Conformation and ion-channeling activity of a 27-residue peptide modeled on the single-transmembrane segment of the IsK (minK) protein.
IsK (minK) protein, in concert with another channel protein KVLQT1, mediates a distinct, slowly activating, voltage-gated potassium current across certain mammalian cell membranes. Site-directed mutational studies have led to the proposal that the single transmembrane segment of IsK participates in the pore of the potassium channel [Takumi, T. (1993) News Physiol. Sci. 8, 175-178]. We present functional and structural studies of a short peptide (K27) with primary structure NH2-1KLEALYILMVLGFFGFFTLGIMLSYI27R-COOH, corresponding to the transmembrane segment of IsK (residues 42-68). When K27 was incorporated, at low concentrations, into phosphatidylethanolamine, black-lipid membranes, single-channel activity was observed, with no strong ion selectivity. IR measurements reveal the peptide has a predominantly helical conformation in the membrane. The atomic resolution structure of the helix has been established by high-resolution 1H NMR spectroscopy studies. These studies were carried out in a solvent comprising 86% v/v 1,1,1,3,3,3-hexafluoro-isopropanol-14% v/v water, in which the IR spectrum of the peptide was found to be very similar to that observed in the bilayer. The NMR studies have established that residues 1-3 are disordered, while residues 4-27 have an alpha-helical conformation, the helix being looser near the termini and more stable in the central region of the molecule. The length (2. 6 nm) of the hydrophobic segment of the helix, residues 7-23, matches the span of the hydrocarbon chains (2.3 +/- 0.25 nm) of fully hydrated bilayers of phosphatidylcholine lipid mixture from egg yolk. The side chains on the helix surface are predominantly hydrophobic, consistent with a transmembrane location of the helix. The ion-channeling activity is believed to stem from long-lived aggregates of these helices. The aggregation is mediated by the pi-pi stacking of phenylalanine aromatic rings of adjacent helices and favorable interactions of the opposing aliphatic-like side chains, such as leucine and methionine, with the lipid chains of the bilayer. This mechanism is in keeping with site-directed mutational studies which suggest that the transmembrane segment of IsK is an integral part of the pore of the potassium channel and has a similar disposition to that in the peptide model system.
Aggeli
A
A
Centre for Self-Organising Molecular Systems, University of Leeds, U.K.
Bannister
M L
ML
Bell
M
M
Boden
N
N
Findlay
J B
JB
Hunter
M
M
Knowles
P F
PF
Yang
J C
JC
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004530:Egg Yolk / Q000737:chemistry; D004553:Electric Conductivity; D008051:Lipid Bilayers / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry; D015221:Potassium Channels / Q000737:chemistry* / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated; D011487:Protein Conformation; D012997:Solvents; D017550:Spectroscopy, Fourier Transform Infrared
D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D010455:Peptides; D010713:Phosphatidylcholines; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D012997:Solvents; C059983:potassium channel protein I(sk)