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 P2X 2 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 P2X 2 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 P2X 2 and ␣ 4 ␤ 2 channels may form a dimer, with the channels ϳ80 Å apart. The measurements also show that P2X 2 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 P2X 2 and ␣ 4 ␤ 2 channels within 100 nm of the plasma membrane of living cells.

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ATP-gated P2X 2 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 P2X 2 channels as they enter a state with increased permeability. P2X 2 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 P2X 2 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.

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