Direct Inhibition of Expressed Cardiac L-Type Ca 2+ Channels by S -Nitrosothiol Nitric Oxide Donors

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Abstract
NO donors have complex effects on Ca2+ currents in native cardiac cells, with reports of direct stimulation and indirect cGMP-mediated inhibition or stimulation. To investigate the molecular basis of these effects, we tested the effects of one class of NO donors, S-nitrosothiols (RSNOs), on expressed cardiovascular L-type Ca2+ channels (α1C±β1a±α2 or α1C±β2a±α2) in human embryonic kidney (HEK293) cells. The RSNO compounds we used were S-nitroso-N-acetylpenicillamine (SNAP, 5 to 10 nmol/L or 100 to 800 μmol/L), S-nitrosocysteine (SNC, 100 μmol/L or 1 mmol/L), and S-nitrosoglutathione (GSNO, 1 mmol/L). Currents were measured using whole-cell patch recordings with 2 to 10 mmol/L Ba2+ as the charge carrier. SNAP reduced the amplitude of barium currents (IBa) through all the subunit combinations, with an EC50 of 360 μmol/L for α1C+β1a channels. SNC or GSNO also inhibited IBa, albeit less potently. The inhibitory effect of SNAP was not affected by methylene blue (10 to 30 μmol/L) or 8-bromo-cGMP (200 to 400 μmol/L). The effects are relatively specific for Ca2+ channels, as expressed cardiac or skeletal muscle Na+ channels, which have a similar overall architecture, were barely affected by SNAP at concentrations as high as 1 mmol/L. We conclude that in the HEK293 expression system, the S-nitrosothiol NO donors inhibit L-type Ca2+ channels by a mechanism independent of cGMP.