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Am J Physiol Heart Circ Physiol 288: H2666-H2676, 2005. First published January 21, 2005; doi:10.1152/ajpheart.00521.2004
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Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

Tiehua Chen, Masashi Inoue, and Michael F. Sheets

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

Submitted 3 June 2004 ; accepted in final form 12 January 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na+ channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of –7 mV in the half point of the voltage-dependent Na+ channel availability curve compared with wild type. In addition, both the time course of decay of Na+ current (INa) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of INa, depending on membrane potential.

gating charge; single channel; voltage sensor; S3-S4 linker; domain IV

THE SCN5A GENE ENCODES the human cardiac Na+ channel (hH1a, NaV1.5), a voltage-gated ion channel that is responsible for the rapid upstroke of the cardiac action potential. The {alpha}-subunit of hH1a is a protein composed of four domains (see Fig. 1), each having six transmembrane-spanning segments with the fourth segment (S4) in each domain responsible for the origin of gating currents (Ig), the small nonlinear capacitance currents resulting from the voltage-dependent movements of the S4 segments (5, 7). Mutations of the human cardiac Na+ channel have been associated with genetic diseases, including the long QT syndrome (LQT3), caused by a gain in Na+ channel function (11, 17), and the Brugada syndrome, associated with a loss of function (1). The prolongation of cardiac repolarization in LQT3 results from the presence of a late component of Na+ current (INa) consistent with a defect in the fast inactivation process (4, 15). In the Na+ channel, one part of the fast inactivation apparatus consists of an inactivation particle, formed by the intracellular linker between domains III and IV, that blocks channel conductance when it becomes bound to its receptor (35). A second part consists of the voltage sensor formed by the S4 in domain IV (S4-DIV) that has been shown to couple Na+ channel activation to inactivation (8, 10, 29, 50, 51). Modification of the Na+ channel by the site-3 peptide toxin anthopleurin-A (ApA) has been shown to inhibit the movement of the S4-DIV, a voltage sensor that contributes a large component (~32%) to the maximal gating charge, Qmax, while at the same time inhibiting fast inactivation (19, 40, 41).



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  		Fig. 1. Mutant delF1617 and wild-type human heart Na+ channel (hH1a) current expressed in fused tsA201 cells. A: family of Na+ current (INa) traces in response to step depolarizations to –60, –40, –20, 0, and 20 mV from a holding potential of –150 mV in wild type (top) and delF1617 (middle). The predicted membrane topology of the Na+ channel is shown (bottom) with 4 domains (D1–DIV), each with 6 transmembrane segments, and the 10-amino acid sequence of the S3-S4 linker in domain IV. The phenylalanine at position 1617 (the 4th residue in the linker) was deleted in delF1617. B: single-exponential fits to the decay of INa in response to step depolarizations for wild-type hH1a and delF1617. Six cells were studied for each Na+ channel, and the data plotted are as means ± SE. The differences between the time constants of decay ({tau}) for the wild type and delF1617 were statistically significant (P < 0.05) at test potentials more negative than –40 mV. C: time to peak INa for delF1617 (n = 6 cells) and wild-type hH1a (n = 6 cells). Data plotted are means ± SE, and the lines connect the points. The values for time to peak INa were significantly different (P < 0.05) at test potentials more negative than –50 mV. D: normalized peak conductance-voltage relationships for delF1617 (n = 6 cells) and wild-type hH1a (n = 6). For each cell, maximum peak conductance (Gmax) was normalized to a value of 1, and the data plotted are means ± SE. The lines represent the mean of the best fit to each individual cell by a Boltzmann distribution (Eq. 1), and the parameters of the best fits to the data are given in Table 1.

 
In previous studies, a LQT3 mutation in which the outermost arginine (a positively charged residue) in the S4-DIV had been neutralized to a glutamine (R1623Q) was shown to interfere with fast inactivation leading to prolonged mean channel open times and to produce a late component of INa (27, 32). Recently, a new mutation in hH1a caused by deletion of a phenylalanine at amino acid position 1617 (delF1617) lying within the extracellular linker between S3 and S4 in domain IV was described and has been tentatively classified as a LQT3 mutation (43). To characterize the phenotype of delF1617, we performed electrophysiological studies in transfected mammalian cells and compared the results with those from wild-type hH1a Na+ channels. Using a combination of whole cell INa, single-channel recordings, and Ig recordings, we found that the shortened S3-S4 linker in delF1617 altered Na+ channel fast inactivation consistent with an apparent reduction in the voltage dependence of the S4-DIV, a voltage sensor associated with inactivation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
cDNA clones. In hH1a (kindly provided by H. Hartmann and A. Brown; Ref. 21), the amino acid phenylalanine at position 1617 (numbering based on hH1) (31) was deleted using a four-primer PCR technique (23, 25). All cDNA inserts were confirmed by sequencing. For expression, the cDNA was subcloned directionally into the mammalian expression vector pRcCMV (Invitrogen, Carlsbad, CA).

Cell preparation. Multiple tsA201 cells (SV40-transformed HEK-293 cells) were fused together by using polyethylene glycol as previously described (42). After fusion, the cells were placed in cell culture for several days to allow for membrane remodeling, and then they were transiently transfected using a calcium phosphate precipitation method (Invitrogen). For single-channel experiments, single tsA201 cells were transiently transfected with cDNA for delF1617 with the use of calcium phosphate, whereas a stable line of HEK-293 cells expressing wild-type hH1a (selected by the addition of G418 to the culture solution) was used for control studies.

Whole cell INa and Ig studies. Recordings of both INa and Ig were made using a large-bore, double-barreled glass suction pipette for both voltage clamp and internal perfusion as previously described (42). Current traces were measured with a virtual ground amplifier (Burr-Brown OPA-101), using a 2.5-M{Omega} feedback resistor with voltage protocols from a 16-bit digital-to-analog 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. An experimental temperature of 13°C was maintained using a TS-4 thermoelectric stage (Physiotemp Instruments, Clifton, NJ) mounted beneath the bath chambers. A cell was placed in the aperture of the pipette, and, after formation of a high-resistance seal, 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 per criteria previously established (20). To allow for full Na+ channel availability, we set the holding membrane potential to either –150 or –160 mV. Ig protocols typically contained four repetitions at each test voltage that were one-quarter of a 60-Hz cycle out of phase to maximize rejection of this frequency and to improve the signal-to-noise ratio.

For most whole cell INa experiments, the control extracellular solution contained (in mM) 15 Na+, 185 Cs+, 2 Ca2+, 200 MES–, and 10 HEPES (pH 7.2), and the intracellular solution contained (in mM) 15 Na+, 185 Cs+, 75 F–, 125 MES–,10 EGTA, and 10 HEPES (pH 7.2). In whole cell experiments investigating late INa, the extracellular solution contained (in mM) 45 Na+, 155 tetramethylammonium (TMA+), 200 MES–, 2 Ca+, and 10 HEPES (pH 7.2), and the intracellular solution contained (in mM) 200 TMA+, 75 F–, 125 MES–, 10 EGTA, and 10 HEPES (pH 7.2). Immediately after control measurements were carried out, the cell was exposed to the same extracellular solution but with the addition of 10 µM saxitoxin (STX; Calbiochem, San Diego, CA). The traces recorded in STX were subtracted from those recorded in control to obtain Na+ current recordings at test potentials at –60, –40, –20, 0, 20, and 40 mV.

For Ig experiments, the control extracellular solution contained (in mM) 200 TMA+, 2 Ca2+, 200 MES–, and 10 HEPES (pH 7.2), and the intracellular solution contained (in mM) 200 TMA+, 75 F–, 125 MES–,10 EGTA, 10 HEPES (pH 7.2), and 10 µM STX. For ionic current experiments with ApA toxin (Sigma, St. Louis, MO), 15 mM Na+ was added to the extracellular solution while the TMA+ concentration was reduced to 185 mM and STX was omitted. ApA toxin was used at a concentration of 1 µM, which is three orders of magnitude greater than the Kd for wild-type hH1a (19, 28).

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 leak corrected by the amount of the calculated time-independent linear leak. Data were capacity corrected by using 4–16 scaled current responses recorded from voltage steps typically between –150 and –190 mV. Ig data were leak corrected by the means of 2–4 ms of data, usually beginning 8 ms after the change in test potential.

To determine time constants of INa decay, we trimmed current traces until the decay phase was clearly apparent and then fit them with a single exponential using DISCRETE software (36). Normalized peak conductance-voltage relationships were fit with a Boltzmann distribution,

(1)
where INa is the peak current in response to a step depolarization, Vt is the test potential, and the fitted parameters are (half point of the relationship), s (slope factor, in mV), and Vrev (reversal potential). For comparison between cells, data were normalized to Gmax, the maximum peak conductance. Steady-state, voltage-dependent Na+ channel availability curves were fit with a Boltzmann distribution,

(2)
where INa is the peak current after a conditioning pulse (Vc). Two-pulse development of inactivation was fit by a double-exponential equation,

(3)
where INa is the recorded normalized peak current at the test potential and the fitted parameters are A (amplitude of the fast time constant), {tau}A (the fast time constant, in ms), B (amplitude of the slow time constant), and {tau}B (the slower time constant, in ms), t is time, and k is a constant. Charge-voltage relationships were fit with a simple Boltzmann distribution,

(4)
where Q is the charge during the depolarizing step and the fitted parameter Qmax is maximum charge. Qmax was normalized to 1 for each cell.

Data were analyzed and graphed on a SUN Sparcstation using SAS (SAS Institute, Cary, NC). Unless otherwise specified, summary statistics are expressed as means ± SD, and figures show results expressed as means ± SE. Data for wild-type hH1a and delF1617 were compared using a two-tailed, unpaired t-test and were considered to be significantly different at P < 0.05. Experimental parameters for ApA toxin-modified Na+ channels were compared with those in control solutions by using a paired t-test and were considered significantly different when P < 0.05.

Single-channel studies. For single-channel, cell-attached recordings, single cells were grown on glass coverslips and then transferred to the bath chamber containing a bath solution with (in mM) 150 K+, 2 Mg2+, 1 Ca2+, 156 Cl–, and 10 HEPES to depolarize the membrane potential to ~0 mV. Single-suction pipettes were pulled on a Flaming/Brown puller (model P-87; Sutter Instruments, Novato, CA) from 1.65-mm borosilicate glass (A&M Systems, Carlsborg, WA), coated with Sylgard 184 (Dow Corning, Midland, MI), and fire-polished. The pipette solution contained (in mM) 280 Na+, 2 Ca2+, 284 Cl–, and 10 HEPES (pH 7.4 adjusted with NaOH), resulting in pipette resistances of 2–6 M{Omega}. All single-channel currents were recorded at room temperature (24 ± 2°C). Single-channel recordings were made with an Axopatch 200B amplifier (Axon Instruments, Union City, CA) using pCLAMP (v. 8.2; Axon Instruments) on a personal computer running Windows 98 (Microsoft). The holding potential was set to –120 mV, and 100-ms step depolarizations at 1 Hz were made to test potentials between –80 and 20 mV. Data were filtered with a single-pole Bessel filter at 5 kHz and digitized at 10 kHz with a Digidata 1321A (Axon Instruments).

Single-channel data were analyzed using Fetchan (Axon Instruments). For each trace, the baseline trace was determined by Fetchan and confirmed by eye. To eliminate both the capacity transient and simultaneous single-channel openings, the data from the first 20 ms in each 100-ms depolarization were deleted from all data sets (see RESULTS). The mean amplitude of single-channel currents was determined from an all-points histogram fit by a Gaussian distribution for each patch at each test potential. Individual traces were then idealized using a half-amplitude criterion with a two-point detection algorithm, and open times were binned into 100-µs bins and fit with a single-exponential function.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Whole cell ionic currents of delF1617. To investigate delF1617, we compared INa recorded during step depolarizations to test potentials between –130 and 80 mV from a holding potential of –150 mV in fused tsA201 cells studied at 13°C. Comparison of individual current traces between the two Na+ channels showed that the overall kinetics appeared similar (Fig. 1A), although on closer inspection delF1617 appeared to have a slightly faster decay at more negative test potentials. To better compare the decay of INa, we trimmed the current traces until the decay phase was clearly evident and then fit them with a single exponential (see METHODS). As anticipated, the time constants of INa decay were significantly shorter for delF1617 at test potentials more negative than –40 mV, whereas the small differences in decay time constants were not statistically significant at potentials >0 mV (Fig. 1B). A similar relationship was found for the time-to-peak INa plots for delF1617 and wild-type hH1a. At negative test potentials, the time to peak INa was shorter for delF1617 (Fig. 1C). However, the peak conductance-voltage relationships were similar for the two Na+ channels (Fig. 1D) with no significant difference between their half points or slope factors (see Table 1), suggesting that channel activation is similar between delF1617 and wild-type hH1a.


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  		Table 1. Comparison of Boltzmann parameters to fits of G-V and voltage-dependent Na+ channel availability relationships for delF1617 mutation and wild-type hH1a

 
If there were little or no change in the activation process between the two channels, then deactivation, when open channels return to a closed, rested state(s) as the result of a sudden membrane hyperpolarization, also should be unchanged. To determine deactivation time constants without contamination by inactivation from the open state, we recorded INa tail currents (Fig. 2A) at hyperpolarized test potentials between –90 and –150 mV after a conditioning potential of –20 mV to open Na+ channels. Figure 2B shows that there were no differences between the time constants of the INa tail current relaxations of delF1617 and wild-type hH1a. These data suggest that the activation kinetics of both delF1617 and wild-type hH1a are similar, and, consequently, the differences noted in the decay of INa and in time to peak INa were not likely to have resulted from changes in activation kinetics.



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  		Fig. 2. Time constants ({tau}) of INa decay tail currents in delF1617 and wild-type hH1a. A: representative INa tail-current recordings (capacity and leak corrected) for both wild type and delF1617. The membrane potential was stepped to a conditioning potential at –20 mV until near peak INa, and then it was stepped to hyperpolarized test potentials between –90 and –150 mV to record tail currents (see inset). B: single-exponential fits to tail-current traces after capacity correction and trimming until the decay phase is evident for delF1617 and wild type. Data plotted are means ± SE for 6 cells in each group. There was no significant difference between the time constants of wild-type hH1a and delF1617 at each test potential.

 
Because the mutation in delF1617 occurs in the S3-S4 linker that is adjacent to S4-DIV, a voltage sensor that has been shown to participate in coupling channel activation to fast inactivation (8, 10, 29, 30, 40, 41, 50, 51), it is possible that the deletion of a phenylalanine may have an effect on channel inactivation. We investigated this possibility by comparing the voltage-dependent Na+ channel availability (steady-state inactivation) plots between delF1617 and wild-type hH1a. Figure 3 shows that the half point of the Boltzmann curve for delF1617 was shifted to a more negative potential by about –7 mV, consistent with an enhancement of inactivation, particularly closed-state inactivation, because Na+ channel threshold occurred near –80 mV (see Fig. 1D). To minimize any difference between the curves resulting from a time-dependent shift in background Na+ channel kinetics of the experimental preparation (39), we performed voltage protocols at similar times after disruption of the cell membrane inside the suction pipette that initiated internal perfusion of the cell. The differences in the mean cell "lifetime" after cell membrane rupture was only 1 ± 4 min, which would be predicted to result in a difference of only 0.1–0.2 mV (39).



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  		Fig. 3. Voltage-dependent Na+ channel availability (steady-state inactivation, or SSI) plots for delF1617 and wild-type hH1a. Cells were stepped to conditioning potentials for 500 ms from a holding potential of –150 mV before being stepped to a test potential of –20 mV (see inset). Peak INa was normalized to the peak INa in the absence of a conditioning step. Data plotted are means ± SE, and each point represents the mean of 6 cells. The lines represent the mean of the best fits to each cell by a Boltzmann distribution (Eq. 2), and parameters of the best fits are given in Table 1.

 
To further investigate the inactivation kinetics of delF1617, we recorded two-pulse development of inactivation protocols for delF1617 and wild-type hH1a. Figure 4 shows the results for conditioning potentials from –120 to 40 mV, and Table 2 shows the results of double-exponential fits to the data (see Eq. 3). The most apparent difference is the increased fraction of delF1617 channels that become inactivated at conditioning potentials of –120 and –80 mV compared with wild-type hH1a. The greatest difference occurred at –80 mV, where both the short and long time constants of inactivation were significantly faster for delF1617 compared with wild-type hH1a. At –120 and –40 mV, there was a trend for the time constants to be faster for delF1617 compared with wild type, although they were not statistically different. At 0 mV, both short and long time constants for delF1617 and wild-type hH1a were almost identical, whereas at 40 mV, the time constants for delF1617 started to become slower than those for wild-type hH1a. These results are consistent with those for INa decay (see Fig. 1B), where inactivation for delF1617 was more rapid at negative potentials compared with wild-type hH1a, whereas inactivation tended to be slower for delF1617 at positive potentials, although the differences were not significant.



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  		Fig. 4. Comparison of 2-pulse development of inactivation between delF1617 and wild-type hH1a. The holding membrane potential of –150 mV was stepped to conditioning potentials between –120 and 40 mV for variable times up to 1 s, stepped back to –130 mV for 2 ms to deactivate any open channels (see inset at bottom right), and then stepped to a test potential of –20 mV. Peak currents following conditioning steps were normalized to peak INa measured in the absence of a conditioning step, and the data plotted are means ± SE for each conditioning potential. Insets in each graph show the development of inactivation for the first 50 ms. The data from individual cells were fit using double-exponential fits (see Eq. 3), and parameters of the best fits are given in Table 2. Vc, conditioning potential.

 

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  		Table 2. Comparison of parameters for development of inactivation in delF1617 and wild-type hH1a

 
An important characteristic of Na+ channel mutations that have been associated with LQT3 is the presence of an increased magnitude in the late component of INa compared with that in wild-type Na+ channels (11, 17). Typically, a late component is measured at the end of a long depolarizing pulse by subtracting a recording obtained in the presence of a Na+ channel blocker (such as tetrodotoxin or STX) from one made in the absence of a Na+ channel blocker (33, 49). To determine whether a late component of INa was associated with delF1617, we measured the magnitude of the STX-sensitive INa at the end of a 100-ms step depolarization after each trace was normalized to its peak INa (Fig. 5). Note that wild-type hH1a showed a voltage-dependent decrease in the magnitude of the late component, as was anticipated if inactivation became more complete as the test potentials became more positive. By comparison, the magnitude of the late component in delF1617 was smaller at –60 mV but larger at +40 mV. In between these two potentials, there was no significant difference in magnitudes of the late component; however, the values for wild-type and delF1617 appeared to cross over near 0 mV.



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  		Fig. 5. Comparison of the late component of INa for delF1617 and wild-type hH1a. Current traces were recorded during 100-ms step depolarizations before and after the addition of 10 µM saxitoxin (STX). After subtraction of the leak trace obtained in STX, each INa trace was normalized by dividing by its peak INa, and the last 1 ms of the 100-ms trace was averaged for each cell. A: STX-sensitive INa for a step depolarization to –60 mV from a holding potential of –150 mV for wild type and delF1617. Each trace was digitally filtered at 5 kHz. Note that the normalized current traces are positive (i.e., outward) and have a peak value of 1. Below each sweep is plotted the last 25 ms on an expanded scale. B: as a function of test potential, the data are plotted as means ± SE for 6 wild-type cells and 4 delF1617 cells. The differences at –60 and 40 mV were statistically significant (P < 0.05).

 
Effects of delF1617 mutation on single-channel mean open times. Because Na+ channels can be approximated as having a single open state (18, 38, 52), the mean open time (MOT) can be represented as

(5)
where KO->C is the rate constant of deactivation and KO->I is the rate constant of inactivation. Previously, we showed that deactivation was unchanged between delF1617 and hH1a; therefore, any change in mean open time should reflect only differences in channel inactivation. Because we were interested in measuring open time intervals and not closed-state intervals, we recorded single-channel events from patches that contained a large number of channels (typically 10–30 channels/patch) and analyzed only the last 80 ms of a 100-ms depolarization, discarding the first 20 ms of the step depolarization, which contained the capacity transient and nearly all of the simultaneous channel openings. This approach allowed us to record single-channel openings while minimizing simultaneous channel openings (see Fig. 6), but it does assume that the mean channel openings discarded in the first 20 ms were representative of the subsequent channel openings. Single-channel current amplitude histograms (data not shown) showed no significant differences between wild type and delF1617 at potentials of –60 mV (1.37 ± 0.07 pA for wild type, n = 5, vs. 1.38 ± 0.02 pA for delF1617, n = 5) and 0 mV (0.63 ± 0.06 pA for wild type, n = 5, vs. 0.66 ± 0.08 pA for delF1617, n = 5). Mean open times were calculated by fitting a single exponential to the open-time histograms of each patch (see Fig. 7) and are plotted in Fig. 8. Inspection of the plot of mean open times vs. test potential shows findings typical for wild-type cardiac Na+ channels, with the longest mean opening occurring at test potentials between –60 and –40 mV, whereas the mean open time becomes shorter at more negative and more positive test potentials (13, 38, 52). We found that delF1617 had a similar appearance except that the mean open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at test potentials >0 mV.



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  		Fig. 6. Single-channel recordings of delF1617 and wild-type hH1a. Top: 200 superimposed sweeps of non-capacity-corrected single Na+ channel traces recorded from wild-type (WT) and delF1617 at a test potential of –40 mV. The holding potential was –120 mV, and the duration of the step depolarization was 100 ms. Note that the first 20 ms contains the residual capacity transient and almost all of the simultaneous channel openings. Only the last 80 ms were included in the analysis for mean channel open times because they did not typically contain simultaneous channel openings. Bottom: representative single sweep recordings after the first 20 ms were discarded.

 


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  		Fig. 7. Representative open channel duration histograms for single patches expressing either wild type or delF1617 at step potentials from –60 to 20 mV. The lines represent single-exponential fits to the binned data.

 


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  		Fig. 8. Mean channel open times for wild-type hH1a and delF1617 at test potentials from –80 to 20 mV. Data plotted are means ± SE for 5 cells at each test potential, except delF1617 had 4 cells at –60 and –40 mV and 3 cells at –80 mV. The mean open times at –60, 10, and 20 mV were significantly different (P < 0.05). The line for the wild type shows the best fit using Eq. 9 (see text), whereas the line superimposed on delF1617 uses the same parameters as those from the wild type, except the voltage dependence of QO->I for delF1617 was set equal to 0.63 x QO->I for wild type. The parameters from the fit to the mean open times of wild-type hH1a from Eq. 9 were KO->C at 0 mV = 0.02 ms–1, KO->I at 0 mV = 7.0 ms–1, QO->C = 1.67 eo, and QO->I = 0.46 eo. (See text for definitions.)

 
Because the O->C kinetic transition was shown to be the same for both channels at hyperpolarized membrane potentials (see Fig. 2), any differences between the mean open times for delF1617 compared with hH1a should result from changes in the O->I kinetic transition, assuming that the O->C kinetic transition remains identical for both channels at depolarized potentials. By applying Erying rate theory to a Markov model of Na+ channel kinetic transitions (see review, Ref. 5), the rate constant for the O->I transition, KO->I, can be represented as

(6)
where KO->I at 0 mV is the rate constant at 0 mV, QO->I is the gating charge associated with the O->I transition, Vm is the membrane potential, F is Faraday’s constant, R is the gas constant, and T is temperature (F/RT approximates a value of 25 mV at 20°C). Considering that the mean channel open times for delF1617 and wild-type hH1a appear almost identical at 0 mV, inspection of Eq. 6 suggests that the voltage dependence between the two channels must be different, with delF1617 having less gating charge (QO->I) than the wild type.

Effects of delF1617 on gating charge contributed by S4-DIV. Because Ig measurements reflect the voltage dependence of kinetic transitions, we measured the gating charge for delF1617 and compared it with previous measurements obtained for wild-type hH1a. As mentioned above, the S4-DIV in Na+ channels has a unique role in coupling channel activation to fast inactivation (8–10, 29, 30, 50, 51), and it is largely responsible for imparting voltage dependence to the O->I transition. ApA toxin, a site-3 toxin, has been shown to specifically block the movement of the S4-DIV, causing a reduction of nearly 32% in the Qmax of wild-type hH1a (39–41). If the change in the mean open time for delF1617 resulted from the S4-DIV contributing less voltage dependence to the O->I transition, then ApA toxin should cause a smaller reduction in the Qmax of delF1617 compared with the wild type. To investigate this possibility, we constructed gating charge-voltage relationships for delF1617 both before and after modification by ApA toxin. Figure 9A demonstrates that ApA toxin was able to modify delF1617 and cause characteristic slowing of INa decay during a step depolarization. Figure 9B shows the gating charge-voltage relationships for four cells before and after modification by ApA toxin. In contrast to our previously reported finding of a 32% decrease in the Qmax of wild-type hH1a, ApA toxin caused only a 23 ± 5% (mean ± SD) decrease in delF1617, suggesting that the deletion of a phenylalanine in the S3-S4 linker in domain IV resulted in a reduction in gating charge contributed by the S4-DIV.



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  		Fig. 9. Effect of anthopleurin A (ApA) toxin, a site-3 toxin, on INa and conductance-voltage relationships of delF1617. A: family of INa traces recorded before (control) and after modification by 1 µM ApA toxin from a holding potential of –160 mV to step depolarizations between –100 and 20 mV in 15 mM extracellular Na+. Note the characteristic slowing of INa decay by ApA toxin for delF1617. The current traces were leak and capacity corrected and were digitally filtered at 5 kHz. B: mean conductance-voltage relationships are shown for delF1617 in control and after modification by 1 µM ApA toxin. Data plotted are means ± SE (n = 4 cells), and gating charge in toxin was normalized to the Qmax determined for each cell in control. The mean of the fits to each cell by a Boltzmann distribution (Eq. 4) is represented by the solid lines and shows that Qmax was significantly (P < 0.5) reduced by 23 ± 5% (SD), whereas both of the slope factors were similar at –12 ± 2 mV (control) vs. –13 ± 3 mV (toxin), as were the half points at –63 ± 6 mV (control) vs. –60 ± 5 mV (toxin).

 
Knowing that the ratio of gating charge in the S4-DIV to that in the other three domains for wild-type hH1a is

(7)
and that the ratio for delF1617 is

(8)
where the gating charge from S4-DI for the wild type is represented by QwtD1 and so on, and the gating charge from S4-DI for delF1617 is represented by QdelD1 and so on. Assuming that the gating charge from domains I, II, and III remained the same for both wild-type hH1a and delF1617, we can solve for the ratio of QdelD4/QwtD4, which equals 0.63. This corresponds to a 37% reduction in the gating charge from the S4-DIV in delF1617 compared with that in wild-type hH1a. Substituting Eq. 6 for the rate constant KO->I into Eq. 5 and including a similar equation for KO->C, the mean open time over a range of test potentials can be represented as (52)

(9)
where the variables are defined as in Eq. 6 with KO->C and QO->C representing the corresponding values for the deactivation transition. Consequently, mean open times can be described by four variables: the rate constants for both the O->C and O->I transitions at 0 mV and the voltage dependencies of QO->C and QO->I. Using Eq. 9, we first fit the mean open time vs. voltage relationship for wild-type hH1a (see Fig. 8). Using the same parameters obtained from the fit to wild-type hH1a but setting QO->I in delF1617 equal to 0.63 x QO->I for wild-type hH1a, the mean open times for delF1617 can be predicted, and they have been plotted as the line overlaying the mean open times for delF1617 (Fig. 8). Note that the line representing the predicted mean open time appears to describe the data well.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We studied the SCN5A mutation delF1617, a deletion of a phenylalanine in the extracellular linker between the S3 and S4 segments in domain IV of the human heart Na+ channel. Whole cell current recording showed similar peak conductance-voltage curves and tail-current relaxations (i.e., deactivation time constants) for delF1617 and wild-type hH1a, suggesting that channel activation was unchanged by the mutation. However, measurements that depend, in large part, on fast inactivation, such as the time course of decay of INa and the two-pulse development of INa inactivation, differed between delF1617 and wild-type hH1a. Accordingly, the voltage-dependent Na+ channel availability curve that depends on both channel activation and inactivation (primarily from closed states) was significantly shifted by –7 mV in delF1617 compared with wild-type hH1a.

To further investigate the effects of delF1617 on inactivation, we compared single channel mean open times between the mutant channel and wild-type hH1a. Because mean open times in Na+ channels can be well described by two rate constants, one for deactivation (O->C) and the other for inactivation (O->I) (38, 52), differences between the O->I rate constants will be directly reflected by changes in mean open time if the O->C rate constants are the same, as we have shown for delF1617 and wild-type hH1a (see Fig. 2). Consistent with whole cell measurements, the mean open times for delF1617 were shorter than those for hH1a at negative potentials, but they were longer at positive potentials. At 0 mV they appeared to be equal. These results suggested that the O->I rate transition in delF1617 may have a reduced voltage dependence compared with wild-type hH1a and led us to measure the fractional reduction in Qmax caused by ApA toxin. ApA toxin is a site-3 toxin that has been shown to inhibit movement of the S4-DIV, a voltage sensor associated with fast inactivation (9, 41). If the S4-DIV in delF1617 were to contribute less gating charge than that from wild-type hH1a, then the reduction in Qmax after modification by ApA toxin should be less than the 32% reduction found for wild-type Na+ channels (39–41). We found that ApA toxin caused only a 23% reduction in Qmax in delF1617, an amount that is equivalent to a 37% reduction in the gating charge contributed by the S4-DIV in delF1617 compared with that for hH1a, assuming that the gating charge from the other S4 segments remain unchanged for both channels. Furthermore, this reduction in gating charge occurred even though the S4 segments in DIV had not been mutated in either channel.

Comparison with other cardiac Na+ channels with mutations in S3-S4 linker of domain IV. The LQT3 channel R1623Q, in which the outermost arginine in the S4-DIV is mutated to a glutamine, also has been shown to affect fast inactivation (10, 26, 32). The mutation in R1623Q is also similar to the mutant human skeletal muscle Na+ channel (hSkM1, NaV1.4), R1448C, in which the outermost positively charged residue, an arginine, in the S4-DIV is mutated to a cysteine and causes the human disease paramyotonia congenita (37). In both of these two Na+ channel mutations, neutralization of the outermost charged residue in S4-DIV would be expected to decrease the gating charge associated with the S4-DIV, and, for hH1a, this has been shown to be the case (41). However, in both R1623Q and R1448C, the decay of INa in response to step depolarizations was slowed across the full potential range (8, 10) in contrast to delF1617, in which decay of INa was faster at negative potentials but tended to be slower at positive test potentials (see Fig. 1B). Furthermore, the single-channel mean open time for R1623Q has been shown to be prolonged at –20 mV (27) compared with a shortening of mean open times at negative potentials for delF1617 (see Fig. 8). As a result, R1623Q causes a gain in Na+ channel function across all test potentials and is characteristic of SCN5A mutations associated with LQT3 (see review, Ref. 48). However, it is not clear that delF1617 will necessarily result in a gain of function, because any change in the magnitude of INa will depend on the membrane potential.

Although both delF1617 and R1623Q (41) have been shown to reduce the gating charge contribution from the S4-DIV, they have different phenotypic characteristics, suggesting additional differences between the two channels. An ion channel’s kinetic transition can be described as a rate constant at 0 mV that is modified by a voltage-dependent factor, a factor that is dependent on the transition’s gating charge (see Eq. 6, and for review see Ref. 24). If only the voltage-dependent factor were decreased, then the rate constant at 0 mV would remain unchanged, and any differences between rate constants would become manifest as the membrane potential varied from 0 mV. This appears to be the case for delF1617, whereas both the rate constant at 0 mV and the voltage-dependent factor appear to be decreased for R1623Q (27, 41).

Although the S3-S4 linker in domain IV has been postulated to be 10 amino acids long (34), it is possible that the last 3 amino acids (threonine, leucine, and phenylalanine) of the linker should be included as part of the presumed {alpha}-helix formed by the S4-DIV, thereby shortening the linker to 7 residues terminating with a proline (see Fig. 1A). In such a shortened linker, the deletion of a phenylalanine may alter its flexibility such that it opposes the force of the applied voltage field on the S4-DIV, thereby making it more difficult for the voltage sensor to return to both a fully closed, rested position during membrane hyperpolarization and to a fully extended position during a strong depolarization. The impaired movement of the S4-DIV during hyperpolarization could become manifest as a leftward shift in the Na+ channel availability curve, whereas the reduced flexibility of the S4-DIV at positive membrane potentials could result in a slowing of inactivation. A second possibility is that the shortened S3-S4 linker in delF1617 may affect the conformation of any water-filled crevices surrounding the S4-DIV, thereby altering the fraction of the electric field that is sensed by the S4-DIV with a resultant decrease in gating charge (16). Either of these two models would decrease the gating charge contribution by the S4-DIV and would be expected to significantly affect INa. In contrast, other mutations involving the S3-S4 linker that do not affect the S4-DIV might be expected to have less effect on INa. Such may be the case for the Brugada mutation, T1620M, in which the threonine (position 8 in Fig. 1A) in the S3-S4 linker of domain IV has been substituted by a methionine. It appears that T1620M has only minor effects on Na+ channel kinetics (3, 47) unless specific conditions such as an increased experimental temperature is present (14). Interestingly, the presence of a second mutation, R1232W, in addition to T1620M severely impairs expression in mammalian cells when the {beta}1-subunit is coexpressed (2, 46), suggesting that the S3-S4 linker in domain IV may play a role during coassembly with {beta}1.

Comparison with studies of S3-S4 linker in other ion channels. Only a few studies have investigated the effects of the S3-S4 linker in domain IV on Na+ channel kinetics. In sensory neuron-specific Na+ channels (SNS, NaV1.8), the S3-S4 linker has 13 amino acids compared with 10 residues for nearly all other Na+ channels (34); however, the additional 3 residues appear to have minimal effects on kinetic measurements of INa (12). Two other Na+ channel mutations in the S3-S4 linker in domain IV have been studied. One mutation (associated with the congenital myasthenic syndrome) has a neutral valine changed to a negatively charged glutamate (V1442E) in the S3-S4 of domain IV in hSkM1(45). Whole cell INa studies showed that V1442E enhanced fast inactivation near the resting membrane potential, although none of the voltage-clamp protocols investigated positive potentials. If inactivation remained enhanced at potentials >0 mV in V1442E, it is possible that the substitution of a glutamate caused an electrostatic attraction between it and the positively charged arginine residues in the S4-DIV similar to the electrostatic mechanisms recently reported in the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel (22, 44). In Shaker K+ channels, shortening the length of the S3-S4 linker from the normal 31-amino acid segment to a 5-amino acid segment had only a minor effect on the magnitude of gating charge, reducing it by 2% from 12.6 eo to 12.3 eo (16). When all 31 amino acids were deleted from the S3-S4 linker of the Shaker K+ channel, the gating charge was reduced by 56% to 5.6 eo. This result is qualitatively similar to our results; however, we found that only a single amino acid deletion in S3-S4 linker in hH1a reduced the gating charge of S4-DIV by 37%.

Potential mechanisms of disease of delF1617 in LQT3. Na+ channels have been shown to cause LQT3 by a gain in function typically associated with a defect in inactivation that causes an increase in the late component of INa (11, 17), whereas a loss of Na+ channel function has been associated with the Brugada Syndrome (1). Although delF1617 has been tentatively classified as a LQT3 mutation (43), our results (see Fig. 5) suggest a more complicated interpretation. delF1617 demonstrated an increase in the late component of INa only at positive test potentials that are close to those of an action potential plateau, whereas at negative test potentials, its late component was smaller than that of the wild type. There are previously reported examples of a single SCN5A mutation causing both the LQT3 syndrome and the Brugada syndrome (6), consequently the overall effect of delF1617 on the clinical presentation of patients may depend on whether the conducted action potential results in an overall augmentation of INa or its diminution.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants P50 HL-52338 and R01 HL-44630.


   ACKNOWLEDGMENTS
 
We thank Dr. Wenqing Yu for excellent technical assistance and Dr. D. A. Hanck for excellent comments.


   FOOTNOTES
 

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.


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