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, me diates a distinct, slowly activating, voltage-gated potassium current across certain mammalian cell membranes. Site-directed mutational stud ies have led to the proposal that the single transmembrane segment of IsK participates in the pore of the potassium channel [Takumi, T. (199 3) News Physiol. Sci. 8, 175-178]. We present functional and structura l studies of a short peptide (K27) with primary structure NH2-(1)KLEAL YI-LMVLGFFGFFTLGIMLSYI(27)R-COOH, corresponding to the transmembrane s egment of IsK (residues 42-68). When K27 was incorporated, at low conc entrations, into phosphatidylethanolamine, black-lipid membranes, sing le-channel activity was observed, with no strong ion selectivity. IR m easurements reveal the peptide has a predominantly helical conformatio n in the membrane. The atomic resolution structure of the helix has be en established by high-resolution H-1 NMR spectroscopy studies. These studies were carried out in a solvent comprising 86% v/v 1,1,1,3,3,3-h exafluoro-isopropanol-14% v/v water, in which the IR spectrum of the p eptide was found to be very similar to that observed in the bilayer. T he NMR studies have established that residues 1-3 are disordered, whil e residues 4-27 have an alpha-helical conformation, the helix being lo oser near the termini and more stable in the central region of the mol ecule. The length (2.6 nm) of the hydrophobic segment of the helix, re sidues 7-23, matches the span of the hydrocarbon chains (2.3 +/- 0.25 nm) of fully hydrated bilayers of phosphatidylcholine lipid mixture fr om egg yolk. The side chains on the helix surface are predominantly hy drophobic, 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 o f phenylalanine aromatic rings of adjacent helices and favorable inter actions of the opposing aliphatic-like side chains, such as leucine an d methionine, with the lipid chains of the bilayer. This mechanism is in keeping with site-directed mutational studies which suggest that th e 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.