The ATP-sensitive potassium (K-ATP(+)) channel is crucial for the regulation of insulin secretion from the pancreatic beta -cell, and mutations in either the sulfonylurea receptor:type 1 (SUR1) or Kir6.2 subunit of this channel can cause persistent hyperinsulinemic hypoglycemia of infancy (PHHI), We analyzed the functional consequences of the PHHI missense mutation R1420C, which lies in the second nucleotide-binding fold (NBF2) of SUR1, Mild tryptic digestion of SUR1 after photoaffinity labeling allowed analysis of the nucleotide-binding properties of NBF1 and NBF2, Labeling of NBF1 with 8-azido-[alpha-P-32]ATP was inhibited by MgATP and MgADP with similar K-i for wild-type SUR1 and SUR1-R1420C. However, the MgATP and MgADP affinities of NBF2 of SUR1-R1420C were about 5-fold lower than those of wild-type SUR1, MgATP and MgADP stabilized 8-azido-ATP binding at NBF1 of wild-type SUR1 by interacting with NBF2, but this cooperative nucleotide binding was not observed for SUR1-R1420C. Studies on macroscopic currents recorded in inside-out membrane patches revealed that the SUR1-R1420C mutation exhibits reduced expression but does not affect inhibition by ATP or tolbutamide or activation by diazoxide, However, co-expression with Kir6.2-R50G, which renders the channel less sensitive to ATP inhibition, revealed that the SUR1-R1420C mutation increases the EC50 for MgADP activation from 74 to 197 muM. We suggest that the lower expression of the mutant channel and the reduced affinity of NBF2 for MgADP may lead to a smaller K-ATP(+) current in R1420C-PHHI beta -cells and thereby to the enhanced insulin secretion. We also propose a new model for nucleotide activation of K-ATP(+) channels.