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

The modulation of large conductance Ca 2+ -activated K + (BK Ca ) 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 m m K + and 15 n m Ca 2+ , few BK Ca channel openings were recorded at potentials negative to 0 mV. However, the current amplitude and open probability ( NP o ) of these BK Ca channels increased with patch depolarization. A plot of ln( NP o ) against the membrane potential ( V ) fitted with a straight line revealed a voltage at half-maximal activation ( V 0.5 ) of 9.4 mV and a slope ( K ) indicating an e-fold increase in NP o with 12.9 mV depolarization. As the cytosolic Ca 2+ was raised to 150 n m , V 0.5 shifted 11.5 mV in the negative direction, with little change in K (13.1 mV). NOCys (10 μ m ) and SNP (100 μ m ) transiently increased NP o 16- and 3.7-fold, respectively, after a delay of 2–5 min. This increase in NP o was associated with an increase in the number of BK Ca channel openings evoked at positive potentials by ramped depolarizations (between −60 and +60 mV). Moreover, this NOCys-induced increase in NP o was still evident in the presence of 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 μ m ), the specific blocker of soluble guanylyl cyclase. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 μ m ) and reduced glutathione (GSH; 1 m m ) also significantly increased NP o (at 0 mV) 7- to 9-fold, as well as increasing the number of BK Ca channel openings evoked during ramped depolarizations. Sulfhydryl oxidizing agents thimerosal (10 μ m ) and 4,4′-dithiodipyridine (4,4DTDP; 10 μ m ) and the thiol-specific alkylating agent N -ethylmaleimide (NEM; 1 m m ) significantly decreased NP o (at 0 mV) to 40–50 % of control values after 5–10 min. Ramped depolarizations to +100 mV evoked relatively few BK Ca channel openings. The effects of thimerosal on NP o 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 α subunit of the BK Ca channel protein can alter channel gating.