The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of CaV2.3 Channels

The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in CaV1 and CaV2 families. Herein we show that glycine substitution of the VAVIM motif in CaV2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G ≈ V1720G), similar (V1718G), or faster (I1721G ≈ M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a ≈+10 mV shift in its voltage dependence of activation (E0.5,act). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈-20 mV in their E0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val 349 in IS6, Ile701 in IIS6, and Leu1420 in IIIS6 at positions predicted to face Val1720 in IVS6 also produced slow inactivating currents with hyperpolarizing shifts in the activation and inactivation potentials, again pointing out a decrease in the stability of the channel closed state. Mutations to other hydrophobic residues at these positions nearly restored the channel gating. Altogether these data indicate that residues at positions equivalent to 1720 exert a critical control upon the relative stability of the channel closed and open states and more specifically, that hydrophobic residues at these positions promote the channel closed state. We discuss a three-dimensional homology model of CaV2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val1720 in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in CaV2.3.