The hydrophobic locus VAVIM is conserved in the S6 transmembrane segment of domain IV (IVS6) in Ca V 1 and Ca V 2 families. Herein we show that glycine substitution of the VAVIM motif in Ca V 2.3 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 (E 0.5,act ). In contrast, the slow kinetics of A1719G and V1720G were accompanied by a significant shift of ≈؊20 mV in their E 0.5,act indicating that the relative stability of the channel closed state was decreased in these mutants. Voltage-dependent Ca 2ϩ channels (VDCC) 3 are membrane proteins that play a key role in promoting Ca 2ϩ influx in response to membrane depolarization in excitable cells. VDCCs arise from the multimerization of distinct subunits: Ca V ␣1, Ca V ␤, and Ca V ␣2␦, and sometimes Ca V ␥ (1). To this date, molecular cloning has identified the primary structures for 10 distinct calcium channel Ca V ␣ 1 subunits (2-8) that are classified into three main subfamilies according to their gating properties (Ca V 1, Ca V 2, and Ca V 3). The Ca V ␣1 subunit is the main pore-forming subunit that carries the channel activation gating among other functions. The Ca V ␣1 subunits of VDCCs are evolutionarily related to the ␣ subunit of Kv channels with a single polypeptidic chain carrying four domains of six transmembrane segments (S1-S6) (9). Although the overall identity at the primary sequence level is very low between Ca V and Kv channels, it goes up to 10 -25% when comparing the S6 transmembrane segments. As in Kv channels, the S6 transmembrane segments of Ca V ␣1 are believed to line the channel pore and form the channel inner vestibule. It was inferred from the three-dimensional structures of KcsA, MthK, KvAP, KirBac, and Kv1.2 channels that the M2/S6 transmembrane segments include the activation gate that controls channel opening (10 -14). In the Shaker K ϩ channels, the residue hydrophobicity in this region could alter the channel closed-open equilibrium (15,16).To study the functional importance of S6 residues in the gating of Ca V 2.3, we searched for conserved motifs of hydrophobic residues. The VAVIM motif is conserved in the S6 transmembrane segment of domain IV (IVS6) of high voltageactivated Ca V 1 and Ca V 2 families (Fig. 1A). Numerous algorithms align the PVPVIV activation locus in Shaker Kv channels with the FVAVIM (50% identity) (Fig. 1B) suggesting that this locus could play a role in the activation gating of HVA VDCCs. Precious clues regarding the role of the VAVIM motif in channel function came from genetic diseases. Mutation of the conserved Ile to Leu in Ca V 2