Biophysical Journal 72: 85-96 (1997)
© 1997 the Biophysical Society

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

Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT

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.

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