The Role of the GX sub(9)GX sub(3)G Motif in the Gating of High Voltage-activated Ca super(2+) Channels

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) Ca sub(V) channels, the mid-S6 glycine residue is only present in IS6 and IIS6, corresponding to G422 and G770 in Ca sub(V)1.2. Two additional glycine residues are found in the distal portion of IS6 (Gly super(432) and Gly super(436) in Ca sub(V)1.2) to form a triglycine motif unique to HVA Ca sub(V) channels. Lethal arrhythmias are associated with mutations of glycine residues in the human L-type Ca super(2+) channel. Hence, we undertook a mutational analysis to investigate the role of S6 glycine residues in channel gating. In Ca sub(V)1.2, alpha -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 Ca sub(V)1.2 wild type > G770A > G422A approximately G436A >> G432A (from the fastest to the slowest). Mutations at position Gly super(432) produced mostly nonfunctional mutants. Macroscopic inactivation kinetics were markedly reduced by mutations of Gly super(436) to Ala, Pro, Tyr, Glu, Arg, His, Lys, or Asp residues with stronger effects obtained with charged and polar residues. Mutations within the distal GX sub(3)G residues blunted Ca super(2+)-dependent inactivation kinetics and prevented the increased voltage-dependent inactivation kinetics brought by positively charged residues in the I-II linker. In Ca sub(V)2.3, mutation of the distal glycine Gly super(352) impacted significantly on the inactivation gating. Altogether, these data highlight the role of the GX sub(3)G motif in the voltage-dependent activation and inactivation gating of HVA Ca sub(V) channels with the distal glycine residue being mostly involved in the inactivation gating.