Isoform-Specific Modulation of Voltage-Gated Na + Channels by Calmodulin

International audienceCalmodulin (CaM) is a calcium-sensing protein that binds to Na(+) channels, with unknown functional consequences. Wild-type CaM produced a hyperpolarizing shift in the steady-state availability of expressed skeletal muscle (micro1) but not cardiac (hH1) Na(+) channels. Mutant CaM(1234) did not alter the voltage dependence or the kinetics of gating of either micro1 or hH1. Mutation of the highly conserved IQ motif in the carboxyl terminus of both isoforms (IQ/AA) slowed the kinetics of current decay and abolished the effect of wild-type CaM on micro1, but did not alter hH1 currents. The IQ/AA mutation eliminated CaM binding to the carboxyl terminus of both micro1 and hH1 channels. Inhibition of Ca(2+)/CaM kinase (CaM-K) slowed the current decay, the rate of entry into inactivation, and shifted the voltage dependence of hH1 in the depolarizing direction independent of CaM overexpression with no effect on micro1 Na(+) channels. CaM signaling modulates Na(+) currents in an isoform-specific manner, via direct interaction with skeletal muscle Na(+) channels and through CaM-K in the case of the cardiac isoform. The full text of this article is available at http://www.circresaha.org

Mechanisms of a Human Skeletal Myotonia Produced by Mutation in the C-Terminus of Na_V1.4: Is Ca²⁺ Regulation Defective?

<div><p>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 (hNa_V1.4_F1705I) has been linked to cold aggravated myotonia. The functional effects of altered regulation of hNa_V1.4_F1705I are incompletely understood. The location of the hNa_V1.4_F1705I in the CT prompted us to examine the role of Ca²⁺ 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 Na_V1.4 channel regulation by Ca²⁺ and CaM. hNa_V1.4_F1705I inactivation gating is Ca²⁺-sensitive compared to wild type hNa_V1.4 which is Ca²⁺ insensitive and the mutant channel exhibits a depolarizing shift of the V_1/2 of inactivation with CaM over expression. In contrast the same mutation in the rNa_V1.4 channel background (rNa_V1.4_F1698I) eliminates Ca²⁺ sensitivity of gating without affecting the CaM over expression induced hyperpolarizing shift in steady-state inactivation. The differences in the Ca²⁺ sensitivity of gating between wild type and mutant human and rat Na_V1.4 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 Ca²⁺/CaM regulation of the mutant channels that produce myotonia. The myotonia mutation F1705I slows I_Na decay in a Ca²⁺-sensitive fashion. The combination of the altered voltage dependence and kinetics of I_Na decay contribute to the myotonic phenotype and may involve the Ca²⁺-sensing apparatus in the CT of Na_V1.4.</p></div

Ca²⁺-sensitivity of human Na_V1.4_F1705I inactivation.

<p>(<b>A</b>) A schematic of the structured region of the C-terminus of hNa_V1.4 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 hNa_V1.4 (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>B</b>) Representative whole-cell currents through wild type and mutant hNa_V1.4_F1705I channels expressed in HEK293 cells in [Ca²⁺]_i free conditions. Na⁺ currents were elicited by the protocol in the inset. (<b>C</b>) [Ca²⁺]_i does not alter the I–V relationship of hNa_V1.4_F1705I. (<b>D</b>) Representative steady-state inactivation currents from different holding potentials through mutant hNa_V1.4_F1705I channels in the presence of 0.5 µM or absence of Ca²⁺. (<b>E</b>) Activation and steady-state inactivation curves of wild type and hNa_V1.4_F1705I channels in the absence and presence of 0.5 µM intracellular Ca²⁺. The V_1/2 of inactivation of hNa_V1.4_F1705I 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 V_1/2 of activation are unaffected by change in [Ca²⁺]_i. (<b>F</b>) and (<b>G</b>) illustrate the window currents through wild type and hNa_V1.4_F1705I in presence (<b>F</b>) and absence (<b>G</b>) 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.</p

Na current decay time constants of hNa_V1.4 and myotonia mutant hNa_V1.4_F1705I.

<p>Values: Mean±S.E(n);</p>#<p>vs. hNa_V1.4 in 0.5 µM Ca²⁺;</p>%<p>vs. hNa_V1.4_F1705I (0.5 µM Ca²⁺);</p>$<p>vs. hNa_V1.4_F1705I (0.5 µM Ca²⁺);</p><p><a href="mailto:@vs" target="_blank">@vs</a>. hNaV1.4 (37°C, 0.5 µM Ca2+);</p>*<p>vs. hNa_V1.4_F1705I (0.5 µM Ca²⁺).</p

Exchange of human and rat EFL residues in hNa_V1.4.

<p>(<b>A</b>) Amino acid sequence alignment of the proximal CT of rat and human wild type Na_V1.4 channel and hNa_V1.5 demonstrate the similarity of rNa_V1.4 and hNa_V1.5 at key positions in the EFL. In hNa_V1.4_F1705I+GA/SD residues G1613S and A1636D are substituted in the human channel hNa_V1.4_F1705I to match the corresponding residues of rNa_V1.4. (<b>B</b>) Representative families of hNa_V1.4_F1705I+GA/SD activation currents in the presence and absence of [Ca²⁺]_i. (<b>C</b>) Normalized I–V relationships hNa_V1.4_F1705I+GA/SD are not affected by altered [Ca²⁺]_i or CaM and CaM₁₂₃₄ over expression. (<b>D</b>) The steady-state inactivation of hNa_V1.4_F1705I+GA/SD channels are not sensitive to changes in [Ca²⁺]_i. The dotted lines in panel (<b>D</b>) represent hNa_V1.4 in 0.5 µM Ca²⁺. (<b>E</b>) Representative steady-state inactivation currents elicited from different holding potentials through hNa_V1.4_F1705I+GA/SD channels in the presence and absence of CaM over expression. (<b>F</b>) There is a significant (p<0.004) hyperpolarizing shift of the inactivation curve by CaM over expression compared to the expression of hNa_V1.4_F1705I+GA/SD (in dotted line) alone. Over expression of CaM₁₂₃₄ has no significant effect compared with the absence of CaM over expression. (<b>G</b>) Representative steady-state inactivation currents elicited from different holding potentials through hNa_V1.4_GA/SD channels in the presence and absence of [Ca²⁺]_i. (<b>H</b>) Steady-state inactivation of hNa_V1.4_GA/SD channel exhibited sensitivity to changes in [Ca²⁺]_i similar to the wild type rat channel.</p