The role of dynamic palmitoylation in Ca²⁺ channel inactivation

Abstract

N- and P/Q-type Ca²⁺ channels regulate a number of critical physiological processes including synaptic transmission and hormone secretion. These Ca²⁺ channels are multisubunit proteins, consisting of a pore-forming α₁, and accessory β and α₂δ subunits each encoded by multiple genes and splice variants. β subunits alter current amplitude and kinetics. The β_2a subunit is associated with slowed inactivation, an effect that requires the palmitoylation of two N-terminal cysteine residues in β_2a. In the current manuscript, we studied steady state inactivation properties of native N- and P/Q-type Ca²⁺ channels and recombinant N-type Ca²⁺ channels. When bovine α_1B and β_2a and human α₂δ were coexpressed in tsA 201 cells, we observed significant variations in inactivation; some cells exhibited virtually no inactivation as the holding potential was altered whereas others exhibited significant inactivation. A similar variability in inactivation was observed in native channels from bovine chromaffin cells. In individual chromaffin cells, the amount of inactivation exhibited by N-type channels was correlated with the inactivation of P/Q-type channels, suggesting a shared mechanism. Our results with recombinant channels with known β subunit composition indicated that inactivation could be dynamically regulated, possibly by alterations in β subunit palmitoylation. Tunicamycin, which inhibits palmitoylation, increased steady-state inactivation of Ca²⁺ channels in chromaffin cells. Cerulenin, another drug that inhibits palmitoylation, also increased inactivation. Tunicamycin produced a similar effect on recombinant N-type Ca²⁺ channels containing β_2a but not β_2b or β_2a subunits mutated to be palmitoylation deficient. Our results suggest that Ca²⁺ channels containing β_2a subunits may be regulated by dynamic palmitoylation.