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The Journal of Neuroscience, July 20, 2005, 25(29):6911-6920; doi:10.1523/JNEUROSCI.0561-05.2005
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Cellular/Molecular
An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2 and 4 2 Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy
Baljit S. Khakh,1
James A. Fisher,1
Raad Nashmi,2
David N. Bowser,1 and
Henry A. Lester2
1Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom, and 2Division of Biology, California Institute of Technology, Pasadena, California 91125
Structurally distinct nicotinic and P2X channels interact functionally, such that coactivation results in cross-inhibition of one or both channel types. It is hypothesized, but not yet proven, that nicotinic and P2X channels interact at the plasma membrane. Here, we show that plasma membrane 4 2 nicotinic and P2X2 channels form a molecular scale partnership and also influence each other when coactivated, resulting in nonadditive cross-inhibitory responses. Total internal reflection fluorescence and fluorescence resonance energy transfer microscopy between fluorescently labeled P2X2 and 4 2 nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2 and 4 2 channels may form a dimer, with the channels 80 Å apart. The measurements also show that P2X2 subunits interact specifically and robustly with the 2 subunits in 4 2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and 4 2 channels within 100 nm of the plasma membrane of living cells.
Key words: channel; cholinergic; purinergic; acetylcholine receptor; ACh; fluorescence microscopy; P2X
Received Feb 10, 2005;
revised June 13, 2005;
accepted June 14, 2005.
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