Negatively Charged Residues in the N-terminal of the AID Helix Confer Slow Voltage Dependent Inactivation Gating to Ca V1.2

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The E462R mutation in the fifth position of the AID ( α1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type Ca V1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-Ca V β regions in L-type Ca V1.1 and Ca V1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit Ca V1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of Ca V1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R ≫ E462K ≈ E462A > E462N > wild-type ≈ E462Q ≈ E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from Ca V1.2 into a Ca V2.3 background. The fast VDI kinetics of the Ca V1.2 E462R and the CEEE + E462R mutants were abolished by the Ca V β2a subunit and reinstated when using the nonpalmitoylated form of Ca V β2a C3S + C4S (Ca V β2a CS), confirming that Ca V β2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated Ca V1.2 channels.