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

Biophys J. 2014 Mar 4;106(5):1070-8. doi: 10.1016/j.bpj.2014.01.027.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Ion Channel Gating
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism*
  • Phospholipids / metabolism
  • Potassium Channels / metabolism*
  • Spectrometry, Fluorescence / methods*


  • Lipid Bilayers
  • Phospholipids
  • Potassium Channels