S-Nitrosation Controls Gating and Conductance of the α1 Subunit of Class C L-type Ca2+ Channels

Abstract

Modulation of smooth muscle, L-type Ca2+ channels (class C, Cav1.2b) 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 α1C-b or by coexpression of α1C-b plus β2a and α2-δ. 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 thio-nitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on α1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither α1C-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 α1 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 Ca 2+ channel gating and conductance.