EFFECTS OF CHEMICAL MODIFICATION OF AMINO AND SULFHYDRYL-GROUPS ON K-ATP 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 (K-ATP) channel in th e CRI-G1 insulin-secreting cell line. Agents which interact with the s ulfhydryl moiety (including 1 mM N-ethylmaleimide (NEM), 1 mM 5,5'-dit hio-bis-(2-nitrobenzoic acid) (DNTB) and 1 mM o-iodobenzoate) produced an irreversible inhibition of K-ATP activity when applied to the intr acellular surface of excised inside-out patches. This inhibition was s ubstantially reduced when attempts were made to eliminate Mg2+ from th e intracellular compartment. ATP 50 mu M and 100 mu M tolbutamide were each shown to protect against the effects of these reagents. The memb rane impermeable DNTB was significantly less effective when applied to the external surface of outside-out patches. Agents which interact wi th 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 Mg2+-dependent irreversible inhibition of K-ATP channel activity whi ch could be prevented by ATP but not tolbutamide. The irreversible act ivation of the K-ATP channel produced by the proteolytic enzyme trypsi n was prevented only when methyl glyoxal and methyl acetimidate were u sed in combination to inhibit channel activity. Radioligand binding st udies showed that the binding of H-3 glibenclamide was unaffected by a ny of the above agents with the exception of TNBS which completely inh ibited binding with a EC(50) of 307 +/- 6 mu M. These results provide evidence for the presence of essential sulfhydryl (possibly cysteine), and basic amino acid (possibly lysine and arginine) residues associat ed with the normal functioning of the K-ATP channel. Furthermore, we b elieve that the sulfhydryl group in question is situated at the intern al surface of the membrane, possibly near to the channel pore.