Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels contribute to the spontaneous rhythmic activities in cardiac and neuronal cells. Recently, we reported that the S3-S4 linker of HCN1 channels influences activation, and that part of the linker is helical with the determinants G231, M232, and E235 clustered on one side. Here we explored the undefined role of the G(231)E(235)M(232) triplet by systematic substitutions. Replacing G231 or M232 next to the "neighboring" E235 in the S3-S4 helix with an anionic residue (i.e., G231E, M232E) rendered channels non-functional although they were localized on the membrane surface. Interestingly, this loss of function could be readily rescued either by introducing a countercharge at position 235 (G231E/E235R, M232E/E235R) or by interchanging residues 231 or 232 and 235 (G231E/E235G, M232E/E235M). We conclude that residues 231, 232, and 235 are in close spatial proximity to each other, and uniquely interact with one another to shape the phenotypes of HCN channels. (C) 2004 Elsevier Inc. All rights reserved.