The Role of Distal S6 Hydrophobic Residues in the Voltage-dependent Gating of Ca sub(V)2.3 Channels

The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca sub(V)1 and Ca sub(V)2 families. Herein we show that glycine substitution of the VAVIM motif in Ca sub(V)2.3 produced whole cell currents with inactivation kinetics that were either slower (A1719G approximately V1720G), similar (V1718G), or faster (I1721G approximately M1722G) than the wild-type channel. The fast kinetics of I1721G were observed with a approximately +10 mV shift in its voltage dependence of activation (E sub(0.5,act)). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of approximately -20 mV in their E sub(0.5,act) indicating that the relative stability of the channel closed state was decreased in these mutants. Glycine scan performed with Val super(349) in IS6, Ile super(701) in IIS6, and Leu super(1420) in IIIS6 at positions predicted to face Val super(1720) 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 Ca sub(V)2.3 based upon Kv1.2 where hydrophobic residues at positions facing Val super(1720) in IS6, IIS6, and IIIS6 play a critical role in stabilizing the closed state in Ca sub(V)2.3.