The Journal of Neuroscience, September 1, 2002, 22(17):7485-7492
cGMP/Protein Kinase G-Dependent Inhibition of N-Type
Ca2+ Channels Induced by Nitric Oxide in Human
Neuroblastoma IMR32 Cells
Marcello
D'Ascenzo,
Giovanni
Martinotti,
Gian Battista
Azzena, and
Claudio
Grassi
Institute of Human Physiology, Medical School, Catholic University
"S. Cuore", I-00168 Rome, Italy
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 Vh = 
90 mV, the
NO donor, sodium nitroprusside (SNP; 200 µM), 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 µM), or after cell treatment with the
guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a]
quinoxalin-1-one (10 µM). 8-Bromoguanosine-cGMP
(8-Br-cGMP) (400 µM) mimicked the effects of SNP,
reducing Ba2+ currents by 37%
(p < 0.001). Cell treatment with the
protein kinase G (PKG) inhibitor KT5823 (1 µM) or
guanosine 3',5'-cyclic monophosphorothioate,
8-(4-chloro-phenylthio)-Rp-isomer, triethylammonium salt (20 µM) 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.
Key words:
nitric oxide; N-type calcium channels; cGMP; protein kinase G; sodium nitroprusside; human neuroblastoma cells
Copyright © 2002 Society for Neuroscience 0270-6474/02/22177485-08$05.00/0
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