Abstract
Although data from our laboratory and others suggest that nitric oxide (NO) exerts an overall inhibitory action on high-voltage-activated Ca2+ channels, conflicting observations have been reported regarding its effects on N-type channels. We performed whole-cell and cell-attached patch-clamp recordings in IMR32 cells to clarify the functional role of NO in the modulation of N channels of human neuronal cells. During depolarizing steps to +10 mV from V(h) = -90 mV, the NO donor, sodium nitroprusside (SNP; 200 microm), reduced macroscopic N currents by 34% (p < 0.01). The magnitude of inhibition was similar at all voltages tested (range, -40 to +50 mV). No significant inhibition was observed when SNP was applied together with the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium salt (300 microm), or after cell treatment with the guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one (10 microm). 8-bromoguanosine-cGMP (8-Br-cGMP) (400 microm) mimicked the effects of SNP, reducing Ba2+ currents by 37% (p < 0.001). Cell treatment with the protein kinase G (PKG) inhibitor KT5823 (1 microm) or guanosine 3',5'-cyclic monophosphorothioate, 8-(4-chloro-phenylthio)-Rp-isomer, triethylammonium salt (20 microm) virtually abolished the effects of 8-Br-cGMP. At the single-channel level, 8-Br-cGMP reduced the channel open probability by 59% and increased both the mean shut time and the null sweep probability, but it had no significant effects on channel conductance, mean open time, or latency of first openings. These data suggest that NO inhibits N-channel gating through cGMP and PKG. The consequent decrease in Ca2+ influx through these channels may affect different neuronal functions, including neurotransmitter release.