Protein Science, Vol 5, Issue 11 2287-2297, Copyright © 1996 by Cold Spring Harbor Laboratory Press

ARTICLE

Secondary structure, membrane localization, and coassembly within phospholipid membranes of synthetic segments derived from the N- and C-termini regions of the ROMK1 K(+) channel

I. BEN-EFRAIM and Y. SHAI
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, 76100 Israel

The hydropathy plot of the inwardly rectifying ROMK1 K(+) channel, which reveals two transmembrane and a pore region domains, also reveals areas of intermediate hydrophobicity in the N terminus (M0) and in the C terminus (post-M2). Peptides that correspond to M0, post-M2, and a control peptide, pre-M0, were synthesized and characterized for their structure, affinity to phospholipid membranes, organizational state in membranes, and ability to self-assemble and coassemble in the membrane-bound state. CD spectroscopy revealed that both M0 and post-M2 adopt highly {alpha}-helical structures in 1% SDS and 40% TFE/water, whereas pre-M0 is not {alpha}-helical in either 1% SDS or 40% TFE/water. Binding experiments with NBD-labeled peptides demonstrated that both M0 and post-M2, but not pre-M0, bind to zwitterionic phospholipid membranes with partition coefficients of 10(3)-10(5) M(-1). A surface localization for both post-M2 and M0 was indicated by NBD shift, tryptophan quenching experiments with brominated phospholipids, and enzymatic cleavage. Resonance energy transfer measurements between fluorescently labeled pairs of donor (NBD)/acceptor (rhodamine) peptides revealed that M0 and post-M2 can coassemble in their membrane-bound state, but cannot self-associate when membrane-bound. The results are in agreement with recent data indicating that amino acids in the carboxy terminus of inwardly rectifying K(+) channels have a major role in specifying the pore properties of the channels (Taglialatela M, Wible BA, Caporaso R, Brown AM, 1994, Science 264:844-847; Pessia M, Bond CT, Kavanaugh MP, Adelman JP, 1995, Neuron 14:1039-1045). The relevance of the results presented herein to the suggested model for the structure of the ROMK1 channel and to general aspects of molecular recognition between membrane-bound polypeptides are also discussed.
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