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Link to original content: https://pubmed.ncbi.nlm.nih.gov/16873405
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. 2006 Oct 15;576(Pt 2):493-501.
doi: 10.1113/jphysiol.2006.115659. Epub 2006 Jul 27.

Isoform-dependent interaction of voltage-gated sodium channels with protons

A Khan  1 , J W Kyle, D A Hanck, G M Lipkind, H A Fozzard
Affiliations

Isoform-dependent interaction of voltage-gated sodium channels with protons

A Khan et al. J Physiol. .

Abstract

Protons are potent physiological modifiers of voltage-gated Na(+) channels, shifting the voltage range of channel gating and reducing current magnitude (pK(a) approximately 6). We recently showed that proton block of the skeletal muscle isoform (Na(V)1.4) resulted from protonation of the four superficial carboxylates in the outer vestibule of the channel. We concluded that the large local negative electrostatic field shifted the outer vestibule carboxylate pK(a) into the physiological range. However, block was not complete; the best-fit titration curves yielded an acid pH asymptote of 10-15%, suggesting that the selectivity filter carboxylates may not be protonated. Using HEK 293 cells stably expressing different isoforms, each with varying channel density, we demonstrate that a pH-independent current is found in Na(V)1.4, but not in the cardiac isoform (Na(V)1.5). Mutational studies showed that absence of the pH-independent current in Na(V)1.5 could be ascribed to the cysteine in domain I, just above the selectivity filter aspartate (Cys373). We suggest that this cysteine can be protonated in acid solution to produce a positive charge that blocks the pore. Competition between protons and Na(+) did not exist for Na(+) concentrations between 1 and 140 mm. The residual current in acid solution, when the cysteine is absent, confirms that over the range of pH values that can be achieved physiologically, the selectivity filter carboxylates are not protonated. The pH-independent current helps to protect activation of skeletal muscle during the acidosis that occurs during exercise.

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Figures

Figure 1
Figure 1. pH-dependent shift in kinetics
Cells were held at −140 mV and stepped to potentials between −120 and +40 mV in 5 or 10 mV increments. Peak currents were measured, plotted and fit to a Boltzmann function as described in Methods. A, raw currents in step depolarizations for an example cell expressing NaV1.4 studied at pH 7.3, 6.75 and 6.3. Currents are shown for voltage steps beginning at −50 mV without leak correction but with capacity correction. Points between onset of the step and 0.2 ms are omitted because of incomplete capacity correction. B, peak current–voltage relationships from data in A. Data at pH 7.3 (open symbols) were obtained before and between each change in bath pH; data at pH 6.3 (▾) and pH 6.75 (•). Conductance transforms are shown in the inset. Continuous lines represent best-fit parameters for the Boltzmann fit to data between −110 and +10 mV. Fit values (± s.e.m.) for the sequentially measured current parameters are given in Table 2. There was a background hyperpolarizing shift in gating that occurred during the experiment without a change the maximum peak conductance (Gmax). The first measurement of the half-point of the relationship (V½) at pH 7.3 was −34 mV, the second was −39 mV, and the third was −46 mV. This shift was independent of the reversible depolarizing shift produced by exposure to acid solutions. Note that for this cell the combination of hyperpolarization with time, and depolarization during exposure to acid solutions, created the impression that peak currents scaled. This was coincidental (see Table 2 and Fig. 1C inset). C, protons reversibly affected the voltage range over which channels gated. Data were corrected by the amount of background shift in kinetics by linear interpolation, as illustrated in the inset for the cell illustrated in A and B. Shifts were similar for each channel isoform, and under all ionic conditions studied. The graph shows the data for the pH shift in V½ of activation for NaV1.4 (50 mm Na0+, •, n = 23), NaV1.4 (10 mm Na0+, ▴, n = 2), NaV1.4 (140 mm Na0+, ▪, n = 4), NaV1.5 (50 mm Na0+, ⋆, n = 19), NaV1.4(Y401C) (50 mm Na0+, ▸n = 22), and NaV1.5(C373Y) (50 mm Na0+, ⋄, n = 14).
Figure 2
Figure 2. Titration curves of Gmax for NaV1.4 and NaV1.5 in solutions of different Na+ concentrations
For each cell, Gmax was expressed relative to the value at pH 7.3. Continuous lines are based on best-fit values for fitted parameters to the logistic function given in Table 1. The symbols represent measurements made in different Na+ concentrations (mm). A, NaV1.4, 47 values derived from 39 cells. □, 140/10; ^, 50/10; ▿, 50/2; ⋄, 10/10; ▵, 10/2. B, NaV1.5, 32 values derived from 28 cells. •, 50/10; ⋄, 10/10; ▴, 10/2; ▾, 2/0.5; ▪, 1/0.5. See text for discussion.
Figure 3
Figure 3. Titration curves for cysteine-switching mutations with asymptotes determined by fitting
For each cell Gmax was expressed relative to the value at pH 7.3. Continuous lines are based on best-fit values for fitted parameters to the logistic function given in Table 1. A, NaV1.5(C373Y); 28 values derived from 23 cells. B, NaV1.4(Y401C); 26 values derived from 19 cells.
Figure 4
Figure 4. Titration curves for NaV1.4 mutations Y401F and Y401S
For each cell, Gmax was expressed relative to the value at pH 7.3. Continuous lines are based on best-fit values for fitted parameters to the logistic function given in Table 1. A, NaV1.4(Y401F); 20 values derived from 20 cells. B, NaV1.4(Y401S); 31 values derived from 31 cells.
Figure 5
Figure 5. Model of the selectivity filter
Side-view model of the selectivity filter showing the native tyrosine in position 401 (top) and the Y401C mutant (bottom). The selectivity filter residues, and the negative tyrosine and cysteine of the Y401C, are shown as space-filled images. P loops of domains I–III are shown as green ribbons. The models were generated using the Insight and Discover graphical environment, as previously described (Lipkind & Fozzard, 1994, 2000).
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