The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels

Abstract

Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with beta1 subunits prominent in brain, as well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly406. As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at approximately 50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism.

Fig. 1.

The TS (G406R) mutation slows VDI irrespective of the coexpressed β subunit. (A) Secondary structure of the Ca_V1.2 α_1C subunit. The approximate locations of mutations G406R and I1624A are indicated. (B) Exemplar normalized Ba²⁺ currents recorded from WT and G406R channels expressed with either β_2a (Left) or β_1c (Right) together with α₂δ. Currents were elicited by step depolarization to +10 mV from a holding potential of −90 mV. (C) Summary of fraction remaining at 3 s. *, P < 0.001 versus WT; #, P < 0.001 versus β_2a.

Fig. 2.

G406R slows current decay for both WT and I1624A channels regardless of charge carrier. (Left) Normalized whole-cell currents elicited in Ba²⁺ (A) or Ca²⁺ (B). Step depolarizations (400-ms) were applied to +10 mV from a holding potential of −90 mV. Shown are average traces ± SEM. (Right) Summary of fraction remaining after 300 ms. *, P < 0.001 versus WT. Note that G406R and G406R/I1624A currents did not differ significantly (N.S.) with either charge carrier.

Fig. 3.

CDI is spared by mutations that slow or accelerate VDI. (A) (Upper) Exemplar normalized Ba²⁺ and Ca²⁺ currents recorded at +10 mV from WT, G406R, and I1624A channels. (Lower) Ratio plots of normalized I_Ca/I_Ba currents. Shown are average plots ± SEM; the data were not significantly different as determined with either ANOVA or unpaired Student's t test. (B) Exemplar currents elicited using the voltage protocol indicated, with either Ba²⁺ or Ca²⁺ as the charge carrier. The pulses labeled I_g1 and I_g2 were to E_rev (see below). (C) Summary of inactivation and gating charge immobilization with either Ba²⁺ or Ca²⁺. For every cell recorded, E_rev was determined empirically; with Ba²⁺ or Ca²⁺ as the charge carrier, E_rev was 59.4 ± 1.4 mV and 81.9 ± 2.0 mV, respectively. *, P < 0.001 versus Ba²⁺. N.S., not significant.

Fig. 4.

The G406R mutation slows VDI and accelerates CDI. (A) Whole-cell Ba²⁺ or Ca²⁺ currents were elicited at +10 mV and normalized to the peak inward current. Shown are average currents ± SEM. (B) Normalized Ca²⁺ currents from two exemplar cells. The dashed line indicates zero current level, and the solid lines are fits to the product of two exponentials (see Materials and Methods). (C) Summary of the fitting parameters for WT (solid bars) and G406R (hatched) Ca²⁺ currents, as in B. *, P < 0.01 vs. WT. (D) Gating scheme showing transitions through the various proposed states. C, closed; O, open; I, inactivated. In Ba²⁺, channels predominantly display mode 1 gating followed by transitioning to the inactivated state (VDI). With Ca²⁺ as the charge carrier, open channels rapidly transition to mode Ca (39, 40), displaying reduced P_o (CDI).

Fig. 5.

Proposed model to account for our findings with VDI. For simplicity, only the α₁ and β subunits of the channel are shown, and only the I–II loop and C-terminal tail of the α₁ subunit are depicted; other regions have also been reported to contribute to VDI (–54). Approximate positions of Gly⁴⁰⁶ and Ile¹⁶²⁴ are shown by green and red circles, respectively. Shown below are idealized whole-cell Ba²⁺ currents. VDI of Ca_V1.2 L-type calcium channels is mediated by the concerted actions of several converging intrinsic and extrinsic interactions. (Top) WT channels expressed with the cardiac β_2a subunit exhibit slowed VDI because of the presence of a unique palmitoylation moiety on β_2a. (Middle) Replacing β_2a with the brain β₁ subunit speeds up VDI. (Bottom) The IQ motif appears to serve as a Ca²⁺-independent brake against VDI, possibly by association with the hinged lid of the I–II loop. The I1624A mutation may disrupt this interaction, thereby accelerating VDI. Under all conditions, the effect of the TS mutation (G406R, dashed box) is to slow VDI powerfully.