S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca2+ channels

Modulation of smooth muscle, L-type Ca2+ channels (class C, Ca(V)1.2b) by t hionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryo nic kidney 293 expression system at the level of whole-cell and single-chan nel currents. Extracellular administration of GSNO (2 mM) rapidly reduced w hole-cell Ba2+ currents through channels derived either by expression of al pha 1C-b or by coexpression of alpha 1C-b plus beta 2a and alpha2-delta, Th e 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 si gnificantly. Intracellular administration of copper ions, which promote dec omposition of the thionitrite, antagonized its inhibitory effect, and loadi ng 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 n or their modulation by GSNO, Analysis of single-channel behavior revealed t hat GSNO inhibited Ca2+ channels mainly by reducing open probability, The d evelopment of GSNO-induced inhibition was associated with the transient occ urrence of a reduced conductance state of the channel. Our results demonstr ate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca2+ cha nnels by an intracellular mechanism that is independent of NO release and s timulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca2+ channel gati ng and conductance.