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J. Biol. Chem., Vol. 277, Issue 40, 37105-37115, October 4, 2002

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Interaction between Fast and Ultra-slow Inactivation in the Voltage-gated Sodium Channel

DOES THE INACTIVATION GATE STABILIZE THE CHANNEL STRUCTURE?^*

Karlheinz Hilber^§, Walter Sandtner^§, Oliver Kudlacek, Blanca Schreiner, Ian Glaaser^¶, Wolfgang Schütz, Harry A. Fozzard^¶, Samuel C. Dudley, and Hannes Todt^**

From the  Institute of Pharmacology, University of Vienna, A-1090 Vienna, Austria, the ^¶ Cardiac Electrophysiology Laboratories, The University of Chicago, Chicago, Illinois 60637, and the  Division of Cardiology, Emory University, Atlanta, Georgia 30033 and the Atlanta Veterans Affairs Hospital, Decatur, Georgia 30033

Received for publication, June 7, 2002, and in revised form, July 10, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, we reported that mutation A1529D in the domain (D) IV P-loop of the rat skeletal muscle Na⁺ channel µ₁ (DIV-A1529D) enhanced entry to an inactivated state from which the channels recovered with an abnormally slow time constant on the order of ~100 s. Transition to this "ultra-slow" inactivated state (USI) was substantially reduced by binding to the outer pore of a mutant µ-conotoxin GIIIA. This indicated that USI reflected a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide could stabilize the pore structure (Hilber, K., Sandtner, W., Kudlacek, O., Glaaser, I. W., Weisz, E., Kyle, J. W., French, R. J., Fozzard, H. A., Dudley, S. C., and Todt, H. (2001) J. Biol. Chem. 276, 27831-27839). Here, we tested the hypothesis that occlusion of the inner vestibule of the Na⁺ channel by the fast inactivation gate inhibits ultra-slow inactivation. Stabilization of the fast inactivated state (FI) by coexpression of the rat brain ₁ subunit in Xenopus oocytes significantly prolonged the time course of entry to the USI. A reduction in USI was also observed when the FI was stabilized in the absence of the ₁ subunit, suggesting a causal relation between the occurrence of the FI and inhibition of USI. This finding was further confirmed in experiments where the FI was destabilized by introducing the mutations I1303Q/F1304Q/M1305Q. In DIV-A1529D + I1303Q/F1304Q/M1305Q channels, occurrence of USI was enhanced at strongly depolarized potentials and could not be prevented by coexpression of the ₁ subunit. These results strongly suggest that FI inhibits USI in DIV-A1529D channels. Binding to the inner pore of the fast inactivation gate may stabilize the channel structure and thereby prevent USI. Some of the data have been published previously in abstract form (Hilber, K., Sandtner, W., Kudlacek, O., Singer, E., and Todt, H. (2002) Soc. Neurosci. Abstr. 27, program number 46.12).

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Voltage-gated Na⁺ channels mediate the Na⁺ conductance responsible for the rapidly rising phase of the action potential in nerve and muscle cells. Upon repolarization from brief depolarizations (<50 ms), Na⁺ channels recover from inactivation with a single kinetic phase whose time constant is on the order of a few milliseconds. After long depolarizations (seconds to minutes), recovery from inactivation proceeds through multiple kinetic phases whose time constants range over several orders of magnitude up to tens of seconds (1, 2). The most rapid phase of recovery is thought to correspond to the process of exit from the fast inactivated state, which occurs via intracellular occlusion of the ion-conducting pore by a cluster of hydrophobic amino acids in the loop that links DIII and DIV, the so-called "fast inactivation gate" of the Na⁺ channel (3, 4). All other kinetic phases of recovery have been associated with a relatively poorly understood process called "slow inactivation." The physiological importance of slow inactivation was established when defects in slow inactivation were found to underlie the pathophysiology of a number of heritable diseases of skeletal muscle (5-10), heart (11-13), and brain (14).

We recently demonstrated that replacement of alanine 1529 by aspartic acid in the DIV P-loop of the rat skeletal muscle Na⁺ channel µ₁ (DIV-A1529D) enhanced entry to an ultra-slow inactivated state, which is characterized by time constants of entry to and recovery from inactivation of ~100 s (1). A similar type of recovery from inactivation in µ₁ channels with slow time constants on the order of several tens of seconds was reported recently by other authors (e.g. Refs. 9 and 15). In DIV-A1529D channels, transition to this ultra-slow inactivated state was substantially reduced by binding to the outer pore of a mutant µ-CTX.¹ This suggested that ultra-slow inactivation may reflect a structural rearrangement of the outer channel vestibule and that binding to the pore of a peptide can stabilize the pore structure (1, 16).

Although the structural determinants of fast inactivation are distinct from those of slow inactivation (e.g. Refs. 17 and 18), the various inactivation processes seem to be coupled. Fast inactivation inhibits the probability of slow inactivation (7, 17, 19, 20), probably via gating charge immobilization (21). Similarly, ultra-slow inactivation may as well be modulated by other types of inactivation. However, the effects of faster types of inactivation on ultra-slow inactivation have not yet been investigated.

µ₁ Na⁺ channels consist of a pore-forming  subunit and an auxiliary ₁ subunit. Coexpression of ₁ subunits with µ₁  subunits in Xenopus oocytes modulates the gating properties of the channels (22, 23). The most dramatic ₁ effect is an increase in the rate of inactivation, which is reflected by an acceleration of the current decay after channel activation. ₁ exerts its effects by stabilization of the fast inactivated state (e.g. Refs. 24 and 25). In the presence of ₁, the channels favor a "fast-gating mode" in which they open only once or twice per depolarizing prepulse. In the absence of ₁, the fast inactivated state is destabilized, and a "slow-gating mode" predominates that is reflected by bursts of channel openings (23, 26, 27).

In a previous study (1), we found that coexpression of the rat brain ₁ subunit slowed entry of DIV-A1529D channels to the ultra-slow inactivated state during long-lasting depolarizations. This result and the fact that the ₁ subunit stabilizes the fast inactivated state suggested that fast inactivation could inhibit ultra-slow inactivation. This hypothesis was investigated in the present study. Therefore, we explored the effects of stabilization and destabilization of the fast inactivated state on ultra-slow inactivation. We found strong evidence that fast inactivation inhibits entry to the ultra-slow inactivated state, possibly because binding to the inner pore of the fast inactivation gate stabilizes the channel structure.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A detailed description of the experimental procedures is given in our previous work (1).

Mutagenesis of the µ₁-- The oligonucleotide-directed point mutation DIV-A1529D was introduced using four-primer PCR. An oligonucleotide containing the mutation was designed with a change in a silent restriction site to allow rapid identification of the mutant. A vector consisting of the µ₁-coding sequence flanked by Xenopus globin 5'- and 3'-untranslated regions was provided as a gift by R. Moorman. This was used as the template for mutagenesis, and PCR fragments were isolated and subcloned to this template using directional ligations. Incorporation of the mutation was confirmed by DNA sequencing of the entire polymerized regions. The vector was linearized by SalI digestion and transcribed with SP6 DNA-dependent RNA polymerase using reagents from the mCAP RNA capping kit (Stratagene, La Jolla, CA). The rat brain ₁ subunit of the Na⁺ channel was also subcloned into pAlterXG, and transcription was prepared from BamH1-linearized template using SP6 RNA polymerase.

Mutagenesis of DIV-A1529D + I1303Q/F1304Q/M1305Q-- I1303Q/F1304Q/M1305Q was made by oligonucleotide-directed mutagenesis and confirmed by sequencing the polymerized region. DIV-A1529D was introduced in this construct by cloning the SacII-KpnI fragment into the I1303Q/F1304Q/M1305Q construct. The presence of both mutations in the final plasmid was confirmed by restriction digests analyzing silent mutations introduced at the time of mutagenesis.

Stage V and VI Xenopus oocytes were isolated from female frogs (NASCO, Ft. Atkinson, WI), washed with Ca²⁺-free solution (90 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 1 mM NaHPO₄, and 5 mM HEPES titrated to pH 7.6 with 1 N NaOH), and treated with 2 mg/ml collagenase (Sigma) for 1.5 h; follicular cell layers were manually removed. As judged from photometric measurements, ~50-100 ng of cRNA was injected into each oocyte with a Drummond micro-injector (Broomall, PA). Either native or mutant  subunit cRNA alone or mixed with various concentrations of rat brain ₁ subunit were injected. In the case of DIV-A1529D channels, the injected molar cRNA /₁ ratio ranged from 100 to 0.07. Oocytes were incubated at 17 °C for 12 h to 3 days before examination.

Recordings were made in the two-electrode voltage clamp configuration using a TEC 10CD clamp (npi electronic, Tamm, Germany). The clamp amplifier had a series compensation circuit. For accurate adjustment of the experimental temperature (20 ± 0.5 °C) an oocyte bath cooling system (HE 204, Dagan, Minneapolis, MN) was used. Oocytes were placed in recording chambers in which the bath flow rate was about 100 ml/h, and the bath level was adjusted so that the total bath volume was less than 500 µl. Electrodes were filled with 3 M KCl and had resistances of less than 1 megaohm. Using pCLAMP6 (Axon Instruments, Foster City, CA) software, data were acquired at 71.4 kHz after low-pass filtration at 2 kHz (3 decibel). Curve fitting was performed using ORIGIN 5.0 (MicroCal Software, Inc., Northampton, MA). Recordings were made in a bathing solution that consisted of 90 mM NaCl, 2.5 mM KCl, 1 mM BaCl₂, 1 mM MgCl₂, and 5 mM HEPES titrated to pH 7.2 with 1 N NaOH. BaCl₂ was used as a replacement for CaCl₂ to minimize Ca²⁺-activated Cl currents.

Data Evaluation-- If not otherwise specified, recovery from ultra-slow inactivation was tested with the following experimental protocol. From a holding potential of 120 mV, the channels were inactivated by a 300-s depolarizing voltage step. Thereafter, the potential was returned to 120 mV, and recovery from inactivation was monitored by repetitive 25-ms test pulses to 20 mV at 20-s intervals. The first test pulse was applied 3-10 ms after the prepulse to allow for settlement of the capacitive current transients. The time courses of recovery from ultra-slow inactivation of normalized peak inward currents were fit with the double exponential function,

(Eq. 1)

where I₂ is the peak inward Na⁺ current of the test pulse during recovery, I₁ is the peak inward Na⁺ current of a test pulse under fully available conditions, ₁ (always constrained to 10-12 s) and ₂ (always constrained to 100-150 s) are the time constants of distinct components of recovery, A₁ and A₂ are the respective amplitudes of these time constants, and C is the final level of recovery. A₂ was taken as a measure of the fraction of channels that recovered from ultra-slow inactivation (see Ref. 1) and will subsequently be referred to as "ultra-slow fraction." In the case of the mutant µ₁-I1303Q/F1304Q/M1305Q, which did not exhibit ultra-slow inactivation, recovery from slow inactivation was fit with the monoexponential function.

(Eq. 2)

The kinetics of the current decay after activation by a step depolarization was taken as a measure of the fraction of DIV-A1529D channels, which inactivated fast (time constants about 1 ms ("fast-gating channels") or slow (time constants >10 ms ("slow-gating channels")). We consider a large fraction of fast-gating channels to reflect a high degree of stabilization of the fast inactivated state (24, 25). The current decay was analyzed using two different methods.

First, the current decay after channel activation was fit by a bi-exponential function (Equation 1). The two different amplitudes (A₁ and A₂) were taken as a measure of the fraction of channels that gated either fast (A₁) or slow (A₂), respectively. If not otherwise specified, the channels were activated with the voltage step that elicited maximum current. For accurate comparison of the time constants of current decay in DIV-A1529D -only and DIV-A1529D  + ₁ channels, it was essential to precisely adjust the bath temperature to 20 °C (see above). The fraction of fast- and slow-gating channels has previously been estimated from macroscopic current records on the basis of bi-exponential fits to the time course of current decay after channel opening (28, 29). However, we found that this fitting procedure frequently resulted in parameter estimates that were associated with substantial standard errors; also the initial setting of the fit parameters influenced the estimation of the final parameters. To eliminate this potential error source, we introduced a different method to analyze the current decay after channel opening.

Second, the slow-gating mode is characterized by multiple channel reopening during depolarization, which underlies the slowing of macroscopic current decay (27). Hence, the total amount of charge entry should be increased by multiple channel reopening. The integral area between the zero current line and the current decay from the current peak to the current 50 ms after the depolarizing voltage step was used to estimate the amount of total charge entry. Here, big integral values reflect slow current decay as well as a large fraction of slow-gating channels, whereas small integral values reflect rapid current decay and a large fraction of fast-gating channels. Such current-time integrals have previously been used as an indices of the total open channel probability after channel activation (25, 30). To allow a direct comparison of the current-time integrals between single oocytes, the peak of the current traces to be analyzed was always normalized to 1.

Data are expressed as means ± S.E. Statistical comparisons were made using two-tailed Student's t-tests. A p < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recovery from Ultra-slow Inactivation in µ₁ and DIV-A1529D Channels

Fig. 1A shows the growth of inward currents through wild type µ₁ channels with subsequent 25-ms test pulses at 20-s intervals. From a holding potential of 120 mV, the channels were first inactivated by a 300-s depolarizing prepulse to 50 mV. Recovery from inactivation after returning to 120 mV was monitored through repetitive test pulses to 20 mV (see "Experimental Procedures"). In wild type µ₁ channels, about 80% of the current recovered within 20 s, whereas the small remaining fraction, about 20%, took several minutes to completely recover ("ultra-slow recovery," see "Experimental Procedures").

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Ultra-slow inactivation in µ1 and DIV-A1529D channels. Growth of inward current during recovery from ultra-slow inactivation in wild type µ1 (A) and mutant DIV-A1529D (B) Na+ channels expressed in Xenopus laevis oocytes. From a holding potential of 120 mV, the channels were inactivated by a 300-s depolarizing step to 50 mV. Thereafter, the potential was returned to 120 mV, and recovery from inactivation was monitored by repetitive 25-ms test pulses to 20 mV at 20-s intervals. The dotted lines indicate the zero current levels. Recovery from ultra-slow inactivation in mutant DIV-A1529D took considerably longer than in µ1 channels.

In contrast to µ₁ channels, mutant DIV-A1529D exhibited a large ultra-slow recovering component of inactivation (Fig. 1B). About 40% of the current recovered within 20 s, whereas the large remaining fraction, about 60%, took several minutes to completely recover.

In a series of such experiments, the calculated fractions of channels recovering from ultra-slow inactivation were 0.22 ± 0.02 (n = 6) in µ₁ channels and 0.60 ± 0.02 (n = 19) in DIV-A1529D (value for µ₁ taken from Hilber et al. (1)). Thus, significantly more channels recovered from ultra-slow inactivation in DIV-A1529D than in µ₁ channels. A more detailed investigation of ultra-slow inactivation in DIV-A1529D channels was carried out in a previous study (1).

Effects of Stabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels

To investigate the relationship between fast inactivation and ultra-slow inactivation, we used different strategies to stabilize the fast inactivated state and tested the effects of these strategies on ultra-slow inactivation.

Stabilization of the Fast Inactivated State by Coexpression of the Rat Brain ₁ Subunit-- In wild type µ₁ channels, coexpression of the ₁ subunit stabilizes the fast inactivated state (e.g. Refs. 24 and 25). Here, we used coexpression of the ₁ subunit to stabilize the fast inactivated state in DIV-A1529D channels to investigate the effects of fast inactivation on ultra-slow inactivation.

First, we confirmed that the ₁ subunit exhibited similar modulatory effects in DIV-A1529D channels as previously reported for wild type Na⁺ channels (22, 23, 31). We compared the current decay in oocytes that were injected either with DIV-A1529D -only cRNA or with DIV-A1529D  + rat brain ₁ cRNA (molar /₁ ratios 0.30). Fig. 2A shows typical Na⁺ current traces through wild type µ₁ -only and µ₁  + ₁ channels. It can be clearly seen that coexpression of the ₁ subunit substantially accelerated the kinetics of current decay. A similar result was obtained with DIV-A1529D -only and DIV-A1529D  + ₁ channels (Fig. 2B). A bi-exponential function (Equation 1) was fit to the decay currents, and the fractions of channels that inactivated with a fast and a slow time constant were calculated (see "Experimental Procedures"). The mean values of time constants (₁, ₂) and amplitudes (A₁, A₂) ± S.E. of a series of such fits are given in Table I. The table shows that both ₁ and ₂ were similar in DIV-A1529D -only and DIV-A1529D + ₁ channels. In contrast, the amplitudes A₁ and A₂ that we used as a measure of the fraction of channels that inactivated with a fast (fast-gating channels) or a slow (slow-gating channels) time constant (see "Experimental Procedures") were strongly dependent on the coexpression of the ₁ subunit. In DIV-A1529D -only injected oocytes only a small fraction of channels inactivated with a fast time constant. DIV-A1529D + ₁ channels mainly inactivated with a fast time constant, but in all oocytes investigated, a small but discernable fraction of channels, about 0.07, inactivated with a slow time constant (Table I). Control experiments showed that an /₁ ratio of 0.30 was sufficient to obtain a maximum ₁ subunit effect on current decay. There was no significant rise in the fast decay fractions if the ₁ subunit concentration was increased from an /₁ ratio of 0.30 to 0.15. An additional increase to 0.07 in three oocytes also showed no effect. The respective fast decay fractions were 0.93 ± 0.01 (/₁ ratio, 0.30; n = 4), 0.93 ± 0.01 (/₁ ratio, 0.15; n = 4), and 0.92 ± 0.01 (/₁ ratio, 0.07; n = 3).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Current decay with and without coexpression of the rat brain 1 subunit. Typical examples of original traces of inward currents through wild type µ1 and µ1 + 1 (A) channels or mutant DIV-A1529D and DIV-A1529D + 1 (B) channels elicited by test pulses from 120 to 20 mV. The molar ratio of /1 subunit cRNA injected was 0.3. The current amplitudes were normalized to 1. Both in µ1 and mutant DIV-A1529D channels, coexpression of the 1 subunit dramatically accelerated the current decay.

View this table: [in this window] [in a new window]   Table I Parameters of bi-exponential current decay fits Equation 1) of DIV-A1529D -only and DIV-A1529D  + 1 channels expressed in Xenopus oocytes The channels were activated from a holding potential of 120 mV with the voltage step, which elicited maximum current (  20 mV). 1 and 2 are the time constants of distinct components of recovery; A1 is the amplitude that was used as a measure of the channel fraction, recovering with the time constant 1 (fast fraction). The injected molar cRNA /1 ratio was 0.3.

These results show that, as in wild type µ₁ channels (22, 23), coexpression of the ₁ subunit dramatically increased the fraction of fast-gating DIV-A1529D channels, whereas the time constants themselves were not altered. Thus, coexpression of the ₁ subunit exhibits similar effects on the gating properties of DIV-A1529D and µ₁ channels, stabilizing the fast inactivated state in DIV-A1529D channels as well.

Coexpression of the ₁ Subunit Delays Entry to the Ultra-slow Inactivated State in DIV-A1529D Channels-- In a previous study (1) we investigated the effects of coexpression of the rat brain ₁ subunit on ultra-slow inactivation in DIV-A1529D channels. We found that the ₁ subunit delayed entry to the ultra-slow inactivated state but did not alter entry voltage dependence.

Fig. 3 shows the recovery of inward currents through DIV-A1529D + ₁ channels with subsequent pulses at 20-s intervals after being inactivated by a 300-s (A) or a 1200-s (B) depolarizing prepulse to 50 mV. We found that the longer prepulse duration substantially delayed recovery from ultra-slow inactivation in DIV-A1529D + ₁ channels. A summary of the recovery curves of four such experiments is shown in the inset. Bi-exponential curve fits to the data points (solid lines) were used to estimate the fraction of DIV-A1529D + ₁ channels, which had entered the ultra-slow inactivated state (see "Experimental Procedures"). This fraction was 0.37 ± 0.03 after a 300-s prepulse and 0.58 ± 0.03 after a 1200-s prepulse to 50 mV (n = 4). These fractions were statistically different (paired Student's t test). In DIV-A1529D -only channels, the corresponding fraction after a 300-s prepulse to 50 mV was 0.60 ± 0.02 (n = 19); this fraction was not further increased when longer prepulse durations were used (see Ref. 1). These data confirm that the ₁ subunit prolongs entry to the ultra-slow inactivated state in DIV-A1529D channels.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Coexpression of the 1 subunit in DIV-A1529D and ultra-slow inactivation. Growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D + 1 channels after a 300-s (A) or 1200-s (B) depolarizing prepulse to 50 mV (experimental design identical to Fig. 1). The molar ratio of /1 subunit cRNA was 0.3. Inset, normalized time course of recovery from inactivation after prepulses to 50 mV for 300-s (open squares, n = 4) and for 1200-s (filled squares, n = 4).

The use of prepulse durations of >300 s led to unacceptably long experimental durations. Thus, for further experiments, to avoid extremely long prepulse durations (1200 s), we used the channel fraction recovering from ultra-slow inactivation after a 300-s prepulse to 50 mV as a quantitative measure of the modulation of ultra-slow inactivation by the ₁ subunit.

The ₁ Subunit Effect on Ultra-slow Inactivation in DIV-A1529D Channels Depends on the Molar cRNA /₁ Ratio-- To explore the relationship between the ₁-induced stabilization of the fast inactivated state and ultra-slow inactivation in more detail, we compared the amount of ultra-slow inactivation produced by a 300-s prepulse to 50 mV with the fraction of channels that showed a fast current decay after activation (the fast-gating fraction). Here, we consider a large fast-gating fraction to reflect a high degree of stabilization of the fast inactivated state.

Xenopus oocytes were injected either with DIV-A1529D -only cRNA or with both DIV-A1529D  and ₁ subunit cRNA. To generate various fractions of either fast or slow current decay, the molar concentration of the ₁ subunit cRNA was varied (see "Experimental Procedures"). The lower the cRNA /₁ ratio, the greater was the fast-gating channel fraction. In Fig. 4A, the fraction of channels that entered the ultra-slow inactivated state after a 300-s prepulse to 50 mV is plotted against the fraction of channels that showed a fast current decay after activation (see "Experimental Procedures"). Each data point reflects data acquired from experiments on a single oocyte. Fig. 4A shows a linear correlation between the fraction of DIV-A1529D channels exhibiting a fast current decay and the fraction of channels that recovered from ultra-slow inactivation; the larger the fast-gating channel fraction, the smaller was the channel fraction recovering from ultra-slow inactivation. Consequently, stabilization of the fast inactivated state by increasing the ₁ subunit concentration seems to directly inhibit entry to the ultra-slow inactivated state. However, in oocytes showing almost exclusively (93%) fast current decay, a considerable fraction of channels still exhibited ultra-slow inactivation, 0.30 ± 0.02 (n = 6). This suggests that ultra-slow inactivation may occur in predominantly fast-mode-gating channels, albeit with a low likelihood.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Current decay and ultra-slow inactivation in DIV-A1529D + 1 channels. A, 50-ms step depolarizations from a holding potential of 120 mV to the voltage that generated maximum inward current were applied to assess current decay after activation. Decay currents were fit by a bi-exponential function. This procedure revealed a large fraction of channels with a fast current decay (1 ~ 1 ms, fast-gating channels) if high concentrations of the 1 subunit were coexpressed and a large fraction of channels with a slow current decay (2~ 13 ms, "slow-gating channels") in the absence of 1, or when low 1 subunit concentrations were used. Molar /1 ratios from infinite to 0.07 were injected. After assessment of current decay, a 300-s prepulse to 50 mV was applied, and the fraction of channels that recovered from ultra-slow inactivation was estimated as described under "Experimental Procedures." The fraction of channels recovering from ultra-slow inactivation (x axis) is plotted against the fraction of channels that showed a fast current decay (y axis). Each data point reflects measurements performed on a single oocyte. A linear regression fit to the data points indicated a significant negative correlation between the fast-gating fraction and the propensity to enter the ultra-slow inactivated state (y = 1.9x + 1.4, r = 0.85, p < 0.0001). B, same experiments as in A, but in this case the fast-gating fraction of channels was estimated from the area under the current trace, reflecting the total amount of charge entry (see "Experimental Procedures"). Oocytes with current-time integrals >10 were considered to reflect channels with a high slow-gating fraction. Linear regression analysis indicated that channels exhibiting a large slow-mode-gating fraction were significantly more susceptible to entry to ultra-slow inactivation than channels with a high fast-mode-gating fraction (y = 20.6x + 2.9, r = 0.9, p > 0.0001).

Fig. 4B shows a similar plot as Fig. 4A, but a different method of analysis of the current decay was used to obtain an additional measure of the extent of stabilization of the fast inactivated state in single oocytes. The fraction of channels that entered the ultra-slow inactivated state after a 300-s prepulse to 50 mV is plotted against the current-time integral (normalized area, see "Experimental Procedures") after activation by the depolarizing voltage step, which generated maximum current. Similar to Fig. 4A, the plot in Fig. 4B shows a linear correlation between the fraction of fast-gating DIV-A1529D channels (reflected by small current-time integral values) and the fraction of channels that recovered from ultra-slow inactivation; also here, the larger the fraction of fast-gating channels, the smaller was the channel fraction recovering from ultra-slow inactivation. The slightly higher correlation coefficient of the linear regression line given in Fig. 4B compared with that in Fig. 4A might indicate that the use of current-time integrals (see "Experimental Procedures") to estimate the fast-gating channel fraction is superior to the "curve-fitting method" (see "Experimental Procedures"). However, the results obtained with both methods were reasonably similar (compare Fig. 4, A and B).

The ₁ Subunit Reduces Ultra-slow Inactivation Produced by Trains of Brief Depolarizations in DIV-A1529D Channels-- Until now, we have described the effects of ₁ coexpression on ultra-slow inactivation produced by prolonged continuous depolarizations. In a previous study (1), we showed that ultra-slow inactivation could also be produced by brief repetitive depolarizations that enhanced the probability of channels to undergo transitions between closed and open states in DIV-A1529D channels. We concluded that ultra-slow inactivation most likely is attained via transitions from "partially activated" closed states that are accumulated by such pulse trains. Here, we examined whether stabilization of the fast inactivated state by coexpression of the ₁ subunit modulates ultra-slow inactivation generated by pulse trains in a similar way as it modulates ultra-slow inactivation produced by prolonged continuous depolarizations.

Fig. 5 presents a comparison between the fractions of DIV-A1529D channels (n = 8) recovering from ultra-slow inactivation after a 100-s pulse train in which repetitive 2-ms step depolarizations to 20 mV were followed by 28-ms periods at 120 mV (30-2-ms train protocol) and after a 100-s pulse train in which 28-ms step depolarizations to 20 mV were followed by 2-ms interpulse intervals at 120 mV (30-28-ms train protocol). During the 30-2-ms train protocol, even though channels spent only 6.7 s at depolarized potentials (20 mV), a considerable fraction of channels entered the ultra-slow inactivated state (Fig. 5A). This pulse protocol was designed to prevent entry of the channels to the fast inactivated state and to enhance partially activated closed states (1). In contrast, the 30-28-ms train protocol in which the channels spent 93.3 s at depolarized potentials (20 mV) produced very little ultra-slow inactivation. This pulse protocol most likely enhances fast inactivated states, which accumulate during the 28-ms depolarization periods. These experiments support the notion that enhancement of partially activated closed states favors entry, whereas enhancement of the fast inactivated state reduces entry to the ultra-slow inactivated state.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Coexpression of the 1 subunit and ultra-slow inactivation elicited by pulse trains. Comparison between the fraction of DIV-A1529D channels (empty bars) recovering from ultra-slow inactivation after a 100-s pulse train, during which repetitive 2-ms step depolarizations to 20 mV were followed by 28-ms periods at 120 mV (30-2-ms train protocol), and after a 100-s pulse train, during which 28-ms step depolarizations to 20 mV were followed by 2-ms periods at 120 mV. The 30-2-ms train forced a considerable channel fraction to enter ultra-slow inactivation. In DIV-A1529D + 1 channels (hatched bars) (molar ratio of /1 subunit cRNA 0.3) neither the 30-2- nor the 30-28-ms train protocol elicited considerable ultra-slow inactivation.

Coexpression of the ₁ subunit dramatically reduced the fraction of channels that entered the ultra-slow inactivated state after a 30-2-ms train protocol from 0.41 ± 0.04 (n = 8) to 0.13 ± 0.01 (n = 5). These results suggest that functional association of the ₁ subunit with DIV-A1529D  subunits prevents ultra-slow inactivation produced by pulse train protocols. We propose that the ₁ subunit stabilizes the fast inactivated state during the 30-2-ms train protocol and thereby inhibits ultra-slow inactivation. The time course of current decay of the original current traces shown in Fig. 2B indeed suggests that a considerable fraction of DIV-A1529D  + ₁ channels entered the fast inactivated state during a 2-ms depolarization, whereas in DIV-A1529D -only channels, this was not the case.

Naturally Occurring Fast Current Decay Reduces Ultra-slow Inactivation in DIV-A1529D -Only Channels-- The observed kinetic effects of ₁-coexpression are most likely the result of a ₁-induced shift of equilibria between slow-mode-gating and fast-mode-gating channels. Alternatively, ₁-coexpression could result in a new kinetic state that is absent in -only channels. To investigate whether the reduction of ultra-slow inactivation due to the coexpression of the ₁ subunit was caused by stabilization of the "naturally occurring" fast inactivated state as opposed to the induction of a new kinetic state, we compared ultra-slow inactivation in DIV-A1529D -only-injected oocytes, which naturally exhibited different fast-gating channel fractions. -Only-injected oocytes normally showed a rather small fast-gating channel fraction (Table I). However, for unknown reasons, a few -only injected oocytes exhibited a considerable fast-gating channel fraction even though the ₁ subunit was not coexpressed. Such a variation in fast- and slow-gating channel fractions (amplitude ratio of fast to slow component, 0.4-2.9) was also reported for wild type µ₁ channels expressed in oocytes (27).

Current traces recorded from -only injected oocytes were grouped on the basis of current-time integrals. In general, currents of DIV-A1529D -only-injected oocytes had current time integrals >10, which is in excellent agreement with reported data from wild type -only channels (25). Nevertheless, we were able to find some "outliers," which, despite the absence of the ₁ subunit, showed current-time integrals <10. Fig. 6 shows that such -only channels with current-time integrals <10, indicating the presence of a large fraction of "naturally" fast-gating channels, had a significantly smaller likelihood of entry to ultra-slow inactivation than -only channels with current-time integrals >10. This result suggests that the reduction of ultra-slow inactivation due to the coexpression of the ₁ subunit (see above) is caused by a shift from slow-to fast channel gating by stabilization of the fast inactivated state.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Current decay and ultra-slow inactivation in DIV-A1529D -only channels. DIV-A1529D channels were pooled into two groups on the basis of current-time integrals (see Fig. 4B). The current-time integrals of these groups (<10: 7.1 ± 0.5, range = 6.0-8.4; >10: 12.1 ± 0.3, range = 10.3-13.2) were significantly different (p < 0.01). The ordinate indicates the fraction of channels that entered the ultra-slow inactivated state during a 300-s prepulse to 50 mV. DIV-A1529D -only channels with current-time integrals <10, i.e. a large fraction of naturally fast-gating channels, had a significantly smaller likelihood of entry to ultra-slow inactivation than -only channels with current-time integrals >10.

Low Holding Potentials Favor Fast Current Decay and Reduce Ultra-slow Inactivation in DIV-A1529D -Only Channels-- To further explore whether in the absence of the ₁ subunit stabilization of fast inactivation inhibits entry to the ultra-slow inactivated state, we investigated if a slow-to-fast shift in gating forced by a low holding potential (23) also reduces ultra-slow inactivation in DIV-A1529D -only channels. Therefore, we compared recovery from ultra-slow inactivation in single DIV-A1529D -only-injected oocytes at two different holding potentials, 120 and 80 mV. Fig. 7 shows the growth of inward current through DIV-A1529D -only channels with subsequent pulses at 20-s intervals after the channels were first inactivated by a 300-s depolarizing prepulse to 50 mV. In A, the holding and recovery potential was 120 mV; in B it was 80 mV. At 80 mV, the current amplitude was reduced by about 50%. This is due to inactivation of a certain channel fraction that can only be regained when the membrane is hyperpolarized to 120 mV. Moreover, it can be noticed that the current decay was markedly accelerated at 80 mV, indicating that the remaining channel fraction (not inactivated at 80 mV) mainly gated fast. The fraction of channels that recovered from ultra-slow inactivation at a holding potential of 80 mV (0.48 ± 0.04) was significantly reduced (paired t test, n = 7) compared with the corresponding fraction at 120 mV (0.61 ± 0.03). These data further confirm that the reduction of ultra-slow inactivation by coexpression of the ₁ subunit in DIV-A1529D channels is caused by a shift from slow to fast channel gating.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Ultra-slow inactivation at different holding potentials. Growth of inward current during recovery from ultra-slow inactivation in mutant DIV-A1529D channels at 120 mV (A) and 80 mV (B). From a holding potential of 120 mV or 80 mV, the channels were inactivated by a 300-s depolarizing step to 50 mV. Thereafter, the potential was returned to 120 or 80 mV, and recovery from inactivation was monitored by repetitive test pulses to 20 mV at 20-s intervals. Both current decay and recovery from ultra-slow inactivation were markedly accelerated at the lower holding potential of 80 mV.

Ultra-slow Inactivation Is Inhibited at Potentials That Stabilize the Fast Inactivated State-- In a previous study we found that the voltage dependence of ultra-slow inactivation was U-shaped with a local maximum at about 60 mV in DIV-A1529D channels (1). Here, we compared the relationship between the voltage dependence of ultra-slow inactivation and steady-state fast inactivation in DIV-A1529D channels. With this approach, we could directly compare the occupancy of the fast inactivated state with the occupancy of the ultra-slow inactivated state at various voltages. Fig. 8 shows the voltage dependence of ultra-slow inactivation and steady state fast inactivation in DIV-A1529D channels. The plot shows that ultra-slow inactivation already started to occur at weak depolarizing potentials between 90 and 80 mV. A considerable fraction of channels entered the ultra-slow inactivated state (>0.5) at 70 mV, a potential at which hardly any channels (<15%) entered the fast inactivated state during a 50-ms prepulse. When the depolarizing potential was further increased to 60 mV, the fraction of ultra-slow inactivated channels reached a local maximum. At this potential, fast inactivation started to occur more prominently, i.e. more than 30% of the channels entered the fast inactivated state during a 50-ms prepulse. As the depolarizing potential was further increased from 50 to 0 mV, the fast inactivated state was increasingly stabilized. At the same time, entry to the ultra-slow inactivated state was inhibited with increasing depolarization, reaching a minimum at potentials where almost 100% fast inactivation occurred. These experiments strongly suggest that stabilization of the fast inactivated state directly inhibits the transition to the ultra-slow inactivated state in DIV-A1529D channels.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Voltage dependence of ultra-slow inactivation and steady-state fast inactivation. To assess the voltage dependence of ultra-slow inactivation, the membrane potential was depolarized from 120 mV to the indicated prepulse voltages for 300 s, and the time course of recovery at 120 mV was monitored for each prepulse potential as described under "Experimental Procedures." The time course of recovery from ultra-slow inactivation was then fit with a bi-exponential function (Equation 1) to estimate the fraction of channels recovering from ultra-slow inactivation (A2). Channel availability defined as the fraction of channels not recovering from ultra-slow inactivation (1-A2 = Fnoninactivating) is plotted as a function of prepulse voltage (these data are reproduced from Fig. 2 in Ref. 1). Steady-state fast inactivation was assessed by delivery of 20-ms test pulses to 20 mV after 50-ms prepulses to the indicated potentials. The respective test pulse current was normalized to the current under fully available conditions. Each data point reflects the mean of several experiments (n always 4). Whereas steady-state fast inactivation became more complete at prepulse voltages positive to 60 mV, ultra-slow inactivation reached a local minimum at 60 mV.

Effects of Destabilization of the Fast Inactivated State on Ultra-slow Inactivation in DIV-A1529D Channels

The Inactivation-defective DIV-A1529D Channel Mutant I1303Q/F1304Q/M1305Q Enhances Ultra-slow Inactivation-- The strategy of all the experiments presented above was based on the stabilization of the fast inactivated state by different methods. We found that stabilization of the fast inactivated state inhibited ultra-slow inactivation. Consequently, destabilization of the fast inactivated state should enhance ultra-slow inactivation. Fast inactivation can be destabilized by mutations in the I¹³⁰³F¹³⁰⁴M¹³⁰⁵ motif, which is located on the linker between DIII and DIV of the µ₁ Na⁺ channel (3, 4, 32). Fig. 9 shows the growth of inward currents through inactivation-defective DIV-A1529D + I1303Q/F1304Q/M1305Q channels with subsequent pulses at 20-s intervals. From a holding potential of 120 mV, the channels were first inactivated by a 300-s depolarizing prepulse to 50 mV (A) or 20 mV (B). Recovery from inactivation after returning to 120 mV was monitored through repetitive test pulses to 20 mV. In both cases, a considerable fraction of channels recovered with a very slow time constant on the order of ~100 s.

View larger version (30K): [in this window] [in a new window]   Fig. 9.   Ultra-slow inactivation in inactivation-defective DIV-A1529D + I1303Q/F1304Q/M1305Q channels. A, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D + I1303Q/F1304Q/M1305Q. From a holding potential of 120 mV, the channels were inactivated by a 300-s depolarizing step to 50 mV. Thereafter, the potential was returned to 120 mV, and recovery from inactivation was monitored by repetitive test pulses to 20 mV at 20-s intervals. B, like A, but a 300-s depolarizing step to 20 mV was applied. C, comparison of the time courses of recovery from inactivation obtained from a series of such experiments after 300-s prepulses to 50 mV (n = 12) or to 20 mV (n = 5). Peak inward currents were normalized to the final current level attained after full recovery. Recovery from ultra-slow inactivation took slightly longer after 300-s prepulses to 50 mV. For comparison, recovery from 300-s prepulses to 20 mV in the single mutant µ1-I1303Q/F1304Q/M1305Q is shown (n = 7). Whereas the time course of recovery in DIV-A1529D + I1303Q/F1304Q/M1305Q (DIV-ADQQQ) was best fit by a bi-exponential equation (Equation 1), the time course of recovery in µ1- I1303Q/F1304Q/M1305Q was best fit by a mono-exponential function (Equation 2). D, growth of inward current during recovery from ultra-slow inactivation in DIV-A1529D + I1303Q/F1304Q/M1305Q + 1 channels (/1 ratio, 0.3). From a holding potential of 120 mV, the channels were inactivated by a 300-s depolarizing step to 20 mV. Thereafter, the potential was returned to 120 mV, and recovery from inactivation was monitored by repetitive test pulses to 20 mV at 20-s intervals. Recovery from ultra-slow inactivation was similar to DIV-A1529D + I1303Q/F1304Q/M1305Q -only channels (compare with B). Functional association of the 1 subunit with inactivation-defective DIV-A1529D  subunits is confirmed by the acceleration of the current decay (compare with B).

The summaries of the recovery curves of a series of such experiments are shown in Fig. 9C. Bi-exponential curve fits to the data points (solid lines, see "Experimental Procedures") were used to estimate the fraction of DIV-A1529D + I1303Q/F1304Q/M1305Q channels that had entered the ultra-slow inactivated state after a 300-s prepulse to 50 and 20 mV. These fractions were 0.58 ± 0.01 (n = 12) and 0.44 ± 0.01 (n = 5), respectively. In DIV-A1529D channels that were not inactivation-defective, the corresponding fractions were 0.60 ± 0.02 (n = 19) and 0.13 ± 0.02 (n = 7). Comparing these fractions between inactivation-defective DIV-A1529D + I1303Q/F1304Q/M1305Q channels and DIV-A1529D channels, it can be noticed that a prepulse potential of 50 mV produced similar fractions of channels recovering from ultra-slow inactivation in both cases, whereas a prepulse potential of 20 mV produced considerable ultra-slow inactivation only in inactivation-defective channels. Also shown in Fig. 9C is the time course of recovery from a 300-s inactivating prepulse to 20 mV in I1303Q/F1304Q/M1305Q channels that did not carry the additional DIV-A1529D mutation in the selectivity filter (n = 7). In these channels recovery assumed a mono-exponential time course (Equation 2) with a time constant of ~30 s, which is in good agreement with previously reported findings (17). The state from which channels recover with a time constant of ~30 s has been referred to as "slow" inactivation. The fact that adding the "selectivity filter mutation" DIV-A1529D to the "inactivation gate mutation" I1303Q/F1304Q/M1305Q changes the mono-exponential time course of recovery from non-fast inactivation in I1303Q/F1304Q/M1305Q to a bi-exponential time course in DIV-A1529D + I1303Q/F1304Q/M1305Q suggests that the mutation DIV-A1529D created an additional inactivated state, i.e. ultra-slow inactivation. These results support the notion that the ultra-slow inactivated state observed in some selectivity filter mutations (1, 16) is distinct from the enhanced slow inactivated state observed in inactivation-defective channels (17, 20, 33, 34).

Stabilization of the fast inactivated state by a strongly depolarized potential (20 mV) dramatically inhibits entry to the ultra-slow inactivated state in DIV-A1529D channels but not in inactivation-defective DIV-A1529D + I1303Q/F1304Q/M1305Q channels (Fig. 9C). In the latter, the fast inactivated state cannot be efficiently stabilized due to the mutations I1303Q/F1304Q/M1305Q.

If the fast inactivated state cannot be stabilized in inactivation-defective DIV-A1529D + I1303Q/F1304Q/M1305Q channels, coexpression of the ₁ subunit, which stabilizes the fast inactivated state in DIV-A1529D channels should not affect the fraction of DIV-A1529D + I1303Q/F1304Q/M1305Q channels that can be driven to the ultra-slow inactivated state. Fig. 9D shows the growth of inward currents through DIV-A1529D + I1303Q/F1304Q/M1305Q + ₁ channels with subsequent pulses at 20-s intervals. The experimental conditions were identical to those in Fig. 9B. The accelerated current decay in Fig. 9D compared with 9B suggests functional coexpression of the ₁ subunit. The fraction of inactivation-defective  + ₁ channels recovering from ultra-slow inactivation after a 300-s prepulse to 20 mV, 0.43 ± 0.01 (n = 4), was very similar to the corresponding fraction of -only channels, 0.44 ± 0.01 (n = 5). Hence, coexpression of the ₁ subunit with DIV-A1529D + I1303Q/F1304Q/M1305Q channels did not affect ultra-slow inactivation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we investigated the relationship between fast inactivation and ultra-slow inactivation in DIV-A1529D channels. We recently reported that ultra-slow inactivation, which is characterized by time constants of entry to and recovery from inactivation in the order of ~100 s, is enhanced by charge-altering mutations of residues DIII-K1237 and DIV-A1529. Both residues are located in the putative selectivity filter of the channel. Entry to the ultra-slow inactivated state could be reduced by binding to the outer pore of a mutant µ-CTX, suggesting that ultra-slow inact