Volume 141, No. 5, May 6, 2013. Pages 601–618.
In our 2013 paper in the Journal, we investigated the effects of mutations in the S1, S2, and S3 segments for each of the four domains of the voltage-gated sodium channel of skeletal muscle. As part of that work, we reported the effects of mutations on activation using I-V relations from experiments using the nonpermeant cation N-methyl-d-glucamine for internal and external recording solutions.
These conditions do not provide a measure of driving force as needed for a Boltzmann fit to obtain conductance parameters of midpoint and slope factor. Therefore, we have analyzed the original dataset using the asymptote for the relative change in current amplitude to estimate driving force and plot G-V relations. G/GMAX was plotted as a function of voltage, and these curves were fit with a Boltzmann function to yield corrected values for midpoint and slope factor. These values and calculations for the change in free energy (ΔΔGo, nzFV0.5) for mutations are provided in Table 3. Correction to plots for I-V relations given in the original paper are provided as extra panels for Figs. 2, 6, and 10. Fig. 11 has been replotted in its entirety. Table 3 and Figs. 2, 6, 10, and 11 are below.
The corrected parameters for activation of wild-type hNaV1.4 in Table 3 are comparable to conductance measurements for the skeletal muscle sodium channel in other reports. Calculations of the change in free energy associated with activation of hNaV1.4 yielded values of 2–3 kcal/mol for several mutations in the ENC and HCR of domains I–III, and the INC of domains I and II. These values are similar to those calculated for S1 and S2 countercharge mutations in NachBac (DeCaen et al., 2008, 2011). Our calculations of free energy differences are based on the effects of point mutations in a single domain of the skeletal muscle sodium channel, and interpretation of the role of S1–S3 countercharges based on those calculations should be limited.
In our original paper we concluded that negative countercharges in domains I–III are an important determinant of channel activation, most likely interacting with S4 positively charged residues to facilitate the outward movement of the voltage sensors in those domains in response to membrane depolarization. Further experimentation is needed to test our hypotheses about the general role of countercharge as a determinant of activation in NaV1.4. In addition, we speculated that ENC and HCR countercharge residues in domains I and II may interact with outer positive charges in the S4 segments of these domains. A more recent study does show that ENC residues in domains I and II of rNaV1.4 are important for channel activation (Pless et al., 2014). Future work describing the role of HCR or INC residues in activation is also needed to investigate the mechanisms by which S1–S3 countercharges affect sodium channel gating.