Modulation of skeletal and cardiac voltage-gated sodium channels by calmodulin

Abstract

Calmodulin (CaM) has been shown to modulate different ion channels, including voltage-gated sodium channels (NaChs). Using the yeast two-hybrid assay, we found an interaction between CaM and the C-terminal domains of adult skeletal (NaV1.4) and cardiac (NaV1.5) muscle NaChs. Effects of CaM were studied using sodium channels transiently expressed in CHO cells. Wild type CaM (CaM(WT)) caused a hyperpolarizing shift in the voltage dependence of activation and inactivation for NaV1.4 and activation for NaV1.5. Intracellular application of CaM caused hyperpolarizing shifts equivalent to those seen with CaM(WT) coexpression with NaV1.4. Elevated Ca2+ and CaM-binding peptides caused depolarizing shifts in the inactivation curves seen with CaM(WT) coexpression with NaV1.4. KN93, a CaM-kinase II inhibitor, had no effect on NaV1.4, suggesting that CaM acts directly on NaV1.4 and not through activation of CaM-kinase II. Coexpression of hemi-mutant CaMs showed that an intact N-terminal lobe of CaM is required for effects of CaM upon NaV1.4. Mutations in the sodium channel IQ domain disrupted the effects of CaM on NaV1.4: the I1727E mutation completely blocked all calmodulin effects, while the L1736R mutation disrupted the effects of Ca2+-calmodulin on inactivation. Chimeric channels of NaV1.4 and NaV1.5 also indicated that the C-terminal domain is largely responsible for CaM effects on inactivation. CaM had little effect on NaV1.4 expressed in HEK cells, possibly due to large differences in the endogenous expression of beta-subunits between CHO and HEK cells. These results in heterologous cells suggest that Ca2+ released during muscle contraction rapidly modulates NaCh availability via CaM.

Figure 1. Effect of CaM and modulators of CaM on Na⁺ currents in CHO cells transiently transfected with Na_V1.4

A and B, increased Ca²⁺ and CIP had no effect on currents. A, normalized peak current–voltage (I–V) relationships, elicited by 30 ms voltage-clamp steps from –90 to +100 mV in 10 mV increments (mean ± s.e.m.). Na_V1.4 whole-cell currents (▪, n = 21) were unchanged with increased Ca²⁺ (▵, n = 13) or CIP (⋄, n = 6) included in the pipette. Inset shows one example of current traces used to generate the I–V curve. B, voltage dependence of steady-state activation and inactivation. Steady-state inactivation was measured by a two-pulse protocol with 100 ms conditioning pulses from −150 to +20 mV followed by a 30 ms test pulse to −10 mV. The data were fitted using a Boltzmann distribution. There was no shift in either activation or inactivation with 10 μm Ca²⁺ (▵) or 75 pm CIP (⋄). C and D, inclusion of CaM in the recording pipette caused hyperpolarizing shifts in the current. C, normalized I–V relationships for Na_V1.4 alone (▪, n = 21) and with 10 μm CaM (◃, n = 11), 30 μm CaM (▹, n = 4), or 50 μm CaM (⋄, n = 6) included in the pipette (mean ± s.e.m.). D, activation and steady-state inactivation shifted with CaM included in the pipette. Na_V1.4 alone (▪); +10 μm CaM (◃); +30 μm CaM (▹); +50 μm CaM (⋄). E and F, shifts in the V_1/2 of activation (E) and steady-state inactivation (F) of Na_V1.4 relative to the values for Na_V1.4 alone from Fig. 1B and D.

Figure 2. Coexpression of Na_V1.4 with CaM_WT or CaM₁₂₃₄

A, normalized representative sodium currents through Na_V1.4 in the absence (continuous line) and presence of coexpressed CaM_WT (dashed line) or Ca²⁺-binding deficient mutant CaM₁₂₃₄ (dotted line). Currents were elicited by a 30 ms step to −10 mV from a holding potential of −120 mV. Time constants for inactivation (control = 0.67 ± 0.02 ms; + CaM_WT= 0.59 ± 0.03 ms; + CaM₁₂₃₄= 0.61 ± 0.04 ms) were not significantly different. CaM_WT is designated by WT in all panels. B, normalized I–V relationships for Na_V1.4 (▪, n = 21) coexpressed with CaM_WT (□, n = 13) or CaM₁₂₃₄ (▵, n = 8)_, as described in Fig. 1A. C, the voltage dependence of both activation and inactivation for Na_V1.4 shifted significantly with coexpression of CaM_WT (−14.9 mV and −11.3 mV, respectively). CaM₁₂₃₄ caused a significant shift in activation (−11.1 mV), but had no significant effect on inactivation. Na_V1.4 alone (▪); + CaM_WT (□); + CaM₁₂₃₄ (▵). D, modulators of CaM (10 μm Ca²⁺, 25 μm CBP or 75 pm CIP) caused a depolarizing shift in V_1/2 for steady-state inactivation. + CaM_WT+10 μm Ca²⁺ (▵, n = 16); + CaM_WT+ 25 μm CBP (◃, n = 14); + CaM_WT+ 75 pm CIP (⋄, n = 9). E, shifts in the V_1/2 of activation relative to the values for Na_V1.4 coexpressed with CaM_WT (filled bars) or CaM₁₂₃₄ (open bars). Ca²⁺, CBP and CIP included in the pipette were not significantly different from CaM_WT alone. Likewise, the addition of these modulators of CaM to cells expressing CaM₁₂₃₄ did not produce any changes in V_1/2 of activation. CIP_c (75 pm; a non-functional peptide analogue of CIP) and 10 μm KN93 (a blocker of CaM activation of CKII) had no effect. F, shifts in the V_1/2 of steady-state inactivation relative to the values for Na_V1.4 coexpressed with CaM_WT (filled bars) or CaM₁₂₃₄ (open bars)_. Ca²⁺, CBP and CIP caused significant shifts from CaM_WT alone (*). 75 pm CIP_c and 10 μm KN93 had no effect. CaM modulators had no effect on the voltage dependence of inactivation of currents with CaM₁₂₃₄ coexpression.

Figure 3. Coexpression of Na_V1.4 with mutant CaMs lacking Ca⁺ binding in either the N-terminal (CaM₁₂) or C-terminal (CaM₃₄) lobes

A, currents from cells with coexpression of CaM₁₂ with Na_V1.4 were unchanged from Na_V1.4 expressed alone. CaM_WT coexpression is shown for comparison: + CaM_WT (▪, n = 13); + CaM₁₂ (▵, n = 10). Addition of Ca²⁺ (10 μm, ▵, n = 8) or CIP (75 pm, ⋄, n = 9) was not different from CaM₁₂. Cartoons depict mutations in the Ca²⁺-binding sites of CaM (N-terminal for panel A and C-terminal for panel B). B, coexpression of CaM₃₄ caused a hyperpolarizing shift in the voltage dependence of both activation and inactivation, similar to CaM_WT. + CaM_WT (▪); + CaM₃₄ (▵, n = 9); + CaM₃₄+ 10 μm Ca²⁺ (▵, n = 10); + CaM₃₄+ 75 pm CIP (⋄, n = 8). C, shifts in the V_1/2 of activation and inactivation of Na_V1.4 coexpressed with CaM₁₂ or CaM₃₄ relative to coexpression with CaM_WT. Coexpression of CaM₁₂ (filled bars) was significantly different from CaM_WT, and not significantly different from Na_V1.4 expressed alone. The shifts in both activation and inactivation with CaM₃₄ (open bars) were not significantly different compared with CaM_WT. D, Ca²⁺ or CIP induced shifts in the V_1/2 of activation relative to coexpression with either CaM₁₂ (filled bars) or CaM₃₄ (open bars). Ca²⁺ and CIP had no effect on currents with CaM₁₂. The depolarizing shifts with 10 μm Ca²⁺ and 75 pm CIP were significant compared with CaM₃₄ alone. E, Ca²⁺ or CIP induced shifts in the V_1/2 of steady-state inactivation relative to coexpression with either CaM₁₂ or CaM₃₄. Ca²⁺ and CIP had no effect on currents with CaM₁₂. The depolarizing shifts seen with Ca²⁺ and CIP were significant compared with CaM₃₄ alone.

Figure 4. Coexpression of Na_V1.4 IQ-domain mutants with CaM_WT

A, normalized I–V relationships (mean ± s.e.m.). I1727E whole-cell currents (▪, n = 5) were unchanged by coexpression with CaM_WT (○, n = 9). Whole-cell currents from I1727E were not significantly different from native Na_V1.4 (data not shown). CaM_WT is designated WT in all panels. B, the I1727E mutation blocked all shifts in V_1/2 of activation or inactivation seen with coexpression of CaM_WT. Curves for I1727E were not significantly different compared with native Na_V1.4 (data not shown). I1727E (▪); I1727E + CaM_WT (○); I1727E + CaM_WT+ 10 μm Ca²⁺ (▵, n = 8); I1727E + CaM_WT+ 75 pm CIP (⋄, n = 9). C, normalized I–V relationships for L1736R (mean ± s.e.m.). L1736R whole-cell currents (▪, n = 5) were shifted by coexpression with CaM_WT (○, n = 10). Whole-cell currents from L1736R were not significantly different from native Na_V1.4 (data not shown). D, the L1736R mutated channel retained its responsiveness to CaM_WT coexpression but activation and inactivation were no longer affected by Ca²⁺ and CIP with CaM_WT coexpression. The V_1/2 of activation and inactivation for L1736R was not significantly different compared with native Na_V1.4 (data not shown). L1736R (▪); L1736R + CaM_WT (○); L1736R + CaM_WT+ 10 μm Ca²⁺ (▵, n = 8); L1736R + CaM_WT+ 75 pm CIP (⋄, n = 9). E, shifts in the V_1/2 of activation for I1727E (filled bars) and L1736R (open bars) coexpressed with CaM_WT, relative to Na_V1.4. Data for coexpression of native Na_V1.4 coexpressed with CaM_WT (cross-hatched bar) were replotted from Fig. 2 for comparison. The I1727E mutation abolished the effects of CaM_WT coexpression (ϕ). For the L1736R mutation, V_1/2 of activation with CaM_WT was significantly shifted compared with L1736R alone, but not different from native Na_V1.4 coexpressed with CaM_WT. Ca²⁺ and CIP caused no significant shift compared with L1736R + CaM_WT. F, shifts in the V_1/2 of steady-state inactivation for I1727E and L1736R coexpressed with CaM_WT, relative to Na_V1.4. Data for coexpression of native Na_V1.4 coexpressed with CaM_WT were replotted from Fig. 2 for comparison. The I1727E mutation abolished the effects of CaM_WT coexpression (ϕ; P < 0.05). For the L1736R mutation, activation with CaM_WT coexpression was significantly shifted compared with L1736R alone. Addition of Ca²⁺ and CIP caused no significant shift compared with L1736R + CaM_WT.

Figure 5. Coexpression of Na_V1.5 with CaM_WT or CaM₁₂₃₄

A, normalized I–V relationships for Na_V1.5 (▪, n = 19) coexpressed with CaM_WT (○, n = 20) or CaM₁₂₃₄ (▵, n = 11) (mean ± s.e.m.). Inset shows one example of current traces used to generate the I–V curve. CaM_WT is designated WT in all panels. B, coexpression of CaM_WT with Na_V1.5 caused a significant hyperpolarizing shift in the voltage dependence of activation, but not inactivation. CaM₁₂₃₄ had no significant effect on currents. Na_V1.5 (▪); + CaM_WT (○); + CaM₁₂₃₄ (▵). C, modulators of CaM (10 μm Ca²⁺, 25 μm CBP or 75 pm CIP) had no significant effect on activation or steady-state inactivation of Na_V1.5 compared with CaM_WT coexpression. + CaM_WT+ 10 μm Ca²⁺ (▵, n = 17); + CaM_WT+ 25 μm CBP (◃, n = 9); + CaM_WT+ 75 pm CIP (⋄, n = 15). D, blocking CKII with KN93 caused a shift in activation, but had no effect on inactivation of Na_V1.5: + 10 μm KN93 (▵, n = 16); + 10 μm KN92 (*, n = 5). KN92 was unchanged from CaM_WT alone. E, shifts in the V_1/2 of activation for Na_V1.5 relative to CaM_WT coexpression. Coexpression with CaM_WT caused a significant hyperpolarizing shift. Ca²⁺, CBP and CIP had no significant effect compared with CaM_WT alone. 10 μm KN93 caused a further hyperpolarizing shift compared with CaM_WT (σ). F, shifts in the V_1/2 of steady-state inactivation for Na_V1.5 relative to CaM_WT coexpression. Coexpression with CaM_WT had no significant effect on Na_V1.5. CaM modulators had no significant effect compared with CaM_WT alone. KN93 (10 μm) caused a hyperpolarizing shift compared with CaM_WT coexpression alone (Θ).

Figure 6. Coexpression of C-terminal chimeric sodium channels with CaM_WT

A, coexpression of Na_V1.4/CT_1.5 with CaM_WT caused a hyperpolarizing shift in the voltage dependence of activation and inactivation. V_1/2 of inactivation was not shifted by 10 μm Ca²⁺ or 75 pm CIP. Activation was shifted by these CaM modulators. Chimera (▪, n = 6); chimera + CaM_WT (○, n = 16); chimera + CaM_WT+ 10 μm Ca²⁺ (▵, n = 8); chimera + CaM_WT+ 75 pm CIP (⋄, n = 10). Inset shows cartoon of chimeric channels, with portions contributed by Na_V1.4 depicted by thin lines and by Na_V1.5 by thick lines. CaM_WT is designated WT in all panels. B, coexpression of Na_V1.5/CT_1.4 with CaM_WT caused a hyperpolarizing shift in the voltage dependence of activation and steady-state inactivation. V_1/2 of activation was not shifted by 10 μm Ca²⁺ or 75 pm CIP. Steady-state inactivation was shifted by these CaM modulators. Chimera (▪, n = 21); chimera + CaM_WT (○, n = 19); chimera + CaM_WT+ 10 μm Ca²⁺ (▵, n = 14); chimera + CaM_WT+ 75 pm CIP (⋄, n = 11). C, shifts in the V_1/2 of activation of chimeric channels compared with native Na_V1.4 + CaM_WT (filled bars) and Na_V1.5 + CaM_WT (cross-hatched bars). The depolarizing shifts seen with the addition of Ca²⁺ and CIP were significantly different for Na_V1.4/CT_1.5+ CaM_WT (open bars), similar to the activation properties of Na_V1.4 (filled bars). Ca²⁺ or CIP had no effect on Na_V1.5/CT_1.4. Chimeric channels behaved like the parent channel. Lettering at the top of the panel indicates the chimeric constructs. D, shifts in the V_1/2 of steady-state inactivation of chimeric channels compared with native Na_V1.4 (filled bars) and Na_V1.5 (cross-hatched bars). Ca²⁺ and CIP had no effect on inactivation in Na_V1.4/CT_1.5. Ca²⁺ and CIP produced significant depolarizing shifts of V_1/2 compared with Na_V1.5/CT_1.4+ CaM_WT (striped bars), similar to the effects of Ca²⁺ and CIP on the inactivation properties of native Na_V1.4 (filled bars). Chimeric channels behaved like the channel contributing the carboxyl tail. Lettering at the top of the panel indicates the chimeric constructs.

Figure 7. CaM effects and β-subunit mRNA expression in HEK and CHO cells

A, coexpression of CaM with Nav1.4 did not shift the voltage dependence of activation or inactivation in HEK cells. Nav1.4 (▪, n = 12); Nav1.4 + CaM (○, n = 7). B and C, relative abundance of mRNA of each of the four NaCh β-subunits, expressed as the number of copies relative to 1000 copies of HPRT (an internal control) in HEK and CHO cells, respectively. Note the logarithmic scale.

Figure 8. Voltage dependence of steady-state activation and inactivation in C2C12 cells

There was a shift in steady-state inactivation with 75 pm CIP (⋄, n = 9, V_1/2=−75.8 ± 1.1 mV) compared with control (▪, n = 10, V_1/2=−83.2 ± 1.2 mV) (P = 0.009). CIP had no effect on activation (control: ▪, n = 11, V_a=−41.8 ± 1.0 mV; + 75 pm CIP: ⋄, n = 9, V_a=−40.8 ± 1.2 mV).

Figure 9. Model of CaM interactions with the IQ domain

CaM is in an extended conformation with the C lobe bound to the proximal IQ region under low Ca²⁺ conditions (apoCaM). The sequence of the sodium channel IQ domain is similar to that of myosin IQ domains that crystallize with myosin light chain (a protein similar to CaM) in an extended conformation (Terrak et al. 2003). The N lobe interacts with another region of the carboxyl-terminal (or alternatively, other domains of the sodium channel or its subunits) termed the NLBD (N-lobe binding domain). When Ca²⁺ is elevated or CaM-binding peptides are added, the N lobe of CaM is released from the NLBD and, in the case of increased Ca²⁺, binds to the distal IQ domain (since mutation of the second leucine of RYLL abolished the Ca²⁺ effects).