Mechanisms of Presynaptic CaV2.2 (N-type) Modulation

Abstract

Neurotransmitter release at presynaptic terminals is a complex process involving calcium ion influx through voltage-gated calcium channels (CaV). In addition to their role as entry points through which calcium influx may occur, CaV are now understood to be fundamental components of a common release-site complex that is highly adapted for modulation. Consistent with this model, I investigated mechanisms of modulating a presynaptic calcium channel, CaV2.2, via a heterotrimeric G-protein pathway. Using the patch-clamp technique, I demonstrated in chick dorsal root ganglion (DRG) neurons that the slow kinetics of G-protein inhibition of CaV2.2 via GTPgammaS were limited by the rate of GDP dissociation from the G-protein nucleotide binding site. In addition, I investigated the role of G-protein regulation of CaV2.2 currents evoked by action potential-like stimuli. Here, I characterized an inhibited current that was advanced in time with respect to uninhibited controls. These currents exhibited a shorter latency to current activation and faster deactivation. These findings may have important physiological ramifications on signal transduction and timing. In addition to G-protein regulation, presynaptic CaV2.2 have been demonstrated to exhibit a resistance to voltage-dependent inactivation (VDI), a property thought to be important in determining channel availability and synaptic excitability. I demonstrated a role for dynamic palmitoylation in conferring resistance to VDI in presynaptic terminals of the chick ciliary ganglion. Using tunicamycin, an inhibitor of palmitoylation, I induced a hyperpolarizing shift in the steady-state-inactivation (SSI) profile of presynaptic CaV2.2. Finally, I examined the role of a CaV interacting protein, Munc18, as a potential regulator of CaV. I probed for alterations in CaV2.2 function in DRG neurons that had been transfected with Munc18 or Munc18 siRNA. Despite the intimate interaction between Munc18 and CaV2.2, no major effects on the fundamental characteristics of CaV2.2 function were observed. However, a hyperpolarizing shift in the inactivation profile of CaV2.2 was determined in DRG neurons in which Munc18 was knocked down. It is not clear if this was a direct consequence of Munc18 perturbation.