Negatively charged residues in the N-terminal of the AID helix confer slow voltage dependent inactivation gating to CaV1.2

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type Ca(V)1.2 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-Vbeta regions in L-type Ca(V)1.1 and Ca(V)1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit Ca(V)1.2 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(V)1.2. 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 E462Rmuch greater thanE462Kapproximate toE462A>E462N>wild-typeapproximate toE462Qapproximate toE462G>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(V)1.2 into a Ca(V)2.3 background. The fast VDI kinetics of the Ca-V1.2 E462R and the CEEE+E462R mutants were abolished by the Ca(Vbeta)2a subunit and reinstated when using the non-palmitoylated form of Ca(Vbeta)2a C3S+C4S (Ca(Vbeta)2a CS), confirming that Ca(Vbeta)2a 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(V)1.2 channels.