Effects of chemical modification of amino and sulfhydryl groups on KATP channel function and sulfonylurea binding in CRI-G1 insulin-secreting cells.

The effects of several group-specific chemical reagents were examined upon the activity of the ATP-sensitive potassium (KATP) channel in the CRI-G1 insulin-secreting cell line. Agents which interact with the sulfhydryl moiety (including 1 mM N-ethyl-maleimide (NEM), 1 mM 5,5'-dithio-bis-(2-nitrobenzoic acid) (DNTB) and 1 mM o-iodobenzoate) produced an irreversible inhibition of KATP channel activity when applied to the intracellular surface of excised inside-out patches. This inhibition was substantially reduced when attempts were made to eliminate Mg2+ from the intracellular compartment. ATP 50 microM and 100 microM tolbutamide were each shown to protect against the effects of these reagents. The membrane impermeable DNTB was significantly less effective when applied to the external surface of outside-out patches. Agents which interact with peptide terminal amine groups and epsilon amino groups of lysine [1 mM methyl acetimidate and 1 mM trinitrobenzene sulfonic acid (TNBS)] and also the guanido group of arginine (1 mM methyl glyoxal) produced a Mg(2+)-dependent irreversible inhibition of KATP channel activity which could be prevented by ATP but not tolbutamide. The irreversible activation of the KATP channel produced by the proteolytic enzyme trypsin was prevented only when methyl glyoxal and methyl acetimidate were used in combination to inhibit channel activity. Radioligand binding studies showed that the binding of 3H glibenclamide was unaffected by any of the above agents with the exception of TNBS which completely inhibited binding with a EC50 of 307 +/- 6 microM. These results provide evidence for the presence of essential sulfhydryl (possibly cysteine), and basic amino acid (possibly lysine and arginine) residues associated with the normal functioning of the KATP channel. Furthermore, we believe that the sulfhydryl group in question is situated at the internal surface of the membrane, possibly near to the channel pore.