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The Journal of Neuroscience, November 17, 2004, 24(46):10475-10487; doi:10.1523/JNEUROSCI.3250-04.2004

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Cellular/Molecular
Time-Resolved Measurement of State-Specific P2X₂ Ion Channel Cytosolic Gating Motions

James A. Fisher, Gemma Girdler, and Baljit S. Khakh

Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

ATP-gated P2X₂ channels undergo permeability changes through a process that is incompletely understood. In the present study, we used fluorescence resonance energy transfer (FRET) and electrophysiology to measure cytosolic gating motions in P2X₂ channels as they enter a state with increased permeability. P2X₂ channels underwent permeability changes with a time course that was similar to decreases in FRET between cyan fluorescent protein and yellow fluorescent protein attached to the cytosolic domain of P2X₂ channels. Wild-type and mutant channels that did not undergo permeability changes also did not show evidence of cytosolic gating motions. Moreover, immobilizing the cytosolic domain by tethering it to the plasma membrane prevented the switch in permeability and impaired the cytosolic gating motions. Both of these phenotypes were restored when the immobilizing tether was cleaved. The data provide a time-resolved measure of state-specific gating motions and suggest how a cytosolic domain may control ion channel permeability.

Key words: channel; fluorescence; gating; permeability; purinergic; receptor; P2X; FRET; TIRF

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Received May 30, 2004; revised September 7, 2004; accepted September 7, 2004.

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