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The 1B Ca²⁺ channel amino terminus contributes determinants for subunit-mediated voltage-dependent inactivation properties

Gary J. Stephens, Karen M. Page, Yuri Bogdanov and Annette C. Dolphin

1. Co-expression of auxiliary subunits with the 1B Ca²⁺ channel subunit in COS-7 cells resulted in an increase in current density and a hyperpolarising shift in the mid-point of activation. Amongst the subunits, 2a in particular, but also 4 and 1b caused a significant retardation of the voltage-dependent inactivation compared to currents with 1B alone, whilst no significant changes in inactivation properties were seen for the 3 subunit in this system.

2. Prevention of 2a palmitoylation, by introducing cysteine to serine mutations (2a(C3,4S)), greatly reduced the ability of 2a to retard voltage-dependent inactivation.

3. Deletion of the proximal half of the 1B cytoplasmic amino terminus (1B_1-55) differentially affected subunit-mediated voltage-dependent inactivation properties. These effects were prominent with the 2a subunit and, to a lesser extent, with 1b. For 2a, the major effects of this deletion were a partial reversal of 2a-mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full-length 1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of 1B when co-expressed with subunits.

4. Transfer of the whole 1B amino terminus into 1C (1bCCCC) conferred a similar retardation of inactivation on 1C when co-expressed with 2a to that seen in parental 1B.

5. Individual (1B(Q47A) and 1B(R52A)) and double (1B(R52,54A)) point mutations within the amino terminus of 1B also opposed the 2a-mediated retardation of 1B inactivation kinetics.

6. These results indicate that the 1B amino terminus contains determinants for subunit-mediated voltage-dependent inactivation properties. Furthermore, effects were subunit selective. As deletion of the 1B amino terminus only partially opposed subunit-mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete subunit function.