Nitric oxide and thiol reagent modulation of Ca2+-activated K+ (BKCa) channels in myocytes of the guinea-pig taenia caeci.

Abstract

The modulation of large conductance Ca2+-activated K+ (BKCa) channels by the nitric oxide (NO) donors S-nitroso-L-cysteine (NOCys) and sodium nitroprusside (SNP) and agents which oxidize or reduce reactive thiol groups were compared in excised inside-out membrane patches of the guinea-pig taenia caeci. When the cytosolic side of excised patches was bathed in a physiological salt solution (PSS) containing 130 mM K+ and 15 nM Ca2+, few BKCa channel openings were recorded at potentials negative to 0 mV. However, the current amplitude and open probability (NPo) of these BKCa channels increased with patch depolarization. A plot of ln(NPo) against the membrane potential (V) fitted with a straight line revealed a voltage at half-maximal activation (V0.5) of 9.4 mV and a slope (K) indicating an e-fold increase in NPo with 12.9 mV depolarization. As the cytosolic Ca2+ was raised to 150 nM, V0.5 shifted 11.5 mV in the negative direction, with little change in K (13.1 mV). NOCys (10 microM) and SNP (100 microM) transiently increased NPo 16- and 3. 7-fold, respectively, after a delay of 2-5 min. This increase in NPo was associated with an increase in the number of BKCa channel openings evoked at positive potentials by ramped depolarizations (between -60 and +60 mV). Moreover, this NOCys-induced increase in NPo was still evident in the presence of 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ; 10 microM), the specific blocker of soluble guanylyl cyclase. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 microM) and reduced glutathione (GSH; 1 mM) also significantly increased NPo (at 0 mV) 7- to 9-fold, as well as increasing the number of BKCa channel openings evoked during ramped depolarizations. Sulfhydryl oxidizing agents thimerosal (10 microM) and 4,4'-dithiodipyridine (4,4DTDP; 10 microM) and the thiol-specific alkylating agent N-ethylmaleimide (NEM; 1 mM) significantly decreased NPo (at 0 mV) to 40-50% of control values after 5-10 min. Ramped depolarizations to +100 mV evoked relatively few BKCa channel openings. The effects of thimerosal on NPo were readily reversed by DTT, while the effects of NOCys were prevented by NEM. It was concluded that both redox modulation and nitrothiosylation of cysteine groups on the cytosolic surface of the alpha subunit of the BKCa channel protein can alter channel gating.