Convergent signaling events underlie dopamine-mediated inhibition of CaV3.2 T-type calcium channels

Commentary to: Hu C, Depuy SD, Yao J, McIntire ME, Barrett PQ. Protein kinase A activity controls the regulation of T-type CaV3.2 channels by Gβγ dimers. J Biol Chem 2009; 284:7465-73; PMID: 19131331; DOI: 10.1074/jbc. M808049200. In the mid-1980’s, it was demonstrated that the muscarinic acetylcholine receptor-dependent activation of an inwardlyrectifying K current (I K,ACh ) in cardiac atrial cells was directly mediated by a pertussis-toxin-sensitive, heterotrimeric GTP-binding protein (G-protein) acting in a membrane-delimited manner. These landmark studies revealed a new paradigm for receptor-G-protein signal transduction and subsequent work established that both the Gα and Gβγ subunits could interact with and regulate a variety of membrane-associated effector molecules (i.e. ion channels, adenylyl cyclase and phospholipase C). In the presynaptic terminals of central neurons, a variety of GPCR-linked hormones evoke inhibition of Nand P/Q-type calcium channels by stimulating G-protein dissociation and the direct interaction of Gβγ dimers with the channel’s pore-forming α subunit. A similar regulatory mechanism has since been reported for certain low threshold-activated, T-type calcium channels (Ca V 3.x). However, not all published data could be readily explained by such a simple one-step mechanism, and in the present study, Barrett and colleagues hypothesized that an additional molecular process involving cAMP and protein kinase A (PKA) may contribute to the Gβγ-mediated inhibition of Ca V 3.2 channels. To examine such a possibility, this group compared the dopamine-mediated inhibition of both recombinantly expressed T-type channels in HEK 293 cells and endogenous T-type channels in a human adrenal cortex cell line (H295R), using single channel and whole-cell patch clamp recordings. Biochemical assays and site-directed mutagenesis were also used to assess PKA-mediated phosphorylation, and direct binding of Gβγ dimers to the intracellular loop linking domains II and III (DII-III linker) of the Ca V 3.2 poreforming α subunit. In adrenocortical cells, dopaminemediated inhibition of endogenous T-type current (both single channel and macroscopic) required the stimulation of both D1-type receptors (coupled to the stimulatory G-protein G s ) and D2-type receptors (coupled to the inhibitory G-proteins G i/o ), in agreement with earlier studies demonstrating that cAMP elevation and the generation of free Gβγ dimers contributed to T-type channel inhibition. Biophysically, this dopamine-evoked inhibition did not involve changes in single channel conductance, open state kinetics, or the steadystate activation and inactivation properties of channel opening. Rather, dopamine appeared to increase the number of blank or null single channel sweeps observed in response to repetitive depolarizing stimuli, indicating a reduction in the collective open probability of available channels (NPopen). In H295R cells pretreated with membrane-permeable 8-bromo-cAMP, direct application of recombinant Gβγ dimers to excised, inside/out membrane patches reproduced the channel inhibition observed in intact cells exposed to dopamine; omitting cAMP pretreatment prevented this inhibitory effect of applied Gβγ subunits. To investigate this apparent co-requirement of cAMP and Gβγ dimers for channel inhibition, the authors demonstrated that intracellular dialysis of the protein kinase A inhibitor PKI prevented dopamine-mediated channel inhibition in whole-cell recordings, as did transient over-expression of the Gβγ binding Convergent signaling events underlie dopamine-mediated inhibition of Ca V 3.2 T-type calcium channels