DIRECT INHIBITION OF EXPRESSED CARDIAC L-TYPE CA2-NITROSOTHIOL NITRIC-OXIDE DONORS( CHANNELS BY S)

NO donors have complex effects on Ca2+ currents in native cardiac cell s, with reports of direct stimulation and indirect cGMP-mediated inhib ition or stimulation. To investigate the molecular basis of these effe cts, we tested the effects of one class of NO donors, S-nitrosothiols (RSNOs), on expressed cardiovascular L-type Ca2+ channels (alpha(1C)+/ -beta(1a)+/-alpha(2) or alpha(1C)+/-B-2a+/-alpha(2)) in human embryoni c kidney (HEK293) cells. The RSNO compounds we used were S-nitroso-N-a cetylpenicillamine (SNAP, 5 to 10 nmol/L or 100 to 800 mu mol/L), S-ni trosocysteine (SNC, 100 mu mol/L or 1 mmol/L), and S-nitrosoglutathion e (GSNO, 1 mmol/L). Currents were measured using whole-cell patch reco rdings with 2 to 10 mmol/L Ba2+ as the charge carrier. SNAP reduced th e amplitude of barium currents (I-Ba) through all the subunit combinat ions, with an EC50 of 360 mu mol/L for alpha(1C)+beta(1a) channels. SN C or GSNO also inhibited I-Ba, albeit less potently. The inhibitory ef fect of SNAP was not affected by methylene blue (10 to 30 mu mol/L) or 8-bromo-cGMP (200 to 400 mu mol/L). The effects are relatively specif ic for Ca2+ channels,as expressed cardiac or skeletal muscle Na+ chann els, which have a similar overall architecture, were barely affected b y 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.