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, 8 (12), e81063
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Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-terminus of NaV1.4: Is Ca2+ Regulation Defective?

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Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-terminus of NaV1.4: Is Ca2+ Regulation Defective?

Subrata Biswas et al. PLoS One.

Abstract

Mutations in the cytoplasmic tail (CT) of voltage gated sodium channels cause a spectrum of inherited diseases of cellular excitability, yet to date only one mutation in the CT of the human skeletal muscle voltage gated sodium channel (hNaV1.4F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNaV1.4F1705I are incompletely understood. The location of the hNaV1.4F1705I in the CT prompted us to examine the role of Ca(2+) and calmodulin (CaM) regulation in the manifestations of myotonia. To study Na channel related mechanisms of myotonia we exploited the differences in rat and human NaV1.4 channel regulation by Ca(2+) and CaM. hNaV1.4F1705I inactivation gating is Ca(2+)-sensitive compared to wild type hNaV1.4 which is Ca(2+) insensitive and the mutant channel exhibits a depolarizing shift of the V1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNaV1.4 channel background (rNaV1.4F1698I) eliminates Ca(2+) sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca(2+) sensitivity of gating between wild type and mutant human and rat NaV1.4 channels are in part mediated by a divergence in the amino acid sequence in the EF hand like (EFL) region of the CT. Thus the composition of the EFL region contributes to the species differences in Ca(2+)/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows INa decay in a Ca(2+)-sensitive fashion. The combination of the altered voltage dependence and kinetics of INa decay contribute to the myotonic phenotype and may involve the Ca(2+)-sensing apparatus in the CT of NaV1.4.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Ca2+-sensitivity of human NaV1.4F1705I inactivation.
(A) A schematic of the structured region of the C-terminus of hNaV1.4 between amino acids residues 1788 and 2040, the predicted helices are labeled H1–H6. The location of the EFL residues in and around H1 harbors species specific variations in the key Ca2+ sensing residues in hNaV1.4 (G1613S and A1636D) compared with the rat isoform. The CaM binding motif IQ in H6 and, the cold aggravated myotonia mutation F1705I (rat: F1698I) in H5 are illustrated. (B) Representative whole-cell currents through wild type and mutant hNaV1.4F1705I channels expressed in HEK293 cells in [Ca2+]i free conditions. Na+ currents were elicited by the protocol in the inset. (C) [Ca2+]i does not alter the I–V relationship of hNaV1.4F1705I. (D) Representative steady-state inactivation currents from different holding potentials through mutant hNaV1.4F1705I channels in the presence of 0.5 µM or absence of Ca2+. (E) Activation and steady-state inactivation curves of wild type and hNaV1.4F1705I channels in the absence and presence of 0.5 µM intracellular Ca2+. The V1/2 of inactivation of hNaV1.4F1705I is sensitive to [Ca2+]i and significantly shifted in the hyperpolarizing direction in the presence of Ca2+ (p<0.005). The activation relationships are fitted with dotted lines, the V1/2 of activation are unaffected by change in [Ca2+]i. (F) and (G) illustrate the window currents through wild type and hNaV1.4F1705I in presence (F) and absence (G) of intracellular Ca2+, respectively. Dotted lines represent the wild type channel. The symbols and color are the same in plots C, E, F, and G.
Figure 2
Figure 2. Current decay of myotonia mutant channels.
The time constant of decay is altered in hNaV1.4F1705I compared to wild type hNaV1.4 channels. (A) Superimposed normalized currents through wild type and hNaV1.4F1705I channels at test pulses of −30, −20, and −10 mV exhibiting slowing of current decay in the mutant channel. (B) The time constants of current decay of hNaV1.4F1705I channels are significantly different compared to wild type in the absence (p<0.05) or presence of 0.5 µM of Ca2+ (p<0.001). The inset is an expanded view −20 mV to 0 mV. (C) Raw current traces of hNaV1.4F1705I channels at RT and at 37°C. Currents were measured at −20 mV, in the same cell at different temperatures with 0.5 µM of Ca2+ in the pipette. (D) Plot of the time constants of current decay of wild type and hNaV1.4F1705I channels at RT and at 37°C in 0.5 µM of Ca2+. Paired measurements were made at RT and at 37°C. The current decay of hNaV1.4F1705I is significantly different at 37°C compared to RT (p<0.05). (E) Plot of the steady-state inactivation of hNaV1.4F1705I channels at 37°C in 0.5 µM of Ca2+. For comparison the steady-state inactivation of wild type and hNaV1.4F1705I from Figure 1E are re-plotted. The symbols are the same in plots B, D and E.
Figure 3
Figure 3. CaM-induced shift of inactivation in hNaV1.4 channels.
Representative Na+ currents and I–V relationships for wild type (A) and hNaV1.4F1705I channels (B) elicited by the same pulse protocol shown in the inset of Figure 1B. CaM and CaM1234 over expression significantly (p<0.05) shifts the wild type but not the hNaV1.4F1705I I–V in the depolarizing direction. (C) Representative steady-state inactivation currents elicited from different holding potentials. (D, E) Plots of the steady-state inactivation and activation relationships of wild type hNaV1.4 (D) and hNaV1.4F1705I channels (E). The solid lines are the fits to steady-state inactivation data with CaM and CaM1234 over expression. There is a significant (p<0.005) depolarizing shift of the inactivation curve by CaM and CaM1234 compared to the expression of hNaV1.4F1705I alone. The symbols and colors are the same in all panels of the figure.
Figure 4
Figure 4. Ca2+ regulation of the rat NaV1.4.
Whole-cell rNaV1.4 and rNaV1.4F1698I expressed currents (A) are not affected by [Ca2+]i. (B) The voltage dependence of activation (dotted lines) and steady-state inactivation (solid lines) of rNaV1.4. (C) rNaV1.4F1698I significantly (p<0.05) shifts the activation and inactivation curves in the depolarizing direction and eliminates the sensitivity of inactivation to changes in [Ca2+]i. The dotted lines represent the wild type channel in the absence of [Ca2+]i. The symbols are the same in plots B and C.
Figure 5
Figure 5. CaM shifts inactivation of rat NaV1.4F1698I.
(A) Representative families of rNaV1.4F1698I activation currents co-expressed with either CaM or CaM1234. (B) Representative steady-state inactivation currents elicited from different holding potentials through rNaV1.4F1698I channels in presence and absence of CaM over expression. (C) Activation and steady-state inactivation relationships. The solid lines are the fits to steady-state inactivation data of rNaV1.4F1698I with CaM and CaM1234 over expression. There is a significant (p<0.05) shift of the inactivation curve by CaM compared to the expression of rNaV1.4F1698I alone. Over expression of CaM1234 has no significant effect compared with the absence of CaM over expression. In contrast, the V1/2 of activation of rNaV1.4F1698I, is not changed by co-expression of CaM or CaM1234. The dotted lines in panel (C) represent wild type channel in 0.5 µM Ca2+.
Figure 6
Figure 6. Exchange of human and rat EFL residues in hNaV1.4.
(A) Amino acid sequence alignment of the proximal CT of rat and human wild type NaV1.4 channel and hNaV1.5 demonstrate the similarity of rNaV1.4 and hNaV1.5 at key positions in the EFL. In hNaV1.4F1705I+GA/SD residues G1613S and A1636D are substituted in the human channel hNaV1.4F1705I to match the corresponding residues of rNaV1.4. (B) Representative families of hNaV1.4F1705I+GA/SD activation currents in the presence and absence of [Ca2+]i. (C) Normalized I–V relationships hNaV1.4F1705I+GA/SD are not affected by altered [Ca2+]i or CaM and CaM1234 over expression. (D) The steady-state inactivation of hNaV1.4F1705I+GA/SD channels are not sensitive to changes in [Ca2+]i. The dotted lines in panel (D) represent hNaV1.4 in 0.5 µM Ca2+. (E) Representative steady-state inactivation currents elicited from different holding potentials through hNaV1.4F1705I+GA/SD channels in the presence and absence of CaM over expression. (F) There is a significant (p<0.004) hyperpolarizing shift of the inactivation curve by CaM over expression compared to the expression of hNaV1.4F1705I+GA/SD (in dotted line) alone. Over expression of CaM1234 has no significant effect compared with the absence of CaM over expression. (G) Representative steady-state inactivation currents elicited from different holding potentials through hNaV1.4GA/SD channels in the presence and absence of [Ca2+]i. (H) Steady-state inactivation of hNaV1.4GA/SD channel exhibited sensitivity to changes in [Ca2+]i similar to the wild type rat channel.
Figure 7
Figure 7. Ca2+ sensitivity, inactivation gating and calmodulation.
This schematic illustrates the relationship between the Ca2+ sensitivity of inactivation gating and the effect of CaM over expression. Channel variants that exhibit shifts in the voltage dependence of inactivation as a function of changes in intracellular [Ca2+] exhibit a depolarized V1/2 compared with variants insensitive to Ca2+. The Ca2+ sensitivity is associated with the direction of the gating shift induced by CaM over expression. Residues in the EFL are key determinants of the Ca2+ sensitivity and effect of CaM over expression on inactivation gating. The red curves in plots indicate steady-state inactivation in the presence of CaM.

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