MG2+-DEPENDENT INHIBITION OF K-ATP BY SULFONYLUREAS IN CRI-G1 INSULIN-SECRETING CELLS

1 Patch-clamp recording techniques were used to examine the effects of tolbutamide, glibenclamide, meglitinide and thiopentone on K-ATP in CRI-GI insulin-secreting cells in the presence and absence of Mg2+. 2 In the absence of Mg2+ in the intracellular bathing solution, tolbutamide was significantly less effective when applied either to the intracellular or to the extracellular surfaces of cell-free patches. Removal of extracellular Mg2+ did not alter the effectiveness of tolbutamide provided that Mg2+ was present at the intracellular surface of the patch. 3 Tolbutamide was also significantly less effective when applied to the intracellular surface of cell-free patches when Mn2+ was used as a replacement for Mg2+. 4 Both the sulphonylurea, glibenclamide and the non-sulphonylurea derivative, meglitinide also showed Mg2+ dependent inhibitory effects in cell-free patches. In contrast, the barbiturate thiopentone inhibited K-ATP in a Mg2+-independent manner. 5 Whole-cell I-K(ATP) were used to quantify the effects of tolbutamide and glibenclamide in the presence and absence of intracellular Mg2+. Concentration-inhibition curves, in the presence of intracellular Mg2+, resulted in IC50 values of 12.1 mu M and 2.1 nM for tolbutamide and glibenclamide, respectively. In the absence of intracellular Mg2+, the corresponding IC50 values were 25.3 mM and 3.6 mu M, respectively. The values of IC50 for thiopentone in the presence and absence of intracellular Mg2+ were 69.4 mu M and 69.2 mu M, respectively. 6 With respect to the high affinity binding sites for [H-3]-glibenclamide in CRI-GI membranes, no significant differences were found between the dissociation constants for, or the maximal binding capacities of, [H-3]-glibenclamide in the presence or absence of Mg2+. 7 In the CRI-G1 insulin-secreting cell line, it is concluded that intracellular Mg2+ does not influence the affinity of the sulphonylureas for the sulphonylurea receptor but that this ion is critically important for the interaction between the sulphonylurea receptor and K-ATP.