Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

Abstract

The 26-residue peptide of sequence KEALYILMVLGFFGFFTLGILSYIR, which contains the single putative transmembrane domain of a small protein that is associated with slow voltage-gated K+ channels, has been incorporated in bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol to form complexes of homogeneous lipidpeptide ratio. Fourier transform infrared spectroscopy indicates that the peptide is integrated in the lipid bilayer wholly in a @-sheet conformation. The electron spin resonance spectra of spin-labeled lipids in the lipidpeptide complexes contain a component corresponding to lipids whose chains are motionally restricted in a manner similar to those of lipids at the hydrophobic surface of integral transmembrane proteins. From the dependence of the lipid spin label spectra on the lipidpeptide ratio of the complexes, it is found that ca. 2.5 lipids per peptide monomer, independent of the species of spin-labeled lipid, are motionally restricted by direct interaction with the peptide in the bilayer. This value would be consistent with, e.g., a @-barrel structure for the peptide in which the @-strands either are strongly tilted or have a reverse turn at their center. A preferential selectivity of interaction with the peptide is observed for the negatively charged spin-labeled lipids phosphatidic acid, stearic acid, and phosphatidylserine, which indicates close proximity of the positively charged residues at the peptide termini to the lipid headgroups. The saturation-transfer electron spin resonance spectra of the peptide spin-labeled at a cysteine residue replacing Leu18 evidence rather slow rotational diffusion in the lipid complexes. This indicates that the presumably enclosed @-sheet units of the peptide are aggregated in oligomeric assemblies in the lipid bilayer. The results suggest a way in which one type of channel unit may be integrated in the membrane.Ion channels are generally large, often multimeric, proteins containing many transmembrane segments (Stephenson, 1991). In spite of the molecular complexity, it is likely that the channel itself is lined with a limited range of, possibly homologous, transmembrane segments. Correlating with thls, it is found that certain relatively short synthetic peptides, of sequences resembling those of putative pore-lining segments, are able to sustain channel activity when incorporated in lipid bilayers (Montal, 1990;Ben-Efraim et al., 1993). Such synthetic peptides are therefore suitable models for studying particular aspects of channel structure and assembly by using biophysical or biochemical techniques. The direct relevance to native channels depends, of course, on the correct identification of the pore-lining transmembrane segments.In addition to the complex channel proteins mentioned above, the DNA clone encoding a small protein associated with a slowly activating voltage-gated potassium channel has been identified in rat kidney (Takumi et al., 1988). This protein, Z s~, contains only 130 amino acids with a single putative 23-residu...

Secondary Structure of the MiRP1 (KCNE2) Potassium Channel Ancillary Subunit

“…A previous CD-based structural study on MinK CT domain indicated that it gave no signal when taken up in methanol, suggesting insolubility in this solvent [37], as we found for MiRP1 CT domain in aqueous solution. Here we demonstrate that hydrophobic interaction is required for solubility and adoption of ordered secondary structure by the MiRP1 CT domain.…”

“…The role of the N-terminus in KCNE subunits is not well understood, but removal of part of the N-terminus of MinK, residues 4-9, results in loss of channel function [37]. Furthermore, inherited Q9E and T8A polymorphisms in MiRP1 NT are associated with drug-induced arrhythmia [4,10].…”

Neuromolecular computing: a new approach to human brain evolution

“…Thè`p artitioning'' concept appears equivalent to lipid selectivity based on hydrophobic mismatch and minimal free-energy search emphasized in the present model. Finally, spectrochemical analysis of membrane lipid rotational mobility in the presence of a 26-residue K + channel-associated peptide indicated that the greatest mobility restriction is at the peptide hydrophobic surface (Horva th et al 1995). The reduced mobility was attributed to interfacial hydrophobic matching due to``selectivity of interaction'' exercised by the protein.…”

A single-residue deletion alters the lipid selectivity of a K+ channel-associated peptide in the beta-conformation: spin label electron spin resonance studies

“…Much of the available evidence, including mutagenesis studies (Takumi et al, 1991;Goldstein and Miller, 1991;Tsai and Goldstein, 1997), is therefore in favour of IsK being a true channel protein. Because of the relatively simple structure of the IsK protein, this has proved an attractive candidate for the study of synthetic peptides related to the putative transmembrane sequence that are reconstituted in lipid bilayers (Ben-Efraim et al, 1993, 1994Horvath et al, 1995;Aggeli et al, 1996).…”

“…Previously we have studied the assembly in phospholipid bilayers of a 26-residue peptide (peptide of sequence KEALYILMVLGFFGFFTlLGIMLSYIR [i.e., K27(AL2)]; K26) that is a deletion mutant but contains the putative transmembrane sequence of the IsK protein (Horvath et al, 1995). On dialysis from the helix-promoting solvent 2chloroethanol, the K26 peptide at high concentration was found to be incorporated into lipid bilayers in a (3-sheet form.…”