SECONDARY STRUCTURE-ANALYSIS OF THE PUTATIVE MEMBRANE-ASSOCIATED DOMAINS OF THE INWARD RECTIFIER K+ CHANNEL ROMK1

The inward rectifier K+ channels contain two putative membrane-spannin g domains per subunit (M1, M2) and a 'pore' (P) region, which is simil ar to the H5 domain of voltage-gated K+ channels. Here we have used Fo urier transform infrared (FTIR) and CD spectroscopy to analyse the sec ondary structures of synthetic peptides corresponding to the M1, M2 an d P regions of ROMK1 in aqueous solution, in organic solvents and in p hospholipid membranes. A previous CD study was unable to provide any s tructural data on a similar P peptide [Ben-Efraim and Shai (1997) Biop hys. J. 72, 85-96]. However, our FTIR and CD spectroscopic analyses in dicate that this peptide adopts an alpha-helical structure when recons tituted into dimyristoyl phosphatidylcholine vesicles and lysophosphat idyl choline (LPC) micelles as well as in trifluoroethanol (TFE) solve nt. This result is in good agreement with a previous study on a peptid e corresponding to the pore domain of a voltage-gated K+ channel [Hari s, Ramesh, Sansom, Kerr, Srai and Chapman (1994) Protein Eng. 7, 255-2 62]. FTIR spectra of the M1 peptide in LPC micelles displayed a strong absorbance characteristic of an intermolecular beta-sheet structure, suggesting aggregation of the M1 peptide. Sucrose gradient centrifugat ion was used to separate aggregated peptide from peptide incorporated into micelles in an unaggregated manner; subsequent analysis by FTIR s uggested that the M1 peptide adopted an alpha-helical structure when i ncorporated into phospholipid membranes. FTIR and CD spectra of the M2 peptide in phospholipids and high concentrations of TFE suggest that this peptide adopts an alpha-helical structure. The structural data ob tained in these experiments have been used to propose a model for the structure of the membrane-associated core (M1-P-M2) of the inward rect ifier K+ channel protein.