Large subtype-specific differences in the sensitivity of cloned inward-rectifier K' channels of the IRKl, BIRlO and ROMKl subtype to being blocked by intracellular spermine (SPM) are described. It is shown, by site-directed mutagenesis, that the four orders of magnitude larger SPM sensitivity of BIRlO channels compared to ROMKl channels may be explained by a difference in a single amino acid in the putative transmembrane segment TMII. This residue, a negatively charged glutamate in BIRlO, is homologous to the residue in IRK1 and ROMKl which has previously heen shown to change gating properties and Me sensitivity. Differential block by physiological SPM concentrations is suggested as a major functional difference between subtypes of inward-rectifier K' channels.