An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X2and α4β2Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy

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Abstract
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 α4β2nicotinic and P2X2channels 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 P2X2and α4β2nicotinic channels demonstrated close spatial arrangement of the channels in human embryonic kidney cells and in hippocampal neuron membranes. The data suggest that P2X2and α4β2channels may form a dimer, with the channels ∼80 Å apart. The measurements also show that P2X2subunits interact specifically and robustly with the β2subunits in α4β2channels. The data provide direct evidence for the close spatial apposition of full-length P2X2and α4β2channels within 100 nm of the plasma membrane of living cells.