S-nitrosation controls gating and conductance of the α1 subunit of class C L-type Ca2+ channels

Modulation of smooth muscle, L-type Ca2+ channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba2+ currents through channels derived either by expression of alpha 1C-b or by coexpression of alpha 1C-b plus beta 2a and alpha2-delta, The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba2+ currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha 1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha 1C-b channel function nor their modulation by GSNO, Analysis of single-channel behavior revealed that GSNO inhibited Ca2+ channels mainly by reducing open probability, The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca2+ channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca2+ channel gating and conductance.