Complex regulation of Ca _v 2.2 N-type Ca ²⁺ channels by Ca ²⁺ and G-proteins Short title: Ca _v 2.2 modulation by Ca ²⁺ and G-proteins

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.