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

R. J. Lang, J. R. Harvey, G. J. McPhee and M. F. Klemm

1. The modulation of large conductance Ca²⁺-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.

2. When the cytosolic side of excised patches was bathed in a physiological salt solution (PSS) containing 130 mM K⁺ and 15 nM Ca²⁺, 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 Ca²⁺ was raised to 150 nM, V_0.5 shifted 11·5 mV in the negative direction, with little change in K (13·1 mV).

3. NOCys (10 µM) and SNP (100 µM) 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 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 µM), the specific blocker of soluble guanylyl cyclase.

4. The sulfhydryl reducing agents dithiothreitol (DTT; 10 and 100 µM) and reduced glutathione (GSH; 1 mM) 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.

5. Sulfhydryl oxidizing agents thimerosal (10 µM) and 4,4'-dithiodipyridine (4,4DTDP; 10 µM) and the thiol-specific alkylating agent N-ethylmaleimide (NEM; 1 mM) 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.

6. The effects of thimerosal on NP_o were readily reversed by DTT, while the effects of NOCys were prevented by NEM.

7. It was concluded that both redox modulation and nitrothiosylation of cysteine groups on the cytosolic surface of the subunit of the BK_Ca channel protein can alter channel gating.

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