SPECTROSCOPIC AND FUNCTIONAL-CHARACTERIZATION OF THE PUTATIVE TRANSMEMBRANE SEGMENT OF THE MINK POTASSIUM CHANNEL

MinK (I(sk)) is a voltage-dependent K+ channel whose gene has been recently cloned and which consists of 130 amino acids [Takumi, T., Ohkubo, H., & Nakanishi, S. (1988) Science 242, 1042-1045]. The protein contains one putative transmembrane segment by hydropathy analysis. Whether this putative transmembrane segment is involved in the function of the protein was studied. A 32 amino acid peptide (residues 41-72) with the sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing the hypothesized transmembrane domain, designated TM-minK, was synthesized and fluorescently labeled. The alpha-helical content of TM-minK, assessed in methanol using circular dichroism (CD), was 57%. The fluorescent emission spectrum of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed a blue shift upon binding to small unilamellar vesicles (SUV), reflecting a relocation of the fluorescent probe to an environment of increased apolarity, i.e., within the lipid bilayer. The increase in NBD's fluorescence upon mixing NBD-labeled TM-minK with small unilamellar vesicles (SUV) was used to generate a binding isotherm, from which was derived a surface partition coefficient of 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled and rhodamine-labeled analogues suggest that TM-minK aggregates within membranes. In addition, single-channel experiments revealed that TM-minK can form single channels in planar lipid membranes only when a trans negative potential is applied. The findings herein experimentally support a role of the transmembrane segment of minK both in the assembly and as a constituent of the pore formed by the protein.