Calmodulin regulation of calcium channels and neurotransmitter release in bovine adrenal chromaffin cells

Calmodulin is a molecule implicated in regulating voltage-gated calcium channels (VGCCs) and the exocytotic machinery to fine tune neurotransmitter release. I have investigated the role this molecule plays in stimulus-secretion coupling in bovine adrenal chromaffin cells by over-expressing either wild-type (CaMwt), or a mutated calmodulin (CaM1234), rendered incapable of binding calcium by adenoviral infection. Stimulus-evoked secretion was monitored by combined measurements of membrane capacitance (DeltaC m) and voltage-clamp recording of calcium currents in single cells. Cells were clamped in either the perforated patch or whole-cell configuration and calcium-dependent exocytosis evoked by single depolarising voltage steps or trains of depolarisations. I show that the exocytotic efficiency, derived by dividing DeltaCm by the intergral of the calcium current, is reduced for N-type channels compared to P/Q-type channels. Cation substitution experiments revealed that pharmacologically isolated N-type channels displayed the most profound sensitivity to calcium-dependent inactivation. Studies aimed at eluding the molecular mechanisms underlying calcium-dependent inactivation show that inhibiting calcinuerin by 20 mins preincubation with 1muM cyclosporine A or by introducing 30muM calmodulin inhibitory peptides through the patch pipette did not significantly reduce the level of whole cell calcium-dependent inactivation. In contrast, adenoviral mediated expression of a mutant calmodulin deficient in calcium binding resulted in a highly significant reduction in N-type, but not P/Q-type channel inactivation. This is the first time that calmodulin has been shown to regulate endogenously expressed N-type calcium channels. These results are consistent with calmodulin acting directly to control N-type channel inactivation and therefore limit this channel's ability to couple to exocytosis during prolonged stimulation. Ca2+/calmodulin was also found to interact with the secretory machinery. Expression of CaM 1234 significantly reduced the exocytotic efficiency of brief depolarisations (≤ 100ms), however, the exocytotic efficiency to longer depolarisations (≥ 200ms) was not significantly different between cells expressing CaM 1234 and CaMwt. This suggests that Ca2+ -Calmodulin is required for filling and/or release from a rapidly releasable pool of vesicles which is easily depleted, but not from the slowly releasable pool which dominates exocytotic responses measured with prolonged responses.