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

TitleRegulation of ion channel function by the host lipid bilayer examined by a stopped-flow spectrofluorometric assay.
Publication TypeJournal Article
Year of Publication2014
AuthorsRusinova R, Kim DM, Nimigean CM, Andersen OS
JournalBiophys J
Date Published2014 Mar 4
KeywordsIon Channel Gating, Lipid Bilayers, Phospholipids, Potassium Channels, Spectrometry, Fluorescence

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 curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H(+) gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H(+) affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels' lipid bilayer environment or other regulatory processes.

Alternate JournalBiophys. J.
PubMed ID24606931
PubMed Central IDPMC4026792
Grant ListGM021342 / GM / NIGMS NIH HHS / United States
GM088352 / GM / NIGMS NIH HHS / United States
R01 GM021342 / GM / NIGMS NIH HHS / United States