inactivation since the rate of activation of the channel and the rate of entry into the inactivated state are not substantially affected by the mutations in segment IVS6. In addition, our results show that a receptor for the IFM motif becomes available for binding and block of the open Na channel by the peptide KIFMK at approximately the same rate in WT and mutant Na channels. These results appear to rule out major effects of the IVS6 mutations on activation, coupling of activation to inactivation, closure of the inactivation gate, and formation of the hypothetical receptor for the IFM motif. Possibly, these critical amino acid residues in segment IVS6 bind to an unidentified motif in the inactivation gate whose interaction with the pore is also critical for stable fast inactivation of the Na channel. Alternatively, binding of the free KIFMK peptide might not be destabilized by these mutations, whereas binding of the IFM motif in its naturally occurring, tethered form is. Experiments that test these alternatives will further define the mechanisms of Na channel inactivation and the role of segment IVS6 in this process.

  
Table: Voltage dependence of activation and inactivation for selected mutants

For activation and inactivation data, normalized conductance (see legend to Fig. 2A) and current (see legend to Fig. 2B) plots, respectively, were fit with a Boltzmann equation (see ``Experimental Procedures'') to obtain V and k values.



FOOTNOTES

*
This research was supported by National Institutes of Health Grant NS15751, postdoctoral fellowships from the National Institutes of Health (to J. C. M. and D. S. R.), and the W. M. Keck Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviation used is: WT, wild type.


ACKNOWLEDGEMENTS

We thank R. MacKinnon for suggesting the analysis of suppressor mutations using a thermodynamic cycle and S. Love for helpful discussions.


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