The putative hinge point revealed by the crystal structure of the MthK potassium channel is a glycine residue that is conserved in many ion channels. In high voltage-activated (HVA) CaV channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in CaV1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly432 and Gly436 in CaV1.2) to form a triglycine motif unique to HVA CaV channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca2+ channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In CaV1.2, α-helix-breaking proline mutants (G422P and G432P) as well as the double G422A/G432A channel did not produce functional channels. The macroscopic inactivation kinetics were significantly decreased with CaV1.2 wild type > G770A > G422A ≅ G436A >> G432A (from the fastest to the slowest). Mutations at position Gly432 produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly436 to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX3G residues blunted Ca2+-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In CaV2.3, mutation of the distal glycine Gly352 impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX3G motif in the voltage-dependent activation and inactivation gating of HVA CaV channels with the distal glycine residue being mostly involved in the inactivation gating.
This work was supported by Canadian Institutes of Health Research Grant MOP13390 and a grant from the Canadian Heart and Stroke Foundation (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.