Charge immobilization of the voltage sensor in domain IV is independent of sodium current inactivation
1 The Nora Eccles Harrison Cardiovascular Research & Training Institute and Department of Internal Medicine, University of Utah, Salt Lake City, UT 84112, USA
2 Department of Medicine, University of Chicago, Chicago, IL 60637, USA
Abstract
Recovery from fast inactivation in voltage-dependent Na+ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICMMTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na+ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes 20% to total gating charge (Qmax), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICMMTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICMMTSET were recorded before and after modification with MTSEAo. Prior to MTSEAo, the time course of the gating charge during repolarization (OFF-charge) was best described by a single fast time constant. After MTSEA, the OFF-charge had both fast and slow components, with the slow component accounting for nearly 35% of Qmax. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.
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Introduction
Fast inactivation of Na+ channels, the primary process by which Na+ channels become non-conductive during an action potential, has been associated with the intracellular linker between domains III and IV acting as an inactivation particle (Vassilev et al. 1988; Stuhmer et al. 1989; West et al. 1992). Fast inactivation coincides with the appearance of a slow component in gating current during repolarization (i.e. charge immobilization), which is attributable to channels recovering from inactivation (Armstrong & Bezanilla, 1977). The immobilizable fraction of gating charge in Na+ channels has been found to account for almost 60% of total gating charge (Qmax) in a variety of Na+ channel preparations (Meves & Vogel, 1977; Nonner, 1980; Starkus et al. 1981; Greeff et al. 1982; Sheets et al. 2000). Gating currents in voltage-gated channels, including Na+ channels, arise principally from movements of the fourth transmembrane spanning segments (out of a total of six segments) in each of four domains that together form the subunit (see review by Bezanilla, 2000). In studies using site-directed fluorescent labelling of human skeletal muscle sodium channels both the S4 segments in domains III and IV, but not those in domains I and II, were found to be responsible for the immobilized gating charge associated with fast inactivation (Cha et al. 1999).
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Evidence for the role of the domain III–IV linker in the modulation of movement of voltage sensors in Na+ channels comes from experiments in which fast inactivation has been removed. Early studies in squid giant axon showed that internal perfusion of the non-specific proteolytic enzyme pronase removed fast inactivation and all charge immobilization (Armstrong & Bezanilla, 1977). Based on these and other experiments (Tanguy & Yeh, 1989), it was reasonable to expect that disruption of fast inactivation by specific mutagenesis of the inactivation particle would also eliminate charge immobilization. Unexpectedly, when fast inactivation was specifically removed by the substitution of a cysteine for the phenylalanine in the isoleucine, phenylalanine, methionine (IFM) motif in the domain III–IV linker and then modified by intracellular MTSET (ICMMTSET) (Kellenberger et al. 1996; Chahine et al. 1997; Vedantham & Cannon, 1998); however, charge immobilization was not eliminated but only reduced by about one-half (Sheets et al. 2000).
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To determine whether the S4-DIV was responsible for the remaining gating charge that could be immobilized after specific removal of fast inactivation, we studied a mutant Na+ channel that combined neutralization of the outermost arginine, R1, in the S4-DIV with ICMMTSET (R1C-DIV-ICMMTSET). If the S4-DIV was responsible for the residual gating charge immobilization in ICMMTSET, then neutralization of R1C-DIV should eliminate nearly all charge immobilization because R1 contributes approximately two-thirds of the gating charge originating from that S4 (Sheets et al. 1999). Furthermore, charge immobilization should be restored if extracellular MTSEA could add back a positive charge to the S4 in R1C-DIV-ICMMTSET, because methanothiosulphonate (MTS) reagents such as MTSET (2-trimethylammonium ethyl methanothiosulphonate bromide) and MTSEA ((2-aminoethyl methanothiosulphonate, hydrobromide) act by adding a single positive charge through a disulphide bond to an otherwise neutral cysteine residue (Holmgren et al. 1996). Our results demonstrated that charge immobilization of R1C-DIV-ICMMTSET could be restored, indicating that the movement of the S4-DIV is independent of the normal binding of the inactivation particle.
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Methods
cDNA clones
In the human heart Na+ channel (hH1a, NaV1.5) (kindly provided by H. Hartmann and A. Brown, see Hartmann et al. 1994), the phenylalanine in the IFM motif at amino acid position 1485 (Fig. 1) and the outermost arginine at position 1623 (numbering is based upon hH1, see Makielski et al. 2003) were both mutated to a cysteine by 4-primer PCR (Higuchi et al. 1988), and the entire inserts containing the mutated sites were sequenced. In addition, the cysteine within the external vestibule of the pore at position 373 was mutated to a tyrosine (C373Y) so that the sensitivity to block by tetrodotoxin (TTX) would be increased (Satin et al. 1992; Chen et al. 1996), and so that permeation would not be blocked by MTS reagents. In this report, wild-type hH1a refers to the wild-type channel with the C373Y mutation. For expression of the triple-mutated channel R1C-DIV-ICM in mammalian cells, the cDNA was subcloned directionally into the mammalian expression vector pRcCMV (Invitrogen, Carlsbad, CA, USA). In addition, we attempted the study of two additional mutant channels in which the second-outermost arginine in the S4-DIII (position 1303) was mutated to either a cysteine (R2C-DIII-ICM) or to a glutamine (R2Q-DIII-ICM). However, their level of expression was too low to accurately measure ‘OFF’ gating currents (OFF-Ig) even when they were cultured in TTX or TTX with lidocaine (lignocaine) at either 37°C or 29°C.
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A, families of ionic currents during step depolarizations between –70 and +20 mV. B, upper panel, decay time constants of single exponential fits to INa. At least 4 cells are included in each group. Note the absence of an effect on wild-type hH1a (with the pore mutation C373Y) by MTSEAo. Wild-type, ; wild-type + MTSEAo, ; R1C-DIV, ; R1C-DIV + MTSETo, ; R1C-DIV + MTSEAo, . Lower panel, predicted membrane topology of the sodium channel showing the four domains each with six transmembrane segments and the charged residues in the fourth transmembrane (S4) segments.
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Cell preparation
Multiple tsA201 cells (SV40-transformed human embryonic kidney-derived HEK293 cells) were fused together using polyethylene glycol as previously described (Sheets et al. 1996). After fusion the cells were placed in cell culture for several days to allow for membrane remodelling, and then they were transiently transfected using a calcium phosphate precipitation method (Invitrogen). Because anecdotal evidence suggested that cell survival of inactivation-impaired Na+ channel mutants may be increased by blocking conductance of sodium channels during cell culture, 300 nM TTX and 100 μM lidocaine were added to the culture solution after transfection. Three to six days after transfection fused cells were detached from culture dishes with trypsin-EDTA solution (Gibco, Grand Island, NY, USA) and studied electrophysiologically.
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Recording technique, solutions and experimental protocols
Recordings were made using a large bore, double-barrelled glass suction pipette for both voltage clamp and internal perfusion as previously described (Sheets et al. 1996). INa and gating currents (Ig) were measured with a virtual ground amplifier (Burr-Brown OPA-101) using a 2.5 M feedback resistor. Voltage protocols were imposed from a 16-bit DA converter (Masscomp 5450, Concurrent Computer, Tinton Falls, NJ, USA) over a 30/1 voltage divider. Data were filtered by the inherent response of the voltage-clamp circuit (corner frequency near 125 kHz) and recorded with a 16-bit AD converter on a Masscomp 5450 at 200 kHz. A fraction of the current was fed back to compensate for series resistance. Cells were studied at 13°C, and temperature was controlled using a Sensortek TS-4 thermoelectric stage (Physiotemp Instruments, Inc., Clifton, NJ, USA) mounted beneath the bath chambers.
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A cell was placed in the aperture of the pipette, and after a high resistance seal formed the cell membrane inside the pipette was disrupted with a manipulator-controlled platinum wire. Voltage control was assessed by evaluating the time course of the capacitive current and the steepness of the negative slope region of the peak current–voltage relationship according to criteria previously established (Hanck & Sheets, 1992). To allow for full sodium channel availability, the holding membrane potential was set between –150 and –170 mV. Ig protocols typically contained four repetitions at each test voltage that were 1/4 of a 60 Hz cycle out of phase to maximize rejection of this frequency and to improve the signal to noise ratio.
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The control extracellular solution for INa measurements contained (mM): 15 Na+, 185 TMA+, 2 Ca2+, 200 Mes– and 10 Hepes (pH 7.2), and the intracellular solution contained 200 TMA+, 75 F–, 125 Mes–, 10 EGTA, and 10 Hepes (pH 7.2). For the measurement of Ig the intracellular solution remained unchanged, while the extracellular solution had Na+ replaced by TMA+ and 1 μM saxitoxin (Calbiochem Corp., San Diego, CA, USA) was added. Both MTSET and MTSEA (Toronto Research Chemicals, Canada) were dissolved in solutions about 1 min prior to their use. For intracellular MTSET modification 2.5 mM was added to the intracellular solution for about 5 min before changing back to control solution. Depolarization to –30 mV for 50 ms from –150 mV at 0.5 Hz allowed for assessment of the modification of the cysteine in the ICM motif. Modification was recognized by the readily apparent further slowing of INa decay that was noticeable within 3 min and complete by 8–10 min (see Sheets et al. 2000). For modification by extracellular MTS reagents, the cell and the pipette were transferred to a bath chamber that contained 1.0 mM of the reagent dissolved in control extracellular solution containing 15 mM Na+. The cell was typically exposed to MTSEAo or MTSETo for 4–5 min while being periodically depolarized to –30 mV at 0.5 Hz, by which time R1C-DIV demonstrated a faster INa decay. R1C-DIV-ICMMTSET showed no obvious change in INa time course by extracellular MTS modification, although modification was apparent when recording gating currents (see Results).
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Data analysis
Leak resistance was calculated as the reciprocal of the linear conductance between –190 and –110 mV. Peak INa was taken as the mean of four data samples clustered around the maximal value of data digitally filtered at 5 kHz, and it was leak corrected by the amount of the calculated time-independent linear leak. Data were capacity corrected using 4–16 scaled current responses recorded from voltage steps typically between –150 and –190 mV. All Ig were leak corrected by the mean of 2–4 ms of data, usually beginning 8 ms after the change in test potential for ON-Ig, and after 10 ms for OFF-Ig.
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To determine time constants of Ig decay, current traces were trimmed until the decay phase was clearly apparent and then fitted by a sum of up to two exponentials with DISCRETE (Provencher, 1976), a program that provides a modified F-statistic in order to evaluate the number of exponential components that best describe the data. To calculate the fraction of gating charge associated with each of the exponential components, it was necessary to take into account the fact that the voltage clamp was not instantaneous. We first calculated the total OFF-charge during each repolarization step, and then extrapolated the exponential curve backwards from the first point used for curve fitting until the total charge from the fitted curve equalled the total OFF-charge measured for that voltage step. This method was a compromise between extrapolation of the data back to the start of the repolarization step, which would have resulted in over-estimation of the contribution of the fast time constant, and no extrapolation at all, which would have over-estimated the contribution of the slow time constant.
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Q–V relationships were fitted with a simple Boltzmann distribution:
where Q is the fractional gating charge during a depolarizing step, Vt is the test potential, and the fitted parameters are: Qmax, the maximum charge; V1/2, the half-point of the relationship; and s, the slope factor in millivolts. Fractional Q was calculated as Q/Qmax for each cell in the control solution and was plotted versus Vt.
Data were analysed and graphed on a SUN Ultra using SAS (Statistical Analysis System, Cary, NC, USA). Unless otherwise specified, summary statistics are expressed as means ± one standard deviation (S.D.). Figures show means ± standard error of the mean (S.E.M.). A paired t test was used to determine statistical significance at the P < 0.05 level.
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Results
Restoration of charge in R1C-DIV by extracellular MTSEA
Previous studies have shown that neutralization of the outermost arginine by cysteine (R1C-DIV) in the S4-DIV of voltage-gated Na+ channels dramatically slowed the decay of INa in response to step depolarizations (Yang & Horn, 1995; Sheets et al. 1999). Figure 1A shows representative sodium currents for R1C-DIV both before and after extracellular modification by either MTSEA or MTSET. Similar to a previous report (Yang & Horn, 1995) for the human skeletal muscle Na+ channel hSkM1, INa decay was accelerated after modification with MTSETo. Furthermore, we found that Na+ channel kinetics of R1C-DIV after MTSEAo were more like wild-type than those after MTSETo (Fig. 1B), although neither MTS reagent totally normalized INa decay. Although MTSEAo had a large effect on INa decay of R1C-DIV, it had only minor effects on both the peak current–voltage (I–V) relationship and the time-to-peak INa relationship (Fig. 2A), similar to the minimal effects of MTSEAo on wild-type hH1a (Fig. 2B). In contrast, the effects of modification by MTSETo were more variable (note the large error bars in Fig. 1B) and produced large voltage shifts in channel kinetics. One example of MTSET modification of R1C-DIV is shown in the insets to Fig. 2A, where the I–V relationship and time-to-peak INa demonstrated a large leftward shift of about –18 mV. The restoration of INa in R1C-DIV towards more normal ionic current kinetics was consistent with the MTS reagents adding back a positive charge on the S4-DIV. However, the two reagents were not entirely equivalent, which may have been because MTSEA, when covalently bound to cysteine, has a smaller volume than, and somewhat different charge characteristics from, MTSET (see Discussion). The best test of whether MTS reagents add back a positive charge is to measure the gating charge of R1C-DIV after modification by MTS reagents.
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A, peak I–V (left) and time-to-peak INa (right) relationships for four cells expressing R1C-DIV before () and after modification with MTSEAo (). The insets to both panels show a representative R1C-DIV cell modified with MTSETo. B, peak I–V (left) and time-to-peak INa (right) relationships for four wild-type cells before () and after modification with MTSEAo (). Details of the modification are given in the text. C, normalized Q–V relationships for four cells expressing R1C-DIV before () and after modification by MTSEAo (). The ON-gating charge was measured during step depolarizations from a holding membrane of –160 mV (or –170 mV) to various test potentials. Data for each cell were normalized to its own Qmax, and the continuous lines are the means of the best fits to each cell by a Boltzmann distribution (eqn (1)). The Qmax was significantly increased by 25 ± 4% after MTSEAo. The parameters from the best fits before MTSEAo showed the V1/2 to be –51 ± 3 mV with a slope factor of –14.4 ± 1.2 mV, and after modification by MTSEAo the V1/2 was –63 ± 3 mV with a slope factor of –17.2 ± 0.7 mV. The negative shift in the Q–V relationship after MTSEAo was due, in part, to the longer time delay between the measurements in control and after MTSEAo compared to the Q–V relationships in Fig. 3D.
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We have previously reported that the voltage sensor in domain IV contributes approximately 30% to the total gating charge (Qmax), with the outermost arginine, R1, contributing 19%, R2 contributing 9%, and R3 contributing 4% (Sheets et al. 1999). Consequently, if positively charged MTS reagents could restore the missing charge in R1C-DIV, Qmax would be predicted to increase by 23% (i.e.% increase = 0.19/0.81). Figure 2C shows the mean gating charge–voltage (Q–V) relationship for four cells expressing R1C-DIV before and after modification by extracellular MTSEA. Consistent with the prediction that MTSEAo could restore charge to the voltage sensor, Qmax increased by 25 ± 4%. Results on gating charge by MTSETo were variable compared to those with MTSEAo probably because of the accompanying large negative shift in Na+ channel kinetics (see Fig. 2A) making it difficult to maintain full channel availability.
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Ionic current recordings of R1C-DIV-ICM
By combining the ability to restore gating charge in R1C-DIV by MTSEAo with a mutation to remove fast inactivation, we were able to investigate the role of the S4-DIV in the origin of the remaining gating charge that could be immobilized in the absence of fast inactivation. Previous studies have shown (Kellenberger et al. 1996; Chahine et al. 1997; Vedantham & Cannon, 1998; Sheets et al. 2000) that modification by MTSETi of a cysteine substituted for the phenylalanine in the IFM motif of the linker between domains III and IV resulted in the removal of fast inactivation. Figure 3A shows a family of ionic current traces of R1C-DIV-ICM after modification by 2.5 mM MTSETi (R1C-DIV-ICMMTSET) with near complete inhibition of INa decay. Previously, it has been shown that MTSETi does not affect wild-type INa (Yang et al. 1996; Sheets et al. 1999). Contrary to the speeding-up of INa decay when R1C-DIV was modified by MTSEAo (see Fig. 1), there was no obvious change in the decay of INa in R1C-DIV-ICMMTSET after MTSEAo. We noted only a small time-dependent shift (Sheets & Hanck, 1999) in the peak I–V relationship of R1C-DIV-ICMMTSET after modification by MTSEAo (Fig. 3B), which was similar in magnitude to that observed when R1C-DIV was modified by MTSEAo.
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A, families of leak- and capacity-corrected INa during step depolarizations from –110 to 10 mV from a holding potential of –150 mV for a cell expressing R1C-DIV-ICMMTSET (upper panel) and after modification by MTSEAo (lower panel). B, the mean peak I–V relationships for four cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). INa was normalized to the maximum peak inward INa for each cell expressing R1C-DIV-ICMMTSET. C,ON-gating currents (upper panels) and their integrals (lower panels) in a cell expressing R1C-DIV-ICMMTSET before (left) and after MTSEAo (right) for step depolarizations between –110 and 40 mV from a holding potential of –160 mV. Data shown are capacity and leak corrected, digitally filtered at 15 kHz, and every fourth point plotted. D, Q–V relationships for six cells expressing R1C-DIV-ICMMTSET normalized to the Qmax for each cell before () and after MTSEAo (). Continuous lines represent the means of the best fits to each cell by a Boltzmann distribution (eqn (1)). The parameters from the best fits for R1C-DIV-ICMMTSET showed that the V1/2 was –58 ± 4 mV and the slope factor was –15.0 ± 2 mV. After MTSEAo the V1/2 was –60 ± 5 mV, the slope factor was –16 ± 2 mV, and the normalized Qmax increased by 24 ± 5% to a normalized value of 1.24 ± 0.05. Only the change in Qmax was statistically significant.
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Extracellular MTSEA increases ON-gating charge of R1C-DIV-ICMMTSET
Even though the presumed positive charge added by MTSEAo to the cysteine at the R1 position in R1C-DIV-ICMMTSET did not affect INa, it did affect gating charge (ON-charge) in response to step depolarizations. Figure 3C shows there was an obvious increase in both the maximal amplitude of the ON-gating currents and in their integrals after MTSEAo. The mean increase in Qmax obtained from the Boltzmann fits to Q–V relationships for six R1C-DIV-ICMMTSET cells after modification by MTSEAo was 24% when compared to the Qmax in control. As anticipated, the magnitude of increase in the Qmax after MTSEAo was similar to that observed after MTSEAo modification of R1C-DIV (Fig. 2C) suggesting that MTSEAo added a full positive charge, eo, to the mutant channel.
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Off-gating charge studies
Internal perfusion of the squid giant axon with the proteolytic enzyme pronase has been shown to remove fast inactivation and to eliminate all charge immobilization (Armstrong & Bezanilla, 1977). On the other hand, we have previously found (Sheets et al. 2000) that specific removal of fast inactivation in ICMMTSET did not eliminate all charge immobilization but only reduced its magnitude to about 30% from a magnitude of 50–60% in native Na+ channels. If the remaining immobilizable gating charge originated from the S4-DIV in ICMMTSET, then the mutant channel R1C-DIV-ICMMTSET should exhibit almost no charge immobilization because the outermost arginine is largely responsible for the gating charge arising from the S4-DIV (Sheets et al. 1999). Figure 4A shows an example of OFF-Ig and OFF-charge in R1C-DIV-ICMMTSET before and after MTSEAo recorded at a repolarization potential of –180 mV following conditioning steps to 0 mV for various lengths of time up to 15 ms. The potential of –180 mV was selected because previous studies on wild-type hH1a have shown that the fast and slow time constants of OFF-Ig are different by about an order of magnitude permitting separation during two exponential fits. In addition, the OFF-charge at –180 mV returned fast enough so that all of it could be measured (Sheets et al. 2000). In control solutions the OFF-charge in R1C-DIV-ICMMTSET appeared to have only a fast component as can be seen by the similar time courses of all four of the integrals. However, after modification by MTSEAo the OFF-charge in R1C-DIV-ICMMTSET developed a slow component. For comparison, wild-type OFF-charge integrals are also shown before and after exposure to MTSEAo (Fig. 4B), where the increased magnitude of the slow components are readily apparent and appear unchanged by MTSEAo.
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A, families of OFF-gating currents and their integrals for a cell expressing R1C-DIV-ICMMTSET before (left) and after MTSEAo (right). The smallest integral in each panel corresponds to a conditioning step for 0.7 ms. Also shown for each cell is an ON-Ig in response to a step depolarization to 0 mV from a holding potential of –160 mV. B, families of OFF-gating currents and their integrals for wild-type hH1a before (left) and after MTSEAo (right) using the same voltage-clamp protocol as above. Data shown are capacity and leak corrected, digitally filtered at 15 kHz, and every fourth point plotted.
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Consistent with the studies above, the magnitude of OFF-charge after MTSEAo modification of R1C-DIV-ICMMTSET was increased by 21% (Fig. 5A), a value similar to the increase in ON-charge after MTSEAo modification of both R1C-DIV and R1C-DIV-ICMMTSET.
A, the mean OFF-charge at –180 mV for six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo () following conditioning steps to 0 mV for durations up to 44 ms. Gating charge was normalized to the maximal OFF-charge for each cell, calculated from its fitted value of Qmax–OFF. Continuous lines were calculated using the means of fitted parameters from each cell to the following: Normalized Q = Qmax–OFF(1 – e–t/), where the parameters of the fit were the time constant () of the appearance of OFF-charge and Qmax–OFF, the maximal OFF-gating charge. Qmax–OFF for R1C-DIV-ICMMTSET increased to 1.21 ± 0.04 (n = 6 cells) after modification by MTSEAo compared to control, while the was increased to 0.81 ± 0.15 ms after MTSEAo compared to a value of 0.66 ± 0.07 for R1C-DIV-ICMMTSET. Both changes were significantly different (P < 0.05). B and C, the mean fast and mean slow time constants, respectively, from up to two exponential fits to OFF-gating current relaxations during repolarization to –180 mV after conditioning to 0 mV for times from 0.7 ms to 44 ms in six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). C, the mean slow time constants from two exponential fits. For comparison both the fast and slow time constants for wild-type hH1a () are also shown (taken from Sheets et al. 2000). Prior to modification by MTSEAo () there were no slow time constants in the OFF-Ig for R1C-DIV-ICMMTSET until the conditioning duration was 10 ms when one of the six cells developed a slow time constant (see Results). After MTSEAo () all R1C-DIV-ICMMTSET cells developed slow time constants after conditioning durations of 7 ms, while none had slow time constants at times less than 1.5 ms. At times intermediate between the two conditioning durations one R1C-DIV-ICMMTSET cell developed a second time constant by 1.5 ms, one cell by 2 ms, two cells by 3 ms and five cells by 4.5 ms. In contrast, wild-type cells developed a slow time constant by 0.7 ms. D, the fraction of OFF-charge associated with the fast time constant for six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). Also shown are the fractions for wild-type hH1a () (Sheets et al. 2000). Lines connect the points. The fractional charge was calculated as the ratio of gating charge associated with the fast time constant compared to the total OFF-charge associated with each conditioning duration from 0.7 to 44 ms at a conditioning voltage, VC, of 0 mV. The differences for R1C-DIV-ICMMTSET were significantly different (P < 0.05) at all times except for the three earliest times of 0.7, 1.1 and 1.5 ms. For six cells at conditioning times 7.0 ms the mean fraction of OFF-charge in the fast time constant of R1C-DIV-ICMMTSET was 0.98 ± 0.05, and after MTSEAo it decreased to 0.65 ± 0.03. For wild-type the fraction was nearly 0.5.
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To quantify the OFF-Ig relaxation time constants, they were fitted by a sum of up to two exponentials and sorted into fast and slow time constants. When only a single exponential best fitted the OFF-Ig it was assigned as a fast time constant. Figure 5B and C shows the results for six cells expressing R1C-DIV-ICMMTSET before and after modification by MTSEAo. Five of the six R1C-DIV-ICMMTSET cells had only fast time constants of relaxation with a value of nearly 0.3 ms before modification by MTSEAo. However, one cell exhibited exceptionally large gating currents, and it had a slow time constant detected in the OFF-Ig relaxation before modification by MTSEAo, most likely because its large gating currents permitted the remaining residues in the S4-DIV (estimated to be 10% of overall gating charge; Sheets et al. 1999) to make a contribution sufficiently large to be detected by the fitting routine (see Fig. 5C). In contrast, after modification by MTSEAo all cells developed a second, slow time constant at conditioning durations of 7 ms (Fig. 5B).
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Coincident with the development of a slow time constant in OFF-Ig, the fraction of OFF-charge accounted for in the fast component decreased as the conditioning time duration at 0 mV increased. Figure 5D shows the fraction of OFF-charge contained in the fast component compared to the total OFF-charge measured at each conditioning duration from 0.7 to 44 ms for R1C-DIV-ICMMTSET before and after MTSEAo. Before MTSEAo almost all the OFF-charge was contained in the fast time constant, while after MTSEAo the fraction of OFF-charge contributed by the fast time constant decreased as the conditioning duration increased. The reduction in the fraction of OFF-charge accounted for by the fast component was significantly different (P < 0.05) at all times except for the three earliest times of 0.7, 1.1 and 1.5 ms. For all conditioning times 7.0 ms the mean fraction of OFF-charge in the fast time constant of R1C-DIV-ICMMTSET before MTSEAo was 0.98 ± 0.05 (n = 6 cells), while after MTSEAo it decreased to 0.65 ± 0.03. Consequently, the mean fraction of OFF-charge contained in the slow time constant was 35%, a value similar to the 31% contribution made by the S4-DIV to the maximal ON-charge in wild-type hH1a (Sheets et al. 1999). For comparison to wild-type hH1a, charge immobilization was first noted after conditioning times of 0.7 ms and accounted for about 50% of the total OFF-charge at later times (Sheets et al. 2000).
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Discussion
We investigated the molecular origin of the slow recovery of gating charge (i.e. charge immobilization) that remained in hH1a when fast inactivation was specifically removed by MTSETi modification of a cysteine in the ICM motif in the inactivation particle formed by the linker between domains III and IV. Our experiments with charge restoration of R1C-DIV-ICMMTSET demonstrated that the S4-DIV is responsible for the residual charge immobilization when fast inactivation has been specifically removed.
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In fast-inactivated wild-type Na+ channels, gating charge becomes immobilized secondary to the slow return of the S4 segments in domains III and IV during repolarization, while the S4 segments from domains I and II return quickly (Cha et al. 1999). Our results showed that charge immobilization of the S4-DIV occurred regardless of whether or not fast inactivation was present, suggesting that the inactivation particle in the wild-type Na+ channel can modulate the time course of gating charge recovery of only the S4-DIII and not that for the S4-DIV. We cannot rule out the possibility that the removal of fast inactivation using MTSETi modification of the ICM motif in the inactivation particle may still permit partial binding of the altered inactivation particle to occur thereby causing the S4-DIV to become immobilized. However, if partial binding occurs with mutations in the IFM motif they do not appear to alter the magnitude of single channel conductance (Hartmann et al. 1994). Furthermore, it is important to note that S4-DIV has been shown to move more slowly than the S4 segments in the other three domains during step depolarizations (Hanck & Sheets, 1995; Sheets & Hanck, 1995; Sheets et al. 1999; Chanda & Bezanilla, 2002). Consequently, it is likely that the slow movement of the S4-DIV in Na+ channels may be an intrinsic property arising from interactions with adjacent transmembrane segments, their segmental linkers or perhaps adjacent domains (Chanda et al. 2004). These findings contrast with those from Shaker K+ channels where most, if not all, of the gating charge results from channel activation transitions leading to channel opening (Schoppa et al. 1992; Bezanilla et al. 1994; Zagotta et al. 1994; Seoh et al. 1996) with little or no gating charge movement after channel opening.
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Both MTSET and MTSEA are able to add a single positive charge through a disulphide bond to an otherwise neutral cysteine residue. MTSETo has been shown to partially restore fast inactivation as measured by the speeding up of INa decay in the paramyotonia congenita mutant hSkM1 Na+ channel R1448C, that also has the outermost arginine in the S4-DIV mutated to a cysteine (Yang & Horn, 1995). We show that this is also the case for R1C-DIV in hH1a for both MTSETo and MTSEAo. Although both MTSEAo and MTSETo were able to modify the S4-DIV, neither of the MTS agents completely restored the modified channel's kinetics to those of the wild-type phenotype. In particular, MTSETo caused large negative shifts in channel kinetics of hH1a by up to –18 mV similar to that found for R1448C in the hSkM1 Na+ channel (Yang & Horn, 1995). This may not be surprising given the different sizes of the two MTS reagents when linked to a cysteine. MTSEA, the smaller of the two reagents, is estimated to be a molecule with a volume of 138 3 after binding with cysteine (personal communication from Gregory Lipkind, University of Chicago, using calculations from the Search/Compare module in Insight from Accelrys Corp, San Diego, CA, USA), and it is only slightly larger than the estimate for an arginine (with a volume of 129 3) in the wild-type channel. In addition, MTSEA's charged nitrogen is surrounded by hydrogens in an ‘open’ charge configuration while the smaller arginine has a more distributed charge. In contrast, MTSET is a much larger residue with a volume of 183 3. Moreover, its nitrogen is surrounded by methyl groups that screen its positive charge making it more likely to interact with nearby hydrophobic neighbours. Furthermore, the charged, primary amine of MTSEA would be able to undergo hydrogen bonding while this would not be possible for MTSET with its quaternary amine. It is likely that a combination of characteristics permitted MTSEAo to be better at restoring channel kinetics and gating charge at the R1C-DIV position compared to MTSETo.
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MTSEAo not only partially restored fast inactivation of INa in R1C-DIV, but also increased Qmax by 24%, an amount equivalent to that predicted when the outermost arginine residue in the S4-DIV was neutralized (Sheets et al. 1999). Although our gating charge measurements do not allow us to calculate the absolute amount of positive charge added by MTSEA to Qmax, it does allow an estimate to be made. Because MTSEA can add no more than a single eo to the Na+ channel, an increase in Qmax by 24%, would predict a lower limit of charges per channel to be approximately 5.2eo (1eo = (0.24/1.24) x Qmax). However, this estimate may be too small if neutralization of R1 in domain IV had a profound effect on the distribution of other charged residues with respect to the electric field, as has been suggested for the Shaker K+ channel (Seoh et al. 1996). Recently, it has been reported that domains I and IV in the rat skeletal muscle Na+ channel may interact suggesting that the charged residues in the S4 segments may have a complex arrangement (Chanda et al. 2004). Furthermore, an estimate of 12eo per Na+ channel was predicted based on an analysis of the limiting slope conductance of single channel data at threshold potentials (Hirschberg et al. 1995), which is more comparable to the values predicted for Shaker K+ channels (Schoppa et al. 1992; Bezanilla et al. 1994; Zagotta et al. 1994). If Na+ channels are found to have much less total charge per channel than K+ channels this may not be unexpected when one considers that the voltage sensors in Na+ channels are not identical, and they may play more diverse roles in channel kinetics. Future studies will have to resolve this question.
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2 Department of Medicine, University of Chicago, Chicago, IL 60637, USA
Abstract
Recovery from fast inactivation in voltage-dependent Na+ channels is associated with a slow component in the time course of gating charge during repolarization (i.e. charge immobilization), which results from the slow movement of the S4 segments in domains III and IV (S4-DIII and S4-DIV). Previous studies have shown that the non-specific removal of fast inactivation by the proteolytic enzyme pronase eliminated charge immobilization, while the specific removal of fast inactivation (by intracellular MTSET modification of a cysteine substituted for the phenylalanine in the IFM motif, ICMMTSET, in the inactivation particle formed by the linker between domains III and IV) only reduced the amount of charge immobilization by nearly one-half. To investigate the molecular origin of the remaining slow component of charge immobilization we studied the human cardiac Na+ channel (hH1a) in which the outermost arginine in the S4-DIV, which contributes 20% to total gating charge (Qmax), was mutated to a cysteine (R1C-DIV). Gating charge could be fully restored in R1C-DIV by exposure to extracellular MTSEA, a positively charged methanethiosulphonate reagent. The RIC-DIV mutation was combined with ICMMTSET to remove fast inactivation, and the gating currents of R1C-DIV-ICMMTSET were recorded before and after modification with MTSEAo. Prior to MTSEAo, the time course of the gating charge during repolarization (OFF-charge) was best described by a single fast time constant. After MTSEA, the OFF-charge had both fast and slow components, with the slow component accounting for nearly 35% of Qmax. These results demonstrate that the slow movement of the S4-DIV during repolarization is not dependent upon the normal binding of the inactivation particle.
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Introduction
Fast inactivation of Na+ channels, the primary process by which Na+ channels become non-conductive during an action potential, has been associated with the intracellular linker between domains III and IV acting as an inactivation particle (Vassilev et al. 1988; Stuhmer et al. 1989; West et al. 1992). Fast inactivation coincides with the appearance of a slow component in gating current during repolarization (i.e. charge immobilization), which is attributable to channels recovering from inactivation (Armstrong & Bezanilla, 1977). The immobilizable fraction of gating charge in Na+ channels has been found to account for almost 60% of total gating charge (Qmax) in a variety of Na+ channel preparations (Meves & Vogel, 1977; Nonner, 1980; Starkus et al. 1981; Greeff et al. 1982; Sheets et al. 2000). Gating currents in voltage-gated channels, including Na+ channels, arise principally from movements of the fourth transmembrane spanning segments (out of a total of six segments) in each of four domains that together form the subunit (see review by Bezanilla, 2000). In studies using site-directed fluorescent labelling of human skeletal muscle sodium channels both the S4 segments in domains III and IV, but not those in domains I and II, were found to be responsible for the immobilized gating charge associated with fast inactivation (Cha et al. 1999).
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Evidence for the role of the domain III–IV linker in the modulation of movement of voltage sensors in Na+ channels comes from experiments in which fast inactivation has been removed. Early studies in squid giant axon showed that internal perfusion of the non-specific proteolytic enzyme pronase removed fast inactivation and all charge immobilization (Armstrong & Bezanilla, 1977). Based on these and other experiments (Tanguy & Yeh, 1989), it was reasonable to expect that disruption of fast inactivation by specific mutagenesis of the inactivation particle would also eliminate charge immobilization. Unexpectedly, when fast inactivation was specifically removed by the substitution of a cysteine for the phenylalanine in the isoleucine, phenylalanine, methionine (IFM) motif in the domain III–IV linker and then modified by intracellular MTSET (ICMMTSET) (Kellenberger et al. 1996; Chahine et al. 1997; Vedantham & Cannon, 1998); however, charge immobilization was not eliminated but only reduced by about one-half (Sheets et al. 2000).
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To determine whether the S4-DIV was responsible for the remaining gating charge that could be immobilized after specific removal of fast inactivation, we studied a mutant Na+ channel that combined neutralization of the outermost arginine, R1, in the S4-DIV with ICMMTSET (R1C-DIV-ICMMTSET). If the S4-DIV was responsible for the residual gating charge immobilization in ICMMTSET, then neutralization of R1C-DIV should eliminate nearly all charge immobilization because R1 contributes approximately two-thirds of the gating charge originating from that S4 (Sheets et al. 1999). Furthermore, charge immobilization should be restored if extracellular MTSEA could add back a positive charge to the S4 in R1C-DIV-ICMMTSET, because methanothiosulphonate (MTS) reagents such as MTSET (2-trimethylammonium ethyl methanothiosulphonate bromide) and MTSEA ((2-aminoethyl methanothiosulphonate, hydrobromide) act by adding a single positive charge through a disulphide bond to an otherwise neutral cysteine residue (Holmgren et al. 1996). Our results demonstrated that charge immobilization of R1C-DIV-ICMMTSET could be restored, indicating that the movement of the S4-DIV is independent of the normal binding of the inactivation particle.
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Methods
cDNA clones
In the human heart Na+ channel (hH1a, NaV1.5) (kindly provided by H. Hartmann and A. Brown, see Hartmann et al. 1994), the phenylalanine in the IFM motif at amino acid position 1485 (Fig. 1) and the outermost arginine at position 1623 (numbering is based upon hH1, see Makielski et al. 2003) were both mutated to a cysteine by 4-primer PCR (Higuchi et al. 1988), and the entire inserts containing the mutated sites were sequenced. In addition, the cysteine within the external vestibule of the pore at position 373 was mutated to a tyrosine (C373Y) so that the sensitivity to block by tetrodotoxin (TTX) would be increased (Satin et al. 1992; Chen et al. 1996), and so that permeation would not be blocked by MTS reagents. In this report, wild-type hH1a refers to the wild-type channel with the C373Y mutation. For expression of the triple-mutated channel R1C-DIV-ICM in mammalian cells, the cDNA was subcloned directionally into the mammalian expression vector pRcCMV (Invitrogen, Carlsbad, CA, USA). In addition, we attempted the study of two additional mutant channels in which the second-outermost arginine in the S4-DIII (position 1303) was mutated to either a cysteine (R2C-DIII-ICM) or to a glutamine (R2Q-DIII-ICM). However, their level of expression was too low to accurately measure ‘OFF’ gating currents (OFF-Ig) even when they were cultured in TTX or TTX with lidocaine (lignocaine) at either 37°C or 29°C.
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A, families of ionic currents during step depolarizations between –70 and +20 mV. B, upper panel, decay time constants of single exponential fits to INa. At least 4 cells are included in each group. Note the absence of an effect on wild-type hH1a (with the pore mutation C373Y) by MTSEAo. Wild-type, ; wild-type + MTSEAo, ; R1C-DIV, ; R1C-DIV + MTSETo, ; R1C-DIV + MTSEAo, . Lower panel, predicted membrane topology of the sodium channel showing the four domains each with six transmembrane segments and the charged residues in the fourth transmembrane (S4) segments.
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Cell preparation
Multiple tsA201 cells (SV40-transformed human embryonic kidney-derived HEK293 cells) were fused together using polyethylene glycol as previously described (Sheets et al. 1996). After fusion the cells were placed in cell culture for several days to allow for membrane remodelling, and then they were transiently transfected using a calcium phosphate precipitation method (Invitrogen). Because anecdotal evidence suggested that cell survival of inactivation-impaired Na+ channel mutants may be increased by blocking conductance of sodium channels during cell culture, 300 nM TTX and 100 μM lidocaine were added to the culture solution after transfection. Three to six days after transfection fused cells were detached from culture dishes with trypsin-EDTA solution (Gibco, Grand Island, NY, USA) and studied electrophysiologically.
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Recording technique, solutions and experimental protocols
Recordings were made using a large bore, double-barrelled glass suction pipette for both voltage clamp and internal perfusion as previously described (Sheets et al. 1996). INa and gating currents (Ig) were measured with a virtual ground amplifier (Burr-Brown OPA-101) using a 2.5 M feedback resistor. Voltage protocols were imposed from a 16-bit DA converter (Masscomp 5450, Concurrent Computer, Tinton Falls, NJ, USA) over a 30/1 voltage divider. Data were filtered by the inherent response of the voltage-clamp circuit (corner frequency near 125 kHz) and recorded with a 16-bit AD converter on a Masscomp 5450 at 200 kHz. A fraction of the current was fed back to compensate for series resistance. Cells were studied at 13°C, and temperature was controlled using a Sensortek TS-4 thermoelectric stage (Physiotemp Instruments, Inc., Clifton, NJ, USA) mounted beneath the bath chambers.
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A cell was placed in the aperture of the pipette, and after a high resistance seal formed the cell membrane inside the pipette was disrupted with a manipulator-controlled platinum wire. Voltage control was assessed by evaluating the time course of the capacitive current and the steepness of the negative slope region of the peak current–voltage relationship according to criteria previously established (Hanck & Sheets, 1992). To allow for full sodium channel availability, the holding membrane potential was set between –150 and –170 mV. Ig protocols typically contained four repetitions at each test voltage that were 1/4 of a 60 Hz cycle out of phase to maximize rejection of this frequency and to improve the signal to noise ratio.
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The control extracellular solution for INa measurements contained (mM): 15 Na+, 185 TMA+, 2 Ca2+, 200 Mes– and 10 Hepes (pH 7.2), and the intracellular solution contained 200 TMA+, 75 F–, 125 Mes–, 10 EGTA, and 10 Hepes (pH 7.2). For the measurement of Ig the intracellular solution remained unchanged, while the extracellular solution had Na+ replaced by TMA+ and 1 μM saxitoxin (Calbiochem Corp., San Diego, CA, USA) was added. Both MTSET and MTSEA (Toronto Research Chemicals, Canada) were dissolved in solutions about 1 min prior to their use. For intracellular MTSET modification 2.5 mM was added to the intracellular solution for about 5 min before changing back to control solution. Depolarization to –30 mV for 50 ms from –150 mV at 0.5 Hz allowed for assessment of the modification of the cysteine in the ICM motif. Modification was recognized by the readily apparent further slowing of INa decay that was noticeable within 3 min and complete by 8–10 min (see Sheets et al. 2000). For modification by extracellular MTS reagents, the cell and the pipette were transferred to a bath chamber that contained 1.0 mM of the reagent dissolved in control extracellular solution containing 15 mM Na+. The cell was typically exposed to MTSEAo or MTSETo for 4–5 min while being periodically depolarized to –30 mV at 0.5 Hz, by which time R1C-DIV demonstrated a faster INa decay. R1C-DIV-ICMMTSET showed no obvious change in INa time course by extracellular MTS modification, although modification was apparent when recording gating currents (see Results).
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Data analysis
Leak resistance was calculated as the reciprocal of the linear conductance between –190 and –110 mV. Peak INa was taken as the mean of four data samples clustered around the maximal value of data digitally filtered at 5 kHz, and it was leak corrected by the amount of the calculated time-independent linear leak. Data were capacity corrected using 4–16 scaled current responses recorded from voltage steps typically between –150 and –190 mV. All Ig were leak corrected by the mean of 2–4 ms of data, usually beginning 8 ms after the change in test potential for ON-Ig, and after 10 ms for OFF-Ig.
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To determine time constants of Ig decay, current traces were trimmed until the decay phase was clearly apparent and then fitted by a sum of up to two exponentials with DISCRETE (Provencher, 1976), a program that provides a modified F-statistic in order to evaluate the number of exponential components that best describe the data. To calculate the fraction of gating charge associated with each of the exponential components, it was necessary to take into account the fact that the voltage clamp was not instantaneous. We first calculated the total OFF-charge during each repolarization step, and then extrapolated the exponential curve backwards from the first point used for curve fitting until the total charge from the fitted curve equalled the total OFF-charge measured for that voltage step. This method was a compromise between extrapolation of the data back to the start of the repolarization step, which would have resulted in over-estimation of the contribution of the fast time constant, and no extrapolation at all, which would have over-estimated the contribution of the slow time constant.
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Q–V relationships were fitted with a simple Boltzmann distribution:
where Q is the fractional gating charge during a depolarizing step, Vt is the test potential, and the fitted parameters are: Qmax, the maximum charge; V1/2, the half-point of the relationship; and s, the slope factor in millivolts. Fractional Q was calculated as Q/Qmax for each cell in the control solution and was plotted versus Vt.
Data were analysed and graphed on a SUN Ultra using SAS (Statistical Analysis System, Cary, NC, USA). Unless otherwise specified, summary statistics are expressed as means ± one standard deviation (S.D.). Figures show means ± standard error of the mean (S.E.M.). A paired t test was used to determine statistical significance at the P < 0.05 level.
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Results
Restoration of charge in R1C-DIV by extracellular MTSEA
Previous studies have shown that neutralization of the outermost arginine by cysteine (R1C-DIV) in the S4-DIV of voltage-gated Na+ channels dramatically slowed the decay of INa in response to step depolarizations (Yang & Horn, 1995; Sheets et al. 1999). Figure 1A shows representative sodium currents for R1C-DIV both before and after extracellular modification by either MTSEA or MTSET. Similar to a previous report (Yang & Horn, 1995) for the human skeletal muscle Na+ channel hSkM1, INa decay was accelerated after modification with MTSETo. Furthermore, we found that Na+ channel kinetics of R1C-DIV after MTSEAo were more like wild-type than those after MTSETo (Fig. 1B), although neither MTS reagent totally normalized INa decay. Although MTSEAo had a large effect on INa decay of R1C-DIV, it had only minor effects on both the peak current–voltage (I–V) relationship and the time-to-peak INa relationship (Fig. 2A), similar to the minimal effects of MTSEAo on wild-type hH1a (Fig. 2B). In contrast, the effects of modification by MTSETo were more variable (note the large error bars in Fig. 1B) and produced large voltage shifts in channel kinetics. One example of MTSET modification of R1C-DIV is shown in the insets to Fig. 2A, where the I–V relationship and time-to-peak INa demonstrated a large leftward shift of about –18 mV. The restoration of INa in R1C-DIV towards more normal ionic current kinetics was consistent with the MTS reagents adding back a positive charge on the S4-DIV. However, the two reagents were not entirely equivalent, which may have been because MTSEA, when covalently bound to cysteine, has a smaller volume than, and somewhat different charge characteristics from, MTSET (see Discussion). The best test of whether MTS reagents add back a positive charge is to measure the gating charge of R1C-DIV after modification by MTS reagents.
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A, peak I–V (left) and time-to-peak INa (right) relationships for four cells expressing R1C-DIV before () and after modification with MTSEAo (). The insets to both panels show a representative R1C-DIV cell modified with MTSETo. B, peak I–V (left) and time-to-peak INa (right) relationships for four wild-type cells before () and after modification with MTSEAo (). Details of the modification are given in the text. C, normalized Q–V relationships for four cells expressing R1C-DIV before () and after modification by MTSEAo (). The ON-gating charge was measured during step depolarizations from a holding membrane of –160 mV (or –170 mV) to various test potentials. Data for each cell were normalized to its own Qmax, and the continuous lines are the means of the best fits to each cell by a Boltzmann distribution (eqn (1)). The Qmax was significantly increased by 25 ± 4% after MTSEAo. The parameters from the best fits before MTSEAo showed the V1/2 to be –51 ± 3 mV with a slope factor of –14.4 ± 1.2 mV, and after modification by MTSEAo the V1/2 was –63 ± 3 mV with a slope factor of –17.2 ± 0.7 mV. The negative shift in the Q–V relationship after MTSEAo was due, in part, to the longer time delay between the measurements in control and after MTSEAo compared to the Q–V relationships in Fig. 3D.
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We have previously reported that the voltage sensor in domain IV contributes approximately 30% to the total gating charge (Qmax), with the outermost arginine, R1, contributing 19%, R2 contributing 9%, and R3 contributing 4% (Sheets et al. 1999). Consequently, if positively charged MTS reagents could restore the missing charge in R1C-DIV, Qmax would be predicted to increase by 23% (i.e.% increase = 0.19/0.81). Figure 2C shows the mean gating charge–voltage (Q–V) relationship for four cells expressing R1C-DIV before and after modification by extracellular MTSEA. Consistent with the prediction that MTSEAo could restore charge to the voltage sensor, Qmax increased by 25 ± 4%. Results on gating charge by MTSETo were variable compared to those with MTSEAo probably because of the accompanying large negative shift in Na+ channel kinetics (see Fig. 2A) making it difficult to maintain full channel availability.
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Ionic current recordings of R1C-DIV-ICM
By combining the ability to restore gating charge in R1C-DIV by MTSEAo with a mutation to remove fast inactivation, we were able to investigate the role of the S4-DIV in the origin of the remaining gating charge that could be immobilized in the absence of fast inactivation. Previous studies have shown (Kellenberger et al. 1996; Chahine et al. 1997; Vedantham & Cannon, 1998; Sheets et al. 2000) that modification by MTSETi of a cysteine substituted for the phenylalanine in the IFM motif of the linker between domains III and IV resulted in the removal of fast inactivation. Figure 3A shows a family of ionic current traces of R1C-DIV-ICM after modification by 2.5 mM MTSETi (R1C-DIV-ICMMTSET) with near complete inhibition of INa decay. Previously, it has been shown that MTSETi does not affect wild-type INa (Yang et al. 1996; Sheets et al. 1999). Contrary to the speeding-up of INa decay when R1C-DIV was modified by MTSEAo (see Fig. 1), there was no obvious change in the decay of INa in R1C-DIV-ICMMTSET after MTSEAo. We noted only a small time-dependent shift (Sheets & Hanck, 1999) in the peak I–V relationship of R1C-DIV-ICMMTSET after modification by MTSEAo (Fig. 3B), which was similar in magnitude to that observed when R1C-DIV was modified by MTSEAo.
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A, families of leak- and capacity-corrected INa during step depolarizations from –110 to 10 mV from a holding potential of –150 mV for a cell expressing R1C-DIV-ICMMTSET (upper panel) and after modification by MTSEAo (lower panel). B, the mean peak I–V relationships for four cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). INa was normalized to the maximum peak inward INa for each cell expressing R1C-DIV-ICMMTSET. C,ON-gating currents (upper panels) and their integrals (lower panels) in a cell expressing R1C-DIV-ICMMTSET before (left) and after MTSEAo (right) for step depolarizations between –110 and 40 mV from a holding potential of –160 mV. Data shown are capacity and leak corrected, digitally filtered at 15 kHz, and every fourth point plotted. D, Q–V relationships for six cells expressing R1C-DIV-ICMMTSET normalized to the Qmax for each cell before () and after MTSEAo (). Continuous lines represent the means of the best fits to each cell by a Boltzmann distribution (eqn (1)). The parameters from the best fits for R1C-DIV-ICMMTSET showed that the V1/2 was –58 ± 4 mV and the slope factor was –15.0 ± 2 mV. After MTSEAo the V1/2 was –60 ± 5 mV, the slope factor was –16 ± 2 mV, and the normalized Qmax increased by 24 ± 5% to a normalized value of 1.24 ± 0.05. Only the change in Qmax was statistically significant.
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Extracellular MTSEA increases ON-gating charge of R1C-DIV-ICMMTSET
Even though the presumed positive charge added by MTSEAo to the cysteine at the R1 position in R1C-DIV-ICMMTSET did not affect INa, it did affect gating charge (ON-charge) in response to step depolarizations. Figure 3C shows there was an obvious increase in both the maximal amplitude of the ON-gating currents and in their integrals after MTSEAo. The mean increase in Qmax obtained from the Boltzmann fits to Q–V relationships for six R1C-DIV-ICMMTSET cells after modification by MTSEAo was 24% when compared to the Qmax in control. As anticipated, the magnitude of increase in the Qmax after MTSEAo was similar to that observed after MTSEAo modification of R1C-DIV (Fig. 2C) suggesting that MTSEAo added a full positive charge, eo, to the mutant channel.
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Off-gating charge studies
Internal perfusion of the squid giant axon with the proteolytic enzyme pronase has been shown to remove fast inactivation and to eliminate all charge immobilization (Armstrong & Bezanilla, 1977). On the other hand, we have previously found (Sheets et al. 2000) that specific removal of fast inactivation in ICMMTSET did not eliminate all charge immobilization but only reduced its magnitude to about 30% from a magnitude of 50–60% in native Na+ channels. If the remaining immobilizable gating charge originated from the S4-DIV in ICMMTSET, then the mutant channel R1C-DIV-ICMMTSET should exhibit almost no charge immobilization because the outermost arginine is largely responsible for the gating charge arising from the S4-DIV (Sheets et al. 1999). Figure 4A shows an example of OFF-Ig and OFF-charge in R1C-DIV-ICMMTSET before and after MTSEAo recorded at a repolarization potential of –180 mV following conditioning steps to 0 mV for various lengths of time up to 15 ms. The potential of –180 mV was selected because previous studies on wild-type hH1a have shown that the fast and slow time constants of OFF-Ig are different by about an order of magnitude permitting separation during two exponential fits. In addition, the OFF-charge at –180 mV returned fast enough so that all of it could be measured (Sheets et al. 2000). In control solutions the OFF-charge in R1C-DIV-ICMMTSET appeared to have only a fast component as can be seen by the similar time courses of all four of the integrals. However, after modification by MTSEAo the OFF-charge in R1C-DIV-ICMMTSET developed a slow component. For comparison, wild-type OFF-charge integrals are also shown before and after exposure to MTSEAo (Fig. 4B), where the increased magnitude of the slow components are readily apparent and appear unchanged by MTSEAo.
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A, families of OFF-gating currents and their integrals for a cell expressing R1C-DIV-ICMMTSET before (left) and after MTSEAo (right). The smallest integral in each panel corresponds to a conditioning step for 0.7 ms. Also shown for each cell is an ON-Ig in response to a step depolarization to 0 mV from a holding potential of –160 mV. B, families of OFF-gating currents and their integrals for wild-type hH1a before (left) and after MTSEAo (right) using the same voltage-clamp protocol as above. Data shown are capacity and leak corrected, digitally filtered at 15 kHz, and every fourth point plotted.
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Consistent with the studies above, the magnitude of OFF-charge after MTSEAo modification of R1C-DIV-ICMMTSET was increased by 21% (Fig. 5A), a value similar to the increase in ON-charge after MTSEAo modification of both R1C-DIV and R1C-DIV-ICMMTSET.
A, the mean OFF-charge at –180 mV for six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo () following conditioning steps to 0 mV for durations up to 44 ms. Gating charge was normalized to the maximal OFF-charge for each cell, calculated from its fitted value of Qmax–OFF. Continuous lines were calculated using the means of fitted parameters from each cell to the following: Normalized Q = Qmax–OFF(1 – e–t/), where the parameters of the fit were the time constant () of the appearance of OFF-charge and Qmax–OFF, the maximal OFF-gating charge. Qmax–OFF for R1C-DIV-ICMMTSET increased to 1.21 ± 0.04 (n = 6 cells) after modification by MTSEAo compared to control, while the was increased to 0.81 ± 0.15 ms after MTSEAo compared to a value of 0.66 ± 0.07 for R1C-DIV-ICMMTSET. Both changes were significantly different (P < 0.05). B and C, the mean fast and mean slow time constants, respectively, from up to two exponential fits to OFF-gating current relaxations during repolarization to –180 mV after conditioning to 0 mV for times from 0.7 ms to 44 ms in six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). C, the mean slow time constants from two exponential fits. For comparison both the fast and slow time constants for wild-type hH1a () are also shown (taken from Sheets et al. 2000). Prior to modification by MTSEAo () there were no slow time constants in the OFF-Ig for R1C-DIV-ICMMTSET until the conditioning duration was 10 ms when one of the six cells developed a slow time constant (see Results). After MTSEAo () all R1C-DIV-ICMMTSET cells developed slow time constants after conditioning durations of 7 ms, while none had slow time constants at times less than 1.5 ms. At times intermediate between the two conditioning durations one R1C-DIV-ICMMTSET cell developed a second time constant by 1.5 ms, one cell by 2 ms, two cells by 3 ms and five cells by 4.5 ms. In contrast, wild-type cells developed a slow time constant by 0.7 ms. D, the fraction of OFF-charge associated with the fast time constant for six cells expressing R1C-DIV-ICMMTSET before () and after MTSEAo (). Also shown are the fractions for wild-type hH1a () (Sheets et al. 2000). Lines connect the points. The fractional charge was calculated as the ratio of gating charge associated with the fast time constant compared to the total OFF-charge associated with each conditioning duration from 0.7 to 44 ms at a conditioning voltage, VC, of 0 mV. The differences for R1C-DIV-ICMMTSET were significantly different (P < 0.05) at all times except for the three earliest times of 0.7, 1.1 and 1.5 ms. For six cells at conditioning times 7.0 ms the mean fraction of OFF-charge in the fast time constant of R1C-DIV-ICMMTSET was 0.98 ± 0.05, and after MTSEAo it decreased to 0.65 ± 0.03. For wild-type the fraction was nearly 0.5.
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To quantify the OFF-Ig relaxation time constants, they were fitted by a sum of up to two exponentials and sorted into fast and slow time constants. When only a single exponential best fitted the OFF-Ig it was assigned as a fast time constant. Figure 5B and C shows the results for six cells expressing R1C-DIV-ICMMTSET before and after modification by MTSEAo. Five of the six R1C-DIV-ICMMTSET cells had only fast time constants of relaxation with a value of nearly 0.3 ms before modification by MTSEAo. However, one cell exhibited exceptionally large gating currents, and it had a slow time constant detected in the OFF-Ig relaxation before modification by MTSEAo, most likely because its large gating currents permitted the remaining residues in the S4-DIV (estimated to be 10% of overall gating charge; Sheets et al. 1999) to make a contribution sufficiently large to be detected by the fitting routine (see Fig. 5C). In contrast, after modification by MTSEAo all cells developed a second, slow time constant at conditioning durations of 7 ms (Fig. 5B).
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Coincident with the development of a slow time constant in OFF-Ig, the fraction of OFF-charge accounted for in the fast component decreased as the conditioning time duration at 0 mV increased. Figure 5D shows the fraction of OFF-charge contained in the fast component compared to the total OFF-charge measured at each conditioning duration from 0.7 to 44 ms for R1C-DIV-ICMMTSET before and after MTSEAo. Before MTSEAo almost all the OFF-charge was contained in the fast time constant, while after MTSEAo the fraction of OFF-charge contributed by the fast time constant decreased as the conditioning duration increased. The reduction in the fraction of OFF-charge accounted for by the fast component was significantly different (P < 0.05) at all times except for the three earliest times of 0.7, 1.1 and 1.5 ms. For all conditioning times 7.0 ms the mean fraction of OFF-charge in the fast time constant of R1C-DIV-ICMMTSET before MTSEAo was 0.98 ± 0.05 (n = 6 cells), while after MTSEAo it decreased to 0.65 ± 0.03. Consequently, the mean fraction of OFF-charge contained in the slow time constant was 35%, a value similar to the 31% contribution made by the S4-DIV to the maximal ON-charge in wild-type hH1a (Sheets et al. 1999). For comparison to wild-type hH1a, charge immobilization was first noted after conditioning times of 0.7 ms and accounted for about 50% of the total OFF-charge at later times (Sheets et al. 2000).
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Discussion
We investigated the molecular origin of the slow recovery of gating charge (i.e. charge immobilization) that remained in hH1a when fast inactivation was specifically removed by MTSETi modification of a cysteine in the ICM motif in the inactivation particle formed by the linker between domains III and IV. Our experiments with charge restoration of R1C-DIV-ICMMTSET demonstrated that the S4-DIV is responsible for the residual charge immobilization when fast inactivation has been specifically removed.
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In fast-inactivated wild-type Na+ channels, gating charge becomes immobilized secondary to the slow return of the S4 segments in domains III and IV during repolarization, while the S4 segments from domains I and II return quickly (Cha et al. 1999). Our results showed that charge immobilization of the S4-DIV occurred regardless of whether or not fast inactivation was present, suggesting that the inactivation particle in the wild-type Na+ channel can modulate the time course of gating charge recovery of only the S4-DIII and not that for the S4-DIV. We cannot rule out the possibility that the removal of fast inactivation using MTSETi modification of the ICM motif in the inactivation particle may still permit partial binding of the altered inactivation particle to occur thereby causing the S4-DIV to become immobilized. However, if partial binding occurs with mutations in the IFM motif they do not appear to alter the magnitude of single channel conductance (Hartmann et al. 1994). Furthermore, it is important to note that S4-DIV has been shown to move more slowly than the S4 segments in the other three domains during step depolarizations (Hanck & Sheets, 1995; Sheets & Hanck, 1995; Sheets et al. 1999; Chanda & Bezanilla, 2002). Consequently, it is likely that the slow movement of the S4-DIV in Na+ channels may be an intrinsic property arising from interactions with adjacent transmembrane segments, their segmental linkers or perhaps adjacent domains (Chanda et al. 2004). These findings contrast with those from Shaker K+ channels where most, if not all, of the gating charge results from channel activation transitions leading to channel opening (Schoppa et al. 1992; Bezanilla et al. 1994; Zagotta et al. 1994; Seoh et al. 1996) with little or no gating charge movement after channel opening.
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Both MTSET and MTSEA are able to add a single positive charge through a disulphide bond to an otherwise neutral cysteine residue. MTSETo has been shown to partially restore fast inactivation as measured by the speeding up of INa decay in the paramyotonia congenita mutant hSkM1 Na+ channel R1448C, that also has the outermost arginine in the S4-DIV mutated to a cysteine (Yang & Horn, 1995). We show that this is also the case for R1C-DIV in hH1a for both MTSETo and MTSEAo. Although both MTSEAo and MTSETo were able to modify the S4-DIV, neither of the MTS agents completely restored the modified channel's kinetics to those of the wild-type phenotype. In particular, MTSETo caused large negative shifts in channel kinetics of hH1a by up to –18 mV similar to that found for R1448C in the hSkM1 Na+ channel (Yang & Horn, 1995). This may not be surprising given the different sizes of the two MTS reagents when linked to a cysteine. MTSEA, the smaller of the two reagents, is estimated to be a molecule with a volume of 138 3 after binding with cysteine (personal communication from Gregory Lipkind, University of Chicago, using calculations from the Search/Compare module in Insight from Accelrys Corp, San Diego, CA, USA), and it is only slightly larger than the estimate for an arginine (with a volume of 129 3) in the wild-type channel. In addition, MTSEA's charged nitrogen is surrounded by hydrogens in an ‘open’ charge configuration while the smaller arginine has a more distributed charge. In contrast, MTSET is a much larger residue with a volume of 183 3. Moreover, its nitrogen is surrounded by methyl groups that screen its positive charge making it more likely to interact with nearby hydrophobic neighbours. Furthermore, the charged, primary amine of MTSEA would be able to undergo hydrogen bonding while this would not be possible for MTSET with its quaternary amine. It is likely that a combination of characteristics permitted MTSEAo to be better at restoring channel kinetics and gating charge at the R1C-DIV position compared to MTSETo.
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MTSEAo not only partially restored fast inactivation of INa in R1C-DIV, but also increased Qmax by 24%, an amount equivalent to that predicted when the outermost arginine residue in the S4-DIV was neutralized (Sheets et al. 1999). Although our gating charge measurements do not allow us to calculate the absolute amount of positive charge added by MTSEA to Qmax, it does allow an estimate to be made. Because MTSEA can add no more than a single eo to the Na+ channel, an increase in Qmax by 24%, would predict a lower limit of charges per channel to be approximately 5.2eo (1eo = (0.24/1.24) x Qmax). However, this estimate may be too small if neutralization of R1 in domain IV had a profound effect on the distribution of other charged residues with respect to the electric field, as has been suggested for the Shaker K+ channel (Seoh et al. 1996). Recently, it has been reported that domains I and IV in the rat skeletal muscle Na+ channel may interact suggesting that the charged residues in the S4 segments may have a complex arrangement (Chanda et al. 2004). Furthermore, an estimate of 12eo per Na+ channel was predicted based on an analysis of the limiting slope conductance of single channel data at threshold potentials (Hirschberg et al. 1995), which is more comparable to the values predicted for Shaker K+ channels (Schoppa et al. 1992; Bezanilla et al. 1994; Zagotta et al. 1994). If Na+ channels are found to have much less total charge per channel than K+ channels this may not be unexpected when one considers that the voltage sensors in Na+ channels are not identical, and they may play more diverse roles in channel kinetics. Future studies will have to resolve this question.
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