RESUMO
Voltage-dependent Ca(2+) channels are heteromultimers of Ca(V)α(1) (pore), Ca(V)ß- and Ca(V)α(2)δ-subunits. The stoichiometry of this complex, and whether it is dynamically regulated in intact cells, remains controversial. Fortunately, Ca(V)ß-isoforms affect gating differentially, and we chose two extremes (Ca(V)ß(1a) and Ca(V)ß(2b)) regarding single-channel open probability to address this question. HEK293α(1C) cells expressing the Ca(V)1.2 subunit were transiently transfected with Ca(V)α(2)δ1 alone or with Ca(V)ß(1a), Ca(V)ß(2b), or (2:1 or 1:1 plasmid ratio) combinations. Both Ca(V)ß-subunits increased whole-cell current and shifted the voltage dependence of activation and inactivation to hyperpolarization. Time-dependent inactivation was accelerated by Ca(V)ß(1a)-subunits but not by Ca(V)ß(2b)-subunits. Mixtures induced intermediate phenotypes. Single channels sometimes switched between periods of low and high open probability. To validate such slow gating behavior, data were segmented in clusters of statistically similar open probability. With Ca(V)ß(1a)-subunits alone, channels mostly stayed in clusters (or regimes of alike clusters) of low open probability. Increasing Ca(V)ß(2b)-subunits (co-)expressed (1:2, 1:1 ratio or alone) progressively enhanced the frequency and total duration of high open probability clusters and regimes. Our analysis was validated by the inactivation behavior of segmented ensemble averages. Hence, a phenotype consistent with mutually exclusive and dynamically competing binding of different Ca(V)ß-subunits is demonstrated in intact cells.