Ca(V)1.2 I-II linker structure and Timothy syndrome

Abstract

Ca(V) channels are multi-subunit protein complexes that enable inward cellular Ca(2+) currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker's helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca(V)1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant's (human Ca(V)1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.

Figure 1. The TS mutation. (A) Representative Ba²⁺ currents of Ca_V1.2 mutants G436R and G436R/G449R. Currents were recorded during a 0 mV depolarizing step from a holding potential of -80 mV. Traces are presented normalized to their peak value. Dotted traces denote the Ca_V1.2 WT current for comparison. (B) Normalized -I_Ca/I_Ba of averaged currents at +20 mV depolarization. Average values and SEM are plotted. Data were averaged from the following numbers of cells measured in Ba²⁺/Ca²⁺: Ca_V1.2 WT: 11/10, G449R: 14/9, G436R: 5/5, G436R/G449R: 8/6 cells.Ca_V1.2 G449R: p = 0.005 vs. WT, p < 0.001 vs. others; Ca_V1.2 G436R p < 0.001 vs. WT, p < 0.001 vs. G449R; Ca_V1.2 G436R/G449R: p < 0.001 vs. WT, p < 0.001 vs. G449R. (Statistical analysis based on comparing -I_Ca/I_Ba values at 400 ms from depolarization, using a One-way Anova) . (C–D) Voltage-dependence of Ca_V1.2 Ba²⁺ current inactivation kinetics with (C) or without (D) Ca_Vβ2b. Similar to G449R, G436R (n = 5 with and without Ca_Vβ2b) decelerates inactivation kinetics. Note the mutant's shallow voltage dependence. In the measurement range tested, G449R is masked by the G436R background effect of the double mutant (n = 8 with Ca_Vβ2b and n = 9 without). Statistics are presented in Table 1. The symbol legend for panels C and D are found in panelE. (E) CD measurements of α-helical content for Ca_V1.2 Helix-PL-AID proteins with increasing TFE. G436R does not show a marked change from Ca_V1.2 WT while G449R increases the PL's α-helix content. All measurements were repeated at least twice.

Figure 2. The effect of PL conformation on the voltage-dependence of activation. (A) Ca_V1.2 G436R (n = 5/5); (B) Ca_V2.2 G357R (n = 10/10) conductance vs. voltage curves compared with their respective wild types in the presence or absence of Ca_Vβ2b.