Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel

Ba2+ current through the L-type Ca2+ channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the ale subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba2+ current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed similar to45% of the sustained Ba2+ or Ca2+ current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (tau(f) similar to 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca2+-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca2+ sensors of inactivation.