Calcium entry through slow-inactivating L-type calcium channels preferentially triggers endocytosis rather than exocytosis in bovine chromaffin cells

摘要

Calcium (Ca~(2+))-dependent endocytosis has been linked to preferential Ca~(2+) entry through the L-type (α_(1D), Ca_v1.3) of voltage-dependent Ca~(2+) channels (VDCCs). Considering that the Ca~(2+)-dependent exocytotic release of neurotransmit-ters is mostly triggered by Ca~(2+) entry through N-(α_(1B), Ca_v2.2) or PQ-VDCCs (α_(1A), Ca_v2.1) and that exocytosis and endocytosis are coupled, the supposition that the different channel subtypes are specialized to control different cell functions is attractive. Here we have explored this hypothesis in primary cultures of bovine adrenal chromaffin cells where PQ channels account for 50% of Ca~(2+) current (I_(Ca)), 30% for N channels, and 20% for L channels. We used patch-clamp and fluorescence techniques to measure the exo-endocy-totic responses triggered by long depolarizing stimuli, in 1,2, or 10 mM concentrations of extracellular Ca~(2+) ([Ca~(2+)]_e). Exo-endocytotic responses were little affected by ω-conotoxin GVIA (N channel blocker), whereas ω-agatoxin IVA (PQ channel blocker) caused 80% blockade of exocytosis as well as endocytosis. In contrast, nifedipine (L channel blocker) only caused 20% inhibition of exocytosis but as much as 90% inhibition of endocytosis. Conversely, FPL67146 (an activator of L VDCCs) notably augmented endocytosis. Photoreleased caged Ca~(2+) caused substantially smaller endocytotic responses compared with those produced by K+ depolarization. Using fluorescence antibodies, no colocalization between L, N, or PQ channels with clafhrin was found; a 20-30% colocalization was found between dynamin and all three channel antibodies. This is incompatible with the view that L channels are coupled to the endocytotic machine. Data rather support a mechanism implying the different inactivation rates of L (slow-inactivating) and N/PQ channels (fast-inactivating). Thus a slow but more sustained Ca~(2+) entry through L channels could be a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells.