Complex regulation of Cav2.2 N-type Ca2+channels by Ca2+and G-proteins Short title: Cav2.2 modulation by Ca2+and G-proteins

ABSTRACT G-protein coupled receptors inhibit Ca v 2.2 N-type Ca 2+ channels by a fast, voltage-dependent pathway mediated by Gα i / Gβγ and a slow, voltage-independent pathway mediated by Gα q −dependent reductions in phosphatidylinositol 4,5-bisphosphate (PIP2) or increases in arachidonic acid. Studies of these forms of regulation generally employ Ba 2+ as the permeant ion, despite that Ca 2+ -dependent pathways may impinge upon G-protein modulation. To address this possibility, we compared tonic G-protein inhibition of currents carried by Ba 2+ ( I Ba ) and Ca 2+ ( I Ca ) in HEK293T cells transfected with Ca v 2.2. Both I Ba and I Ca exhibited voltage-dependent facilitation (VDF), consistent with Gβγ unbinding from the channel. Compared to that for I Ba , VDF of I Ca was less sensitive to an inhibitor of Gα proteins (GDP-β-S) and an inhibitor of Gβγ (C-terminal construct of G-protein coupled receptor kinase 2). While insensitive to high intracellular Ca 2+ buffering, VDF of I Ca that remained in GDP-β-S was blunted by reductions in PIP2. We propose that when G-proteins are inhibited, Ca 2+ influx through Ca v 2.2 promotes a form of VDF that involves PIP2. Our results highlight the complexity whereby Ca v 2.2 channels integrate G-protein signaling pathways, which may enrich the information encoding potential of chemical synapses in the nervous system.