Sodium (2-sulfonatoethyl) methanethiosulfonate prevents S-nitroso-L-cysteine activation of Ca 2+-activated K + (BK Ca) channels in myocytes of the guinea-pig taenia caeca

Abstract

1. The modulation of large conductance Ca 2+-activated K + (BK Ca) channels by the nitric oxide (NO .) donor, S-nitroso-L-cysteine (NOCys) and three sulfhydryl-specific methanethiosulfonate (MTS) reagents, positively charged 2-aminoethyl MTS hydrobromide (MTSEA C 3H 9NO 2S 2HBr) and [2-(trimethylammonium) ethyl MTS bromide (MTSET C 6H 16NO 2S 2Br), and negatively charged sodium (2-sulfonatoethyl) MTS (MTSES C 3H 7O 5S 3Na) were compared in excised inside-out membrane patches of the guinea-pig taenia caeca. 2. In membrane patches bathed in a low Ca 2+ (15 nM) high K + physiological salt solution, 1-3 BK Ca channels opened with a low probability (N.P o) of 0.019±0.011 at 0 mV. N.P o readily increased with membrane depolarization, raised Ca 2+ concentration or upon the addition of NOCys (10 μM for 2-5 min) such that 5-15 open BK Ca channels were evident. 3. MTSEA (2.5 mM) decreased, while MTSES (2.5 mM) increased N.P o (at 0 mV) and the number of open BK Ca channels at positive potentials. These changes in channel activity remained after a prolonged washout of these two MTS reagents. 4. MTSET (2.5 mM) increased N.P o (at 0 mV) and voltage-dependently decreased BK Ca current amplitudes in a manner readily reversed upon washout. 5. Pre-exposure of excised membrane patches to MTSES or N-ethylmaleimide (NEM 1 mM), a specific alkylating agent of cysteine sulfhydryls, but not MTSEA or MTSET, prevented the excitatory actions of NOCys (10 μM). 6. It was concluded that NOCys-evoked increases in BK Ca channel activity occur via the S-nitrosylation of cysteine residues within basic regions of the channel α subunit that have an accessible water interface.