NEUROPEPTIDE Y-2-TYPE RECEPTOR-MEDIATED ACTIVATION OF LARGE-CONDUCTANCE CA2+-SENSITIVE K+ CHANNELS IN A HUMAN NEUROBLASTOMA CELL-LINE

We have proposed recently that a pertussistoxin-insensitive Ca2+ influx stimulated by Y-2-type receptor activation in CHP-234 human neuroblastoma cells underlies increases in intracellular free Ca2+ concentration ([Ca2+](i)) induced by neuropeptide Y (NPY), which were strictly dependent on extracellular Ca2+ and independent of internal Ca2+ stores. We describe here the actions of NPY in these same cells, using the activity of Ca2+-activated K+ channels as an indicator of [Ca2+](i). The elementary slope conductance of these channels was 110 +/- 3 pS (with an asymmetrical K+ gradient), their activity was greatly increased by application of ionomycin, and they were reversibly blocked by 1 mM tetraethylammonium (TEA) and 100 nM charybdotoxin. Application of 100 nM NPY, in the presence but not in the absence of extracellular Ca2+ increased the channel open probability. ATP applied in the absence of external Ca2+ caused rises both in chan nel open probability and [Ca2+](i). Inositol trisphosphate production was stimulated by ATP but not by NPY. In outside-out patches, NPY increased channel open probability, indicating that NPY-associated Ca2+ influx does not require all the intracellular machinery present in intact cells. Channel activation by NPY was unaffected by the replacement of guanosine 5'-triphosphate (GTP) by (guanosine 5'-O-(2-thiodiphosphate) (GDP[beta S]), a non-hydrolysable GDP analogue, in the pipette internal solution, consistent with the lack of involvement of G-proteins in the coupling of Y-2-type receptors to Ca2+ influx in CHP-234 cells.