Secondary structure and membrane localization of synthetic segments and a truncated form of the IsK (minK) protein.
IsK, also referred to as minK, is a membrane protein consisting of 130 amino acids and localized mainly in epithelial cells but also in human T lymphocytes. Depending on the cRNA concentration that was injected into Xenopus oocytes, IsK and its truncated forms can induce either a K+ current alone or both K+ and Cl- currents [Attali et al. (1993) Nature 365, 850-852]. To obtain information on the secondary structure and the topology of IsK in a membrane-bound state, the synthesis, fluorescent-labeling, and structural and functional characterization of five polypeptides of 20-63 amino acids within the rat IsK protein were examined. The alpha-helical content of the segments, assessed in methanol using circular dichroism, suggests that both the N-terminal and transmembrane segments of IsK adopt alpha-helical structures. Binding experiments and the blue shift of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled peptides suggest that while both the alpha-helical transmembrane segment and the N-terminal of IsK are located within the lipid bilayer, the linking segment between the two segments lies on the surface of the membrane. The fluorescence energy transfer, between donor and acceptor-labeled truncated IsK, suggests that it aggregates within phospholipid membranes. Although a protein whose sequence is similar to that of truncated IsK can induce K+ channel activity when expressed in Xenopus oocytes, the inability of a truncated IsK to form functional K+ channels in planar lipid membranes supports increasing evidence that the protein alone cannot form a K+ channel.
Ben-Efraim
I
I
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Strahilevitz
J
J
Bach
D
D
Shai
Y
Y
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008051:Lipid Bilayers; D008081:Liposomes; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry; D015221:Potassium Channels / Q000032:analysis / Q000737:chemistry*; D024642:Potassium Channels, Voltage-Gated; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; C059983:potassium channel protein I(sk); C032930:7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole; D009327:4-Chloro-7-nitrobenzofurazan