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Link to original content: https://pubmed.ncbi.nlm.nih.gov/12181282
Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block - PubMed Skip to main page content
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. 2002 Aug 15;543(Pt 1):71-84.
doi: 10.1113/jphysiol.2002.021014.

Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block

A Khan  1 , L Romantseva, A Lam, G Lipkind, H A Fozzard
Affiliations

Role of outer ring carboxylates of the rat skeletal muscle sodium channel pore in proton block

A Khan et al. J Physiol. .

Abstract

Voltage-gated Na+ current is reduced by acid solution. Protons reduce peak Na+ conductance by lowering single channel conductance and shift the voltage range of gating by neutralizing surface charges. Structure-function studies identify six carboxyls and a lysine in the channel's outer vestibule. We examined the roles of the superficial ring of carboxyls in acid block of Na(v)1.4 (the rat skeletal muscle Na+ channel isoform) by measuring the effects of their neutralization or their substitution by lysine on sensitivity to acid solutions, using the two-micropipette voltage clamp in Xenopus oocytes. Alteration of the outer ring of carboxylates had little effect on the voltage for half-activation of Na+ current, as if they are distant from the channels' voltage sensors. The mutations did not abolish proton block; rather, they all shifted the pK(a) (-log of the dissociation constant) in the acid direction. Effects of neutralization on pK(a) were not identical for different mutations, with E758Q > D1241A > D1532N > E403Q. E758K showed double the effect of E758Q, and the other lysine mutations all produced larger effects than the neutralizing mutations. Calculation of the electrostatic potential produced by these carboxylates using a pore model showed that the pK(a) values of carboxylates of Glu-403, Glu-758, and Asp-1532 are shifted to values similar to the experimentally measured pK(a). Calculations also predict the experimentally observed changes in pK(a) that result from mutational neutralization or introduction of a positive charge. We propose that proton block results from partial protonation of these outer ring carboxylates and that all of the carboxylates contribute to a composite Na+ site.

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Figures

Figure 1
Figure 1. Effect of acid solutions on the Nav1.4 channel currents
A, uncorrected currents from steps to various voltages from a Xenopus oocyte expressing Nav1.4 wild-type channels at pH 7.08, 5.76 and 8.3. B, current-voltage plot of peak whole-cell currents from the experiments shown in A. C, conductance transforms of the data in A and B for pH 7.08 and pH 5.76. Continuous lines represent the Boltzmann fits to the data. D, dose-response curve of average values for the wild-type channel from all pH values tested. Beside each point is the number of experimental values obtained at that pH. Error bars indicate the s.e.m. Fitted to the data is a single site binding curve with zero asymptote, resulting in a pKa of 5.91 ± 0.08.
Figure 2
Figure 2. Titration curves for mutant channels compared to the wild-type channel
A, domain I mutants. E403Q pKa = 5.82 ± 0.05; E403Q/E758Q pKa = 5.33 ± 0.07; E403K pKa = 5.05 ± 0.07. B, domain II mutants. E758Q pKa = 5.50 ± 0.05; E758K pKa = 4.99 ± 0.03. C, domain III mutant. D1241A pKa = 5.62 ± 0.03. D, domain IV mutants D1532N pKa = 5.72 ± 0.05; D1532K pKa = 5.26 ± 0.03. The derived fit to the wild-type channel illustrated in Fig. 1 is shown in each panel as a dashed line(WT). Average values (with s.e.m.) are fitted to a single site binding curve with zero asymptote. The numbers of experimental values included in the average response at one pH differ. Beginning at the most alkaline pH they are for E403Q: 7, 20, 13, 10, 3, and 3; for E403K: 4, 3, 10, 4, 5, and 4; for E403Q/E758Q: 8, 21, 5, 7, 7, and 4; for E758Q: 4, 6, 5, 3, and 3; for E758K: 16, 38, 12, 11, 7, and 4; for D1241A: 3, 15, 4, 3, 5, and 3; for D1532N: 6, 13, 6, and 3; and for D1532K: 3, 8, 8, 14, 3, 3, and 3.
Figure 3
Figure 3. V1/2 of activation as a function of pH
The V1/2 of activation for each pH and for each mutant is plotted, with its s.e.m. n = 3 or more for each point. The line connects the points for the wild-type Nav1.4 channel. No obvious difference was seen between the shift of the wild-type (wt) channel and those of the outer ring carboxylate mutants.
Figure 4
Figure 4. Electrostatic isopotential surfaces inside a model of the Na+ channel outer vestibule (top view)
Electrostatic calculations were carried out using the DelPhi module of Insight II and the Lipkind & Fozzard (2000) model of the Na+ channel outer vestibule. Contours of isopotential surfaces are shown at the level of −2 and −6 kT and at the level of +6 kT, with yellow and black for negative potentials, respectively, and with blue for positive potentials. The backbones for the channel P loops of domains I-IV are shown by green ribbons. The S5 and S6 α-helices from each domain were included in the calculation, but for clarity they are omitted from the figure. The residues of the selectivity filter (DEKA motif) and the residues of the external charged ring are shown by ball and stick images. This image looks directly into the outer mouth of the pore. Note the arginines (R395 and R750) located on the P loop helices of domains I and II, and the selectivity filter lysine (K1237), which make the vestibule electrostatic field asymmetrical.
Figure 5
Figure 5. Electrostatic potential at the carboxylate of Asp-1532 resulting from other charged residues around the channel vestibule (top view)
Electrostatic potential inside the Na+ channel vestibule calculated with zero charge on the carboxylate group of the side chain of Asp-1532. Its charge was set to zero in order to determine the electrostatic potential at the oxygens of Asp-1532 produced by the other vestibule charged residues. The red contour is −3 kT, the yellow contour is −2 kT, and the blue contour is +3 kT. Note that the two oxygens of Asp-1532 (small red balls) are located close to the −3 kT contour. In this case, the electrostatic potential is less than the one shown in Fig. 4, and the −3 kT contour has a shape and distribution similar to that of the −6 kT contour in Fig. 4. Similar calculations were made for the other carboxylates (see Table 2).
Figure 6
Figure 6. Electrostatic potentials around the side chain of Asp-1532 (side view)
The contours are as described in the legend of Figure 5. Note the positive potential around Lys-1237, as well as the location of Asp-1532 relative to the −3 kT electrostatic potential contour.
Figure 7
Figure 7. Electrostatic difference potential surface for the E758Q mutant
The +1 kT difference between the electrostatic fields calculated for the wild-type channel and for the mutant E758Q (ψE758Q – ψwild-type) is shown in blue. The channel structure is as shown in Fig. 4, except that it is tilted to the side to show the relation between the difference field and the selectivity filter. With neutralization of Glu-758 the potential is altered at the other carboxylate residues located within this surface sufficient to shift their pKa values by −0.40 to −0.45 pH units.
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References

    1. Antosiewicz J, McCammon JA, Gilson MK. Prediction of pH-dependent properties of proteins. Journal of Molecular Biology. 1994;238:415–436. - PubMed
    1. Bashford D, Karplus M. pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990;29:10219–10225. - PubMed
    1. Benitah J-P, Balser JR, Marbán E, Tomaselli GF. Proton inhibition of sodium channels: mechanisms of gating shifts and reduced conductance. Journal of Membrane Biology. 1997;155:121–131. - PubMed
    1. Cai M, Jordan PC. How does vestibule surface charge affect ion conduction and toxin binding in the sodium channel? Biophysical Journal. 1990;57:883–891. - PMC - PubMed
    1. Chang NS, French RJ, Lipkind GM, FoARDZZ HA, Dudley SC., Jr Predominant interactions between μ-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis. Biochemistry. 1998;37:4407–4419. - PubMed

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