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

Abstract

Calcium (Ca(2+))-dependent endocytosis has been linked to preferential Ca(2+) entry through the L-type (α(1D), Ca(V)1.3) of voltage-dependent Ca(2+) channels (VDCCs). Considering that the Ca(2+)-dependent exocytotic release of neurotransmitters is mostly triggered by Ca(2+) entry through N-(α(1B), Ca(V)2.2) or PQ-VDCCs (α(1A), Ca(V)2.1) 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-endocytotic 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 clathrin 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.

Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms

“…These results suggest that calcium influx via VDCCs, including P/Q-type VDCCs, at the plasma membrane triggers endocytosis (49, 129). Consistent with this suggestion, blocking P/Q- and L-type calcium channels inhibits endocytosis in chromaffin cells (144). Given that calcium influx may trigger all forms of endocytosis at secretory cells in which calcium influx through VDCCs triggers exocytosis, we suggest that VDCCs at the plasma membrane trigger endocytosis at neuronal nerve terminals and in nonneuronal secretory cells.…”

Hormone-stimulated modulation of endocytic trafficking in osteoclasts

“…It is thus more likely that it is the thapsigargin-induced calcium release that is relieving the calcitonin-induced block in endocytic trafficking. In neurons and chromaffin cells, a rise in intracellular calcium triggers exocytosis, however, other trafficking steps, such as endocytosis also depend on intracellular calcium (Hosoi et al, 2009; Rosa et al, 2011). These findings are in line with our observations that clamping intracellular calcium with the cell-permeable calcium chelator BAPTA-AM reduced the number of endocytosis-active osteoclasts and did not reverse the effect of calcitonin.…”

Cytosolic organelles shape calcium signals and exo–endocytotic responses of chromaffin cells

“…It is interesting that Ca 2+ -dependent endocytosis triggered by single long depolarising pulses in voltage-clamped bovine chromaffin cells seems to be coupled to L-type VDCCs, whereas N-or PQ-type of calcium channels seem to play little role [210,211]. Lack of co-localisation between VDCC subtypes and clathrin or dynamin suggests a functional, rather than physical coupling between L-type calcium channels and the endocytotic machinery.…”

“…In bovine chromaffin cells, L-type calcium channels undergo a Ca 2+ -dependent inactivation slower than N-or PQ-type of calcium channels [136,212]. It is therefore plausible that a slower but more sustained Ca 2+ entry through slowly inactivating L-type calcium channels, rather than through higher but fast-inactivating Nand PQ-type of calcium channels, is a requirement to trigger endocytosis efficiently, at least in bovine chromaffin cells [211]. This Ca 2+ -dependent endocytotic response is enhanced by sphingosine dialysis, that seems to play a permissive role for endocytosis by acting on an endocytotic pathway different to those of dynamin-and calmodulin-signalling pathways [209].…”