Attenuation of G protein‐mediated inhibition of N‐type calcium currents by expression of caveolins in mammalian NG108–15 cells

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Abstract
1. Caveolins are integral proteins of glycolipid/cholesterol-rich plasmalemmal caveolae domains, where, they may function as a plasma membrane scaffold onto which many classes of signalling molecules, including receptors and heterotrimeric G proteins, can assemble. To ascertain whether caveolins influence G protein-mediated signal transduction, we stably expressed caveolin-1 and -3 isoforms in the neuroblastoma x glioma NG108-15 hybrid cell line, lacking endogenous caveolins. Subsequently, using whole-cell voltage clamp methods, we examined whether the modulation of N-type voltage-gated Ca2+ channels by G(o) protein-coupled, delta-type opioid receptors might be affected by recombinant caveolin expression. 2. In transfected NG108-15 cells, caveolins localized at the plasma membrane and, upon subcellular fractionation on sucrose density gradients, they co-localized in Triton-resistant, low buoyancy fractions, with endogenous G(o) protein alpha-subunits. 3. The voltage-dependent inhibition of omega-conotoxin GVIA-sensitive Ba2+ currents following either activation of delta-opioid receptors by the agonist [o-pen2,o-pen5]-enkephalin (DPDPE), or direct stimulation of G proteins with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was significantly attenuated in caveolin-expressing cells. The kinetics of Ca2+ channel inhibition were also modified by caveolins. 4. Overall, these results suggest that caveolins may negatively affect G protein-dependent regulation of voltage-gated N-type Ca2+ channels, presumably by causing a reduction of the available pool of activated G proteins.
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