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Am J Physiol Heart Circ Physiol 283: H966-H975, 2002. First published May 16, 2002; doi:10.1152/ajpheart.00097.2002
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Vol. 283, Issue 3, H966-H975, September 2002

Enhancement of closed-state inactivation in long QT syndrome sodium channel mutation Delta KPQ

Tiehua Chen and Michael F. Sheets

1 The Nora Eccles Harrison Cardiovascular Research and Training Institute and The Department of Internal Medicine, University of Utah, Salt Lake City, Utah 84112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Delta KPQ, a three amino acid [lysine (K), proline (P), glutamine (Q)] deletion mutation of the human cardiac Na channel (hH1), which is one cause of long QT syndrome (LQT3), has impaired inactivation resulting in a late sodium current. To better understand inactivation in Delta KPQ, we applied a site-3 toxin anthopleurin A, which has been shown to inhibit inactivation from the open state with little or no effect on inactivation from the closed state(s) in wild-type hH1. In contrast to the effect of site-3 toxins on wild-type hH1, inactivation from closed state(s) in toxin-modified Delta KPQ demonstrated a large negative shift in the Na channel availability curve of nearly -14 mV. Recovery from inactivation showed that toxin-modified Delta KPQ channels recovered slightly faster than those in control, whereas development of inactivation at potentials negative to -80 mV showed that inactivation developed much more rapidly in toxin-modified Delta KPQ channels compared with control. An explanation for our results is that closed-state inactivation in toxin-modified Delta KPQ is enhanced by the mutated inactivation lid being positioned "closer" to its receptor resulting in an increased rate of association between the inactivation lid and its receptor.

site-3; anthopleurin; Nav1.5 channel; heart; gating current


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE GENE THAT ENCODES the cardiac Na channel is SCN5A gene, which is a voltage-gated protein that is largely responsible for the rapid upstroke of the cardiac action potential and for propagation of the cardiac impulse in cardiac muscle and Purkinje fibers. Mutations of the cardiac Na channel have been associated with the congenital long QT syndrome (LQT3) causing tosades des pointes (14), the Brugada syndrome linked to ventricular fibrillation (5), and conduction system defects resulting in atrioventricular heart block (29, 34). More recently, single mutations in the cardiac Na channel have been linked to the presentation of multiple, distinct clinical diseases. For example, a mutation at amino acid position 1795 can result in either LQT3 or the Brugada syndrome (28, 38), whereas a mutation at amino acid position 1406 can cause either a cardiac conduction defect or the Brugada syndrome (22). The different clinical presentations result from the mutation altering more than one functional property of Na channel behavior.

The mutant cardiac Na channel with deletions of lysine (K), proline (P), and glutamine (Q) at amino acid positions 1505-1507 (Delta KPQ) within the putative inactivation lid formed by the intracellular linker between domains III and IV was one of the first mutations to be linked to LQT3 (40). It has been shown to result in an abnormal gain in Na channel function caused by a defect in the inactivation process characterized by late channel reopenings leading to a late (or persistent) depolarizing Na current (INa) that prolongs the cardiac action potential (1, 15, 39). However, more recent reports have demonstrated additional abnormalities in the kinetic properties of Delta KPQ, including the presence of a decreased voltage dependence of INa decay rates and small (<3 mV) negative shifts of the voltage-dependent Na channel availability curves, suggesting additional effects on Delta KPQ inactivation properties (10, 24). To further investigate the inactivation process in Delta KPQ, we applied a site-3 toxin (8), Anthopleurin A (APA) toxin, which has been shown to inhibit inactivation from the open state while leaving inactivation from closed state(s) essentially unchanged in native cardiac Na channels (17). Site-3 toxins have been shown to bind to the extracellular surface of the Na channel thereby causing a 31% reduction in the maximum gating charge (Qmax) through inhibition of the normal movement of the S4 in domain IV (32), a voltage sensor that facilitates coupling of inactivation to channel activation (9, 17, 25). In contrast to the dramatic slowing in the decay of INa caused by site-3 toxin modification of wild-type hH1, we found that the decay of INa in Delta KPQ channels was only slightly prolonged after ApA toxin at test potentials greater than -60 mV. The most dramatic effect of ApA toxin on Delta KPQ was noted in its large negative shift of the voltage-dependent Na channel availability relationship compared with the small positive shift (+2 mV) of the Na channel availability curve caused by ApA toxin in native cardiac Na channels (17). Two-pulse development of inactivation and recovery from inactivation protocols further suggested that closed-state inactivation was enhanced in toxin-modified Delta KPQ channels because of an increased rate of association between the mutant inactivation lid and its receptor. Some of these data have been published in abstract form (13).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA clones. In hH1a Na (NaV1.5) channels [kindly provided by H. Hartmann and A. Brown (19)], the three amino acids (lysine, proline, and glutamine) at positions 1504, 1505, and 1506, respectively, were deleted using a four-primer PCR technique (2). The equivalent positions in the hH1 Na channel (16) are 1505, 1506, and 1507. All cDNA inserts were confirmed by sequencing. The cDNA for Delta KPQ and wild-type hH1a were subcloned directionally into the mammalian expression vector pRcCMV (Invitrogen; Carlsbad, CA).

Cell preparation. Multiple tsA201 cells (SV40 transformed HEK293 cells) were fused together by using polyethylene glycol as previously described (33). After fusion, the cells were placed in culture for several days to allow for membrane remodeling, and then they were transiently transfected with either cDNA for Delta KPQ or wild-type hH1a by using a calcium phosphate precipitation method (GIBCO-BRL; Grand Island, NY). Three to six days after transfection, fused cells were detached from culture dishes with trypsin-EDTA solution (GIBCO-BRL) and studied electrophysiologically.

Recording technique, solutions, and experimental protocols. Recordings were made using a large bore, double-barreled glass suction pipette for both voltage clamp and internal perfusion as previously described (33). Currents were recorded with a virtual ground amplifier (Burr-Brown OPA-101) by using a 2.5-MOmega feedback resistor. Voltage protocols were imposed from a 16-bit DA converter (Masscomp 5450, Concurrent Computer; Tinton Falls, NJ) 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 analog-to-digital converter on a Masscomp 5450 at 200 kHz. A fraction of the current was fed back to compensate for series resistance. Temperature was controlled using a Sensortek (Physiotemp Instruments; Clifton, NJ) TS-4 thermoelectric stage mounted beneath the bath chambers, which typically allowed temperature to vary <0.5°C during an experimental set. Cells were studied at 12 to 13°C.

A cell was placed in the aperture of the pipette, and after a high resistance seal formed between the cell and glass pipette, the cell membrane inside the pipette was disrupted with a manipulator-controlled platinum wire. For INa experiments, voltage control was assessed by evaluating the time course of the capacitive current and by the steepness of the negative slope region of the peak current-voltage relationship (18). To allow for full Na channel availability, the holding membrane potential was set between -150 to -180 mV. Gating current (Ig) protocols contained four repetitions at each test voltage that were three of a 60-Hz cycle out of phase to improve the signal-to-noise ratio.

In most ionic current experiments, the control extracellular solution contained (in mM) 15 Na+, 185 tetra-methylammonium (TMA+), 2 Ca2+, 200 2-(N-morpholino)ethanesulfonic acid- (MES), and 10 HEPES (pH 7.2), and the intracellular solution contained 200 TMA+, 200 F-, 10 EGTA, and 10 HEPES (pH 7.2). In some experiments for the construction of conductance-voltage (G-V) relationships, both the intracellular and extracellular solutions contained 15 mM Na+ and 185 mM Cs+. For measurement of Ig the extracellular Na+ was removed and replaced with TMA+, and 10 µM saxitoxin (Calbiochem; San Diego, CA) was added to the extracellular solution. APA toxin (Sigma Chemical; St. Louis, MO) was used at a concentration of 1 µM, which is three orders of magnitude greater than the dissociation constant (KD) (17, 20). To assure full Na channel availability, the holding potential for Delta KPQ recordings in control solutions was -150 mV while it was increased to -180 mV after Na channels were modified by ApA toxin.

Data analysis. Peak INa was taken as the mean of four data samples clustered around the maximal value of current that had been digitally filtered at 5 kHz and leak corrected by the amount of the extrapolated time-independent linear leak. Linear leak currents were calculated from the linear conductance measurements obtained between test potentials from -190 mV to -110 mV. Data were capacity corrected using 4 to 16 scaled current responses recorded from voltage steps of 40 mV negative to the holding potential. To determine time constants of INa decay, the current traces were trimmed after the current peak and were fit by a sum of up to two exponentials by DISCRETE (27), a program that provides a modified F statistic to evaluate the number of exponential components that best describes the data. Normalized peak G-V relationships were fit with a Boltzmann distribution
I<SUB>Na</SUB><IT>=</IT>(<IT>V</IT><SUB>t</SUB><IT>−V</IT><SUB>rev</SUB>)<IT>G</IT><SUB>max</SUB>&cjs1134;[1<IT>+</IT> exp(<IT>V</IT><SUB>t</SUB><IT>−V</IT><SUB>1<IT>/</IT>2</SUB><IT>/S</IT>)] (1)
where INa is the peak Na current in response to a step depolarization, Vt is the test potential and the fitted parameters were V1/2, the half point of the relationship, s, the slope factor (in mV), and Vrev, the reversal potential. For comparison between cells, data were normalized to the maximum peak conductance (Gmax). Steady-state voltage-dependent Na channel availability curves were fit with a Botzmann distribution
I<SUB>Na</SUB><IT>=I</IT><SUB>max</SUB>&cjs1134;[1<IT>+</IT> exp(<IT>V</IT><SUB>C</SUB><IT>−V</IT><SUB>1<IT>/</IT>2</SUB><IT>/S</IT>)] (2)
where INa is the peak Na current after a conditioning pulse, Imax, is the maximal curent, and the fitted parameters were as defined earlier. Two-pulse development of inactivation and recovery from inactivation protocols were fit by a two exponential equation
Fraction of <IT>I</IT><SUB>Na</SUB><IT>=</IT>1<IT>−A</IT>·  exp(−<IT>t/</IT>tau<IT>A</IT>) (3)

<IT>−B</IT>·exp(−<IT>t/</IT>tau<IT>B</IT>)<IT>−k</IT>
where INa is the recorded normalized peak Na current at the test potential, and the fitted parameters are A, the amplitude of the fast time constant (tauA), B, the amplitude of the slow time constant (tauB), and k, a constant.

Ig values were capacity corrected as described above, leak corrected by the mean of 2-4 ms of data typically beginning at 8 ms after the depolarizing step, and then integrated to measure charge. Q-V relationships were fit with a simple Boltzmann distribution as follows
Q=Q<SUB>max</SUB>&cjs1134;[1<IT>+e</IT><SUP>(<IT>V</IT><SUB>t</SUB><IT>−V</IT><SUB>1<IT>/</IT>2</SUB>)<IT>/s</IT></SUP>] (4)
where Q is the charge during depolarizing step, and Qmax is the maximum gating charge. For comparison between cells fractional Q was calculated as Q/Qmax for each cell in control solution.

Data were analyzed and graphed on a SUN SparcStation using SAS (Statistical Analysis System; Cary, NC). Unless otherwise specified, summary statistics are expressed as means ± SD, and figures show means ± SE. In some figures, error bars were obscured by the symbols. Experimental parameters for toxin-modified channels were compared with those in control solutions by using a paired t-test and were considered significantly different when P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ApA toxin binds to Delta KPQ and inhibits movement of gating charge. Site-3 toxins have been shown to bind to the extracellular surface of Na channels in domain IV (2, 36), to selectively inhibit the movement of the S4 in domain IV (32), and to cause a 31% decrease in Qmax (30). Associated with inhibition of gating charge movement, there is prominent slowing in the decay of INa in response to step depolarizations resulting from inhibition of inactivation from the open state while leaving inactivation from the closed state intact (17). Because the three amino acid deletions in Delta KPQ are located in the inactivation lid and not in the putative voltage sensors, the effect of site-3 toxins on the gating charge of Delta KPQ would be expected to be similar to the effects of toxin on wild-type hH1. Figure 1 shows the normalized gating charge-voltage (Q-V) relationships before and after modification by ApA toxin for four cells expressing Delta KPQ. The effects of the toxin on the Q-V relationship of Delta KPQ were comparable to those for native cardiac Na channels (31) and for wild-type hH1 (30). These findings are consistent with the expectation that 1 µM ApA toxin can bind and modify all Delta KPQ channels and suggests that the three amino acid deletions in the inactivation lid of Delta KPQ does not alter the contribution made by the S4 in domain IV to overall Qmax.


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  		Fig. 1.   Effects of Anthopleurin A (ApA) toxin on charge-voltage (Q-V) relationships of the mutant cardiac Na channel with the three amino acid deletion [lysine (K), proline (P), and glutamine (Q) (Delta KPQ)]. Data plotted are means ± SE for cells (n = 4) in control and after modification by 1 µM ApA toxin. Gating charge (Q) in toxin was normalized to the maximum Q (Qmax) determined for each cell in control. The mean of the fits to each cell by a Boltzmann distribution (see Eq. 4 in the text) is represented by the solid line and showed that maximal conductance (Gmax) was significantly reduced (P < 0.5) by 33 ± 3 (±SD)%, whereas the slope factors were similar at -14 ± 1 mV for control and -15 ± 1 mV in toxin. The half point in toxin [-63 ± 2 (±SD) mV] was significantly different (P < 0.5) from control [-58 ± 2 (±SD) mV], reflecting an obligatory leftward shift in the half point, because Q was selectively reduced at the more positive test potentials (31).

Effects of ApA toxin on INa in Delta KPQ. Ionic currents in response to step depolarizations before and after 1 µM ApA toxin are shown in Fig. 2 for a representative cell expressing Delta KPQ. For comparison, examples of wild-type INa are also shown in Fig. 2. After modification by toxin, Delta KPQ demonstrated only a slightly slower decay of INa, which was much less prominent than that seen for wild-type INa (Fig. 2D) or for native Na channels (32). In two cells expressing Delta KPQ (data not shown), the concentration of ApA toxin was increased to 10 µM without any additional slowing of INa decay, suggesting that the small effect on the decay of INa in Delta KPQ by toxin did not result from incomplete modification of channels.


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  		Fig. 2.   Families of capacity-corrected Na current (INa) during step depolarization to -60, -40, -20, 0, and 20 mV. The holding membrane potential (Vh) was -150 mV for Delta KPQ in control (A) and it was -180 mV in 1 µM ApA toxin (B), whereas the Vh was -150 mV for wild-type (WT) in control (C) and in 1 µM ApA toxin (D). Scale bars represent 10 nA and 10 ms. Current traces were digitally filtered at 5 kHz.

To better compare the decay rates of the ionic current traces of Delta KPQ, they were fit by a sum of up to two exponentials (see METHODS). Figure 3 shows the short time constants and the ratios of the amplitude associated with the short time constant compared with the sum of both short and long amplitudes for eight cells. Where decays best fit only a single exponential, that value was combined with the shorter of the two time constants from cells that were fit best by two exponentials. Although INa decays of Delta KPQ were best fit (see METHODS) by two time constants, 76% of the time in control solution and 95% of the time after toxin modification, in both instances the short time constant accounted for the majority of the INa decay with its contribution only slightly decreased by toxin (Fig. 3B). In contrast to wild-type Na channels where ApA toxin dramatically slowed the decay of INa (32), ApA toxin only slightly increased the short time constant (Fig. 3A) except at the most negative test potentials. At potentials less than or equal to -60 mV, where inactivation from closed states has been shown to occur in cardiac Na channels (23), the decay of INa became more rapid after toxin modification. The long time constants for both Delta KPQ in control and after toxin modification typically ranged from minimal values of 15-20 ms up to nearly 40 ms (data not shown), a value too long to be accurately recorded by a 50-ms step depolarizations. However, there were no apparent differences in the long time constants of Delta KPQ in control and after toxin.


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  		Fig. 3.   Time constants of INa decay from two exponential fits for Delta KPQ channels. Data plotted are means ± SE for 8 cells in control and after modification by 1 µM ApA toxin for short time constants (A) and the ratio of the amplitude of the short time constant to the sum of the amplitudes from both the short and long time constants (B). The differences between the short time constants were statistically significant (P < 0.05) at all potentials as were the differences between the ratio of amplitudes except at test potentials of -65 and -70 mV. Long time constants were typically >10 to 20 ms and did not appear different between control and after toxin (see text).

To compare the G-V relationships before and after ApA toxin, cells expressing Delta KPQ were studied with symmetrical intracellular and extracellular concentrations of 15 mM Na+ and with Cs+ as a substitute cation instead of TMA+, because intracellular TMA ions can cause voltage-dependent block of INa (26). Figure 4A shows that there was little effect of ApA toxin on the G-V relationship of Delta KPQ with Gmax increasing by a nonsignificant amount of only 2% (see Table 1) in contrast to the 26% increase in Gmax shown for toxin-modified Na channels in single cardiac cells (17). In cells perfused with our standard solutions used in these studies (i.e., 15 mM extracellular Na+ with no intracellular Na+ and with TMA+ as the replacement cation), Gmax showed a small but statistically significant decrease (11 ± 5%, n = 8 cells) after ApA toxin (data not shown). Consistent with only minimal changes in the G-V relationship after ApA toxin, the time to peak INa measurements were only modestly affected by toxin (Fig. 4B). At more depolarized test potentials (greater than -40 mV) time to peak INa increased as expected if inactivation from the open state were slowed by toxin. However, ApA toxin shortened the time to peak INa at test potentials less than -55 mV, the same test potentials where the primary time constants of INa decays were shortened. Both of these findings suggest that inactivation at more negative test potentials may become augmented after modification of Delta KPQ by site-3 toxins.


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  		Fig. 4.   Effect of ApA toxin on Gmax (A) and time to peak INa (B) for Delta KPQ. A: normalized peak G-V relationships for Delta KPQ in control and after modification by ApA toxin. Data plotted are means ± SE for 3 cells. Lines represent the mean of the best fits to each cell by a Boltzmann distribution (Eq. 1 in the text). Parameters from the best fits to the data are given in Table 1. B: time to peak of INa in Delta KPQ in control and after modification by ApA toxin. Data plotted are means ± SE for 5 cells and the lines connect the points. Time to peak INa values were significantly different (P < 0.05) except at test potentials of -55, -50, -45, and -35 mV.


                              
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  		Table 1.   Comparison of Boltzmann parameters to fits of G-V and voltage-dependent sodium channel availability relationships for Delta KPQ in control and after ApA toxin

If inactivation from closed states were to be altered by site-3 toxins, then steady-state voltage-dependent availability relationships of Delta KPQ should be altered. We have previously shown that site-3 toxins cause a small rightward shift (to more positive potentials) of the steady-state availability curve in native Na channels in single cardiac cells (17). Figure 5 and Table 1 show the results of the steady-state availability curve for five cells expressing Delta KPQ before and after ApA toxin. For comparison, the steady-state availability curves for five cells expressing wild-type hH1a before and after modification by ApA toxin are shown in Fig. 5, inset. In marked contrast to the small positive shift in the half point (from -111 ± 5 mV to -109 ± 4 mV, n = 5) for wild-type Na channels, site-3 toxin resulted in a large leftward shift of the steady-state voltage-dependent availability relationship with the half point shifted by -14 mV. Because the mean difference in time between recording the data in control and after toxin modification was only 10 ± 3 min, any time-dependent background shift of Na channel kinetics would be predicted to be less than 1 or 2 mV (30) and cannot account for the large difference in half points between the two relationships. The large negative shift of the steady-state availability relationship must result from increased inactivation from closed state(s) because Delta KPQ channels do not open until the membrane potential approaches -80 mV (see Fig. 4). Consequently, the increase in closed state(s) inactivation after toxin implies that there was either an increase in the rate of association between the inactivation lid and its receptor and/or a decrease in the rate of dissociation between the two.


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  		Fig. 5.   Normalized voltage-dependent Na channel availability relationships for Delta KPQ in control and after modification by ApA toxin for 5 cells. Inset: results for 5 cells expressing wild-type hH1a in control and after modification by ApA toxin. Data plotted are means ± SE. Lines represent the mean of the best fits to each cell by a Boltzmann distribution (Eq. 2 in the text). Cells were stepped to conditioning potentials for 500 ms before depolarization to a test potential of 0 mV (see protocol inset), and peak INa was normalized to the peak INa in the absence of a conditioning step. Parameters from the best fits to the data for Delta KPQ are given in Table 1 and those for wild-type Na channel are in the text.

To further investigate the kinetics of Delta KPQ, two pulse developments of inactivation protocols were recorded in control solutions and after ApA toxin. Figure 6 shows the results for development of inactivation at potentials from -120 mV to 0 mV, and Table 2 shows the best fits to two exponentials (see Eq. 3). The most obvious difference is the increased fraction of toxin-modified Delta KPQ channels that inactivate at potentials of -120 and -100 mV, potentials where Na channels have a very low probability of opening and where inactivation occurs from closed state(s). Accompanying the greater magnitude of inactivation at those two potentials, the primary, short time constant of inactivation was also faster after modification by toxin. As the conditioning potentials became more positive where inactivation from open states becomes greater (17, 23), the differences between the time courses of development of inactivation for Delta KPQ channels in control and after toxin became minimal.


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  		Fig. 6.   Development of inactivation for Delta KPQ in control (open circle ) and after modification by ApA toxin (). Data plotted are means ± SE for 4-9 cells. Lines represent the mean of the best fits to each cell by two exponentials (Eq. 4 in the text). Inset between panels: pulse protocol where the membrane potential was stepped to conditioning potentials of -120 mV (A), -100 mV (B), -80 mV (C), -40 mV (D), -20 mV (E), and 0 mV (F) for durations from 0.7 to 1,000 ms then clamped back to -130 mV for 2 ms before stepping to a test potential of 0 mV. There were 2.5 s between pulses. Peak currents following conditioning steps were normalized to peak INa measured in the absence of a conditioning step. Insets to each panel show the development of inactivation for the first 50 ms. Parameters from the best fits to the data are given in Table 2.


                              
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  		Table 2.   Comparison of parameters for development of inactivation of Delta KPQ sodium current before and after ApA Toxin


                              
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  		Table 3.   Comparison of parameters for recovery from inactivation for Delta KPQ sodium current before and after ApA toxin

Because the development of inactivation protocols that is performed at negative conditioning potentials such as -100 mV where kinetic transitions may lead both to channel inactivation as well as recovery from inactivated state(s), we measured INa recovery from inactivation at very hyperpolarized potentials where no channel inactivation occurs. Figure 7 shows the time course of recovery from inactivation for Delta KPQ channels in control and after toxin at recovery potentials as negative as -190 mV and their fits by two exponential time constants (Eq. 3). Overall, there were only small differences between recovery from inactivation in control and after toxin at the most negative potentials of -170 and -190 mV with the primary, short time constants of recovery being slightly shorter for Delta KPQ in toxin compared with the control. The primary time constant for Delta KPQ channels in toxin continued to be faster compared with the control at -150 mV, even though the total fraction of Na channels that recovered was smaller for toxin-modified channels compared with control. Comparison of the time constants between control and the toxin-modified channel demonstrate that there was no significant prolongation in the recovery from inactivation that could account for the additional inactivation found on the voltage-dependent Na channel availability curve in ApA-modified Delta KPQ (see Fig. 5). Consequently, ApA toxin must increase the rate of association between the inactivation lid and its receptor leading to enhancement of closed state(s) inactivation.


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  		Fig. 7.   Recovery from inactivation for Delta KPQ in control (open circle ) and after modification by ApA toxin (). Data plotted are means ± SE, and the lines represent the mean of the best fits to each cell by two exponentials (Eq. 3 in the text). Inset in C shows the pulse protocol where the membrane potential was stepped to 0 mV for 500 ms to inactivate Na channels and then stepped back to recovery potentials of -190 mV (A), -170 mV (B), and -150 mV (C) for durations from 0.7 to 1,000 ms before a test potential step at 0 mV. There were 2.5 s between pulses. Peak currents following recovery steps were normalized to peak INa measured in the absence of a conditioning step to 0 mV. Inset to A-C shows the development of inactivation for the first 50 ms. Parameters from the best fits to the data are given in Table 3.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although ApA toxin modifies the gating charge of both the mutant Na channel Delta KPQ and wild-type Na channels similarly (30), we have shown that site-3 toxins have different effects on INa of Delta KPQ channels compared with wild-type hH1a. The decay of INa in Delta KPQ channels was only slightly prolonged after ApA toxin and only at test potentials greater than -60 mV (see Fig. 3) in contrast to the dramatic slowing of INa in native Na channels (17). Similarly, the times to peak INa were only modestly prolonged by toxin modification in Delta KPQ and only at potentials greater than -50 mV in contrast to the dramatic prolongation in time to peak INa in native Na channels (17). Comparison of the normalized G-V relationships before and after toxin modification for Delta KPQ show almost no change, suggesting that the toxin caused minimal changes in channel activation.

The most dramatic difference between toxin-modified Delta KPQ and toxin-modified cardiac Na channels was evident in the voltage-dependent Na channel availability relationships. Previously, we found that site-3 toxins cause the Na channel availability curve in native Na channels to be shifted minimally to more positive potentials with a slightly more shallow slope as a result of inhibition of inactivation from the open state but leaving inactivation from closed state(s) essentially unchanged (17). Similar results were found for wild-type hH1a (see Fig. 5). However, toxin modification of Delta KPQ resulted in a dramatic negative shift of the voltage-dependent Na channel availability curve by nearly -14 mV with a large reduction in its slope factor. Because most of the conditioning steps in the voltage-dependent Na channel availability protocols do not result in channel opening, the shift of the Na channel availability curve resulted from a greater fraction of toxin-modified Delta KPQ channels that inactivated directly from closed state(s) compared with Delta KPQ in control or to toxin-modified wild-type Na channels. For a greater fraction of toxin-modified Delta KPQ channels to undergo closed-state inactivation, either the rate transition from closed to inactivated states must be increased and/or the backward rate transition from inactivated to closed must be decreased.

To further investigate the kinetics of the enhancement of closed-state inactivation, two-pulse development and recovery from inactivation protocols were performed (see Figs. 6 and 7). At conditioning potentials negative to INa, threshold inactivation of toxin-modified Delta KPQ channels developed more rapidly and to a greater degree than Delta KPQ in control. At more positive conditioning potentials where inactivation from the open state starts to predominate (17, 23), the development of inactivation between control and toxin-modified Delta KPQ channels became similar. However, the time constant for the development of closed-state inactivation depends on the balance of both the forward and backward rate constants, and either an increase in the forward rate constant or a decrease in the backward rate constant would result in an enhancement of inactivation. To isolate the backward rate constant from the forward rate constant, we performed two-pulse recovery from inactivation protocols at very negative recovery potentials where toxin-modified Delta KPQ channels do not appreciably inactivate (i.e., on the top of the Na channel availability curve). However, toxin-modified Delta KPQ channels were found to recover from inactivation slightly faster than control measurements similar to the slightly faster recovery from inactivation previously found for site-3 toxin-modified cardiac and neuronal Na sodium channels (3). Consequently, the negative shift of the Na channel availability curve in toxin-modified Delta KPQ channels did not result from a slower recovery from inactivation but from a significant increase in the forward rate constant of the closed state(s) inactivation transition(s).

ApA toxin is a small hydrophilic protein that has been shown to bind to the extracellular surface of the Na channel in domains I and IV (2, 35, 36) and shown to inhibit movement of the putative voltage sensor formed by the S4 in domain IV (32). As a consequence of toxin binding to the channel, inactivation from the open state becomes inhibited presumably by altering the coupling of the voltage sensor in domain IV to channel inactivation (9, 11, 21, 31). Alteration of coupling may result from either changing the "molecular link" between the voltage sensor in domain IV (25), the putative inactivation lid formed by the linker between domains III and IV (19, 41), or by toxin changing the conformation of the receptor for the inactivation lid. In toxin-bound wild-type hH1, altered coupling most likely results from changes in the conformation of the receptor for the inactivation lid and not from changes in the lid or in the molecular link because ApA toxin acts extracellularly while the inactivation lid and its link are intracellular. Furthermore, the identical effect of ApA toxin on both the Q-V relationships of Delta KPQ and wild-type hH1a (see Fig. 1) suggests that the receptor for the inactivation lid associated with the inhibition of movement of the S4 in domain IV is similar for both wild-type hH1a and Delta KPQ. Consequently, the enhanced closed-state inactivation in Delta KPQ likely results from either 1) the mutated lid causing a slower rate of dissociation, or 2) the mutated lid causing a faster rate of association. If the first possibility were correct, then the mutated lid would have a slower recovery from inactivation, whereas if the second possibility were correct, then the development of inactivation would be enhanced in the presence of a normal rate of recovery from inactivation. Our studies of recovery from inactivation and development of inactivation support the second possibility, that binding of the toxin caused an increase in the rate of association between the lid and receptor consistent with the mutant inactivation lid having a conformation that is physically "closer" to the inactivation receptor thereby increasing its rate of association.

Review of previously reported studies comparing Delta KPQ to wild-type Na channels offer additional support for this conclusion. It is possible that binding of site-3 toxins only accentuates a predilection of the inactivation lid of Delta KPQ channels to bind to the inactivation receptor that forms at potentials negative to INa threshold. Support for this possibility is found in previous studies comparing Delta KPQ to wild-type Na channels done under control conditions. Makielski and colleagues (24) found that the voltage-dependent Na channel availability curve for Delta KPQ was significantly shallower than wild-type hH1 accompanied by a small negative shift of the V1/2. Additionally, they found that the decay of INa in Delta KPQ was more rapid than wild-type hH1 channels, particularly at negative test potentials near -40 mV where appreciable closed-state inactivation has been shown to occur (23). Additional support for enhanced closed state inactivation in nontoxin-modified Delta KPQ channels was reported by Grant's laboratory (10), who not only found that the V1/2 and slope of voltage-dependent Na channel availability relationship were also more negative and shallower, respectively, for Delta KPQ compared with wild-type hH1, but that development of inactivation at -80 and -60 mV was faster for Delta KPQ. These additional findings support the likelihood that the mutation in the inactivation lid of Delta KPQ channels facilitates closed-state inactivation even in the absence of channel modification by site-3 toxins.

Consequently, the Delta KPQ mutation has been shown to have different effects on inactivation, one that facilitates closed-state inactivation before channel opening, whereas the other demonstrates an inhibition of normal inactivation after channel opening leading to channel reopenings. Typically, inactivation from closed states is not considered to be absorbing (i.e., requiring repolarization before channels open again), otherwise the voltage-dependent Na channel availability curve would not be a typical sigmoidal curve, because holding potentials that did not allow for all Na channels to be available would result in all channels become completely inactivated if they were held for a long enough time at even a minimally depolarized potential. In contrast, inactivation following a strong depolarization during which channels open is generally thought of as an absorbing state. Presumably, it is the receptor for the inactivation lid that imparts voltage sensitivity through movement of the S4 segments, whereas the inactivation lid is thought to be a rigid structure that stabilizes the projection of the hydrophobic phenylalanine residue within the Ile, Phe, Met motif into the aqueous intracellular solvent (7). The mutated inactivation lid in the Delta KPQ channel has been shown to have a decreased affinity for the inactivation receptor associated with strong depolarizations (1, 15, 39), while at the same time it has an increased affinity for the inactivation receptor associated with partial depolarizations. Furthermore, our studies show that the increased affinity of the mutated lid for the inactivation receptor results from an increased rate of association as if the mutated lid were physically closer to the inactivation receptor compared with the wild-type lid.

Initial studies of the LQT3 mutation Delta KPQ emphasized the presence of delayed Na channel reopenings causing a depolarizing current to occur late in an action potential leading to torsade des pointes (1, 15, 39). Recently, it has been reported (4, 28) that a mutation of a single amino acid in the human cardiac Na channel can result in multiple, distinct clinical presentations such as the long QT syndrome caused by a gain of function (14) and the Brugada syndrome thought to be related to a loss of Na channel function (6, 12). These different clinical presentations result from multiple alterations of functional Na channel behavior caused by a single mutation. Similarly, Delta KPQ channels demonstrate dual effects on channel inactivation: 1) a defect in inactivation causing an increase in late INa in the action potential leading to arrhythmias associated with long QT syndrome, and 2) a facilitation in inactivation resulting from increased closed-state inactivation particularly noticeable in the presence of site-3 toxins. The importance of these dual effects on inactivation may become manifest if antiarrhythmic drugs had a disproportionate effect on the block of Delta KPQ channels that underwent closed-state inactivation compared with open-state inactivation. Furthermore, future studies may find that Delta KPQ interacts with other Na channel polymorphisms, causing a further facilitation of closed-state inactivation thereby resulting in a pathological decrease in Na channel density. Recently, Valdivia et al. (37) reported that the LQT3 mutation M1766L markedly affects expression levels dependent upon the presence of polymorphisms in the human cardiac Na channel. It is likely that future studies will further demonstrate the complex behavior that single Na channel mutations may have on their functional properties leading to multiple clinical presentations of disease.


    ACKNOWLEDGEMENTS

The authors generously thank WenQing Yu for outstanding technical assistance.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Grant P50 HL-52338.

Address for reprint requests and other correspondence: M. F. Sheets, CVRTI, Bldg. 500, 95 South 2000 East, Univ. of Utah, Salt Lake City, UT 84112 (E-mail: michael{at}cvrti.utah.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 16, 2002;10.1152/ajpheart.00097.2002

Received 6 February 2002; accepted in final form 13 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 283(3):H966-H975
0363-6135/02 $5.00 Copyright © 2002 the American Physiological Society



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