Regulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay.

ABSTRACT: To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl(+)) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H(+)). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation c

SUBMITTER: Rusinova R 

PROVIDER: S-EPMC4026792 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature