K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) channels in a tissue-dependent manner by binding to the channel regulatory subunit, the sulfonylurea receptor (SUR), an ATP-binding cassette protein. Residues crucial to this action were previously identified in the last transmembrane helix of SUR, transmembrane helix 17. This study examined the residue at the most important position, 1253 in the muscle isoform SUR2A and the matching 1290 in the pancreatic/neuronal isoform SUR1 (rat numbering). At this position in either isoform, a threonine enables action of openers, whereas a methionine prohibits it. Using single-point mutagenesis, we have examined the physicochemical basis of this phenomenon and discovered that it relied uniquely on side chain volume and not on shape, polarity, or hydrogen-bonding capacity of the residue. Moreover, the aromatic nature of neighboring residues conserved in SUR1 and SUR2A was found necessary for SUR2A to sustain the wild-type levels of channel activation by the openers tested, the cromakalim analog SR47063 [4-(2-cyanimino-1,2-dihydro-1-pyridyl)-2,2-dimethyl-6-nitrochromene] and the pinacidil analog P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N'-3-pyridylguanidine]. These observations suggest that these residues can interact with openers via nonspecific stacking interactions provided that the adjacent 1253/1290 residue does not obstruct access. The smaller Thr1253 of SUR2A would permit activation, whereas the bulky Met1290 of SUR1 would not. This hypothesis is discussed in the context of a simple molecular model of transmembrane helix 17.