Time-resolved measurement of state-specific P2X2 ion channel cytosolic gating motions.
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.
Fisher
James A
JA
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.
Girdler
Gemma
G
Khakh
Baljit S
BS
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000235:genetics / Q000502:physiology*; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D021122:Protein Subunits / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058476:Receptors, Purinergic P2X2
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D005456:Fluorescent Dyes; D007473:Ion Channels; D008164:Luminescent Proteins; C546401:P2RX2 protein, human; D021122:Protein Subunits; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins