Thiol reagents and nitric oxide modulate the gating of BKCa channels from the guinea-pig taenia caeci.
1. The site of the direct modulation of the gating of BKCa channels by the nitric oxide donor s-nitroso-l-cysteine (NOCys) was examined in excised membrane patches of the guinea-pig taenia caeci by the use of various thiol (sulphydryl)-specific reagents, including N-ethylmaleimide (NEM) and three charged methanethiosulphonate (MTS) reagents, namely positively charged 2-aminoethyl MTS hydrobromide (MTSEA) and [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium (2-sulphonatoethyl) MTS (MTSES), which all specifically convert sulphydryls to a disulphide. 2. At 10 micro mol/L, NOCys transiently increased the probability of opening (N.Po) of the BKCa channels (at 0 mV) after a delay of 1-2 min. 3. Disulphide-reducing agents, such as dithiothreitol (10 micro mol/L), increased N.Po in a manner that was reversed by the sulphide-oxidizing agent thimerosal (10 micro mol/L). Both positively charged MTSET (2.5 mmol/L) and negatively charged MTSES (2.5 mmol/L) rapidly increased N.Po. However, only the MTSES-evoked increase in N.Po remained after a prolonged washout period. 4. The specific alkylating agent of cysteine thiols NEM (1 mmol/L) and the positively charged, but membrane permeable, MTSEA (2.5 mmol/L) decreased N.Po (at 0 mV). 5. Pre-exposure of excised membrane patches to NEM or MTSES prevented the excitatory actions of NOCys (10 micro mol/L). 6. We conclude that MTSES and NOCys must modify thiols on cysteine residues within basic regions of the channel protein that would electrostatically exclude MTSEA and MTSET. A consensus sequence of various mammalian alpha-subunits of the BKCa channel reveals two pairs of cysteine residues surrounded by basic amino acids that could be the site of action for NOCys and MTSES.
Department of Physiology, Monash University, Melbourne, Victoria, Australia. rick.lang@monash.edu.au
R J
RJ
Lang
J R
JR
Harvey
D000818:Animals; D006168:Guinea Pigs; D015640:Ion Channel Gating / Q000187:drug effects* / Q000502:physiology; D009569:Nitric Oxide / Q000378:metabolism / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology*
D020030:Nitric Oxide Donors; D024681:Potassium Channels, Calcium-Activated; D013439:Sulfhydryl Reagents; D009569:Nitric Oxide