Inactivation determinants in segment IIIS6 of Cav3.1

Low threshold, T-type, Ca2+ channels of the Ca-v(3) family display the fastest inactivation kinetics among all voltage-gated Ca2+ channels. The molecular inactivation determinants of this channel family are largely unknown. Here we investigate whether segment IIIS6 plays a role in Ca(V)3.1 inactivation as observed previously in high voltage-activated Ca2+ channels. 2. Amino acids that are identical in IIIS6 segments of all Ca2+ channel subtypes were mutated to alanine (F1505A, F1506A, N1509A, F1511A, V1512A, F1519A, FV1511/1512AA). Additionally M1510 was mutated to isoleucine and alanine, 3. The kinetic properties of the mutants were analysed with the two-microelectrode voltage-clamp technique after expression in Xenopus oocytes. The time constant for the barium current (I-Ba) inactivation, tau (inact), of wild-type channels at -20 mV was 9.5 +/- 0.4 ms; the corresponding time constants of the mutants ranged front 9.2 +/- 0.4 ins in V1512A to 45.7 +/- 5.2 ms (4.8-fold slowing) in M1510I Recovery at -80 mV was most significantly slowed by V1512A and accelerated by F1511A. 4. We conclude that amino acids M1510, F1511 and V1512 corresponding to previously identified inactivation determinants in IIIS6 of Ca(V)2.1 (Hering et at. 1998) have a significant role in Ca(V)3.1 inactivation. These data suggest common elements in the molecular architecture of the inactivation mechanism in high and low threshold Ca2+ channels.