Spectroscopic and functional characterization of the putative transmembrane segment of the minK potassium channel.

摘要: MinK (Isk) 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 involved in the function was studied. A 32 acid peptide (residues 41-72) with sequence SKLEALYILMVLGFFGFFTLGIMLSYIRSKKL, containing hypothesized domain, designed TM-minK, synthesized fluorescently labeled. alpha-helical content assessed methanol using circular dichroism (CD), 57%. fluorescent emission spectrum 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled TM-minK displayed blue shift upon binding to small unilamellar vesicles (SUV), reflecting relocation probe an environment increased apolarity, i.e., within lipid bilayer. increase NBD's fluorescence mixing NBD-labeled (SUV) used generate isotherm, from derived surface partition coefficient 5.5 x 10(4) M-1. Fluorescence energy transfer measurements between carboxyfluoresceine-labeled rhodamine-labeled analogues suggest that aggregates membranes. In addition, single-channel experiments revealed can form single channels planar membranes only when trans negative potential applied. findings herein experimentally support role minK both assembly as constituent pore formed protein.