The structure and organization of synthetic putative membranous segments of ROMK1 channel in phospholipid membranes.

The hydropathy plot of ROMK1, an inwardly rectifying K+ channel, suggests that the channel contains two transmembrane domains (M1 and M2) and a linker between them with significant homology to the H5 pore region of voltage-gated K+ channels. To gain structural information on the pore region of the ROMK1 channel, we used a spectrofluorimetric approach and characterized the structure, the organization state, and the ability of the putative membranous domains of the ROMK1 channel to self-assemble and coassemble within lipid membranes. Circular dichroism (CD) spectroscopy revealed that M1 and M2 adopt high alpha-helical structures in egg phosphatidylcholine small unilamellar vesicles and 40% trifluoroethanol (TFE)/water, whereas H5 is not alpha-helical in either egg phosphatidylcholine small unilamellar vesicles or 40% TFE/water. Binding experiments with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled peptide demonstrated that all of the peptides bind to zwitterionic phospholipid membranes with partition coefficients on the order of 10(5) M-1. Tryptophan quenching experiments using brominated phospholipids revealed that M1 is dipped into the hydrophobic core of the membrane. Resonance energy transfer (RET) measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that H5 and M2 can self-associate in their membrane-bound state, but M1 cannot. Moreover, the membrane-associated nonhelical H5 serving as a donor can coassemble with the alpha-helical M2 but not with M1, and M1 can coassemble with M2. No coassembly was observed between any of the segments and a membrane-embedded alpha-helical control peptide, pardaxin. The results are discussed in terms of their relevance to the proposed topology of the ROMK1 channel, and to general aspects of molecular recognition between membrane-bound polypeptides.