An Angstrom Scale Interaction between Plasma Membrane ATP-Gated P2X₂ and α₄β₂ Nicotinic Channels Measured with Fluorescence Resonance Energy Transfer and Total Internal Reflection Fluorescence Microscopy

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 α₄β₂ 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 α₄β₂ 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 α₄β₂ channels may form a dimer, with the channels ∼80 Å apart. The measurements also show that P2X₂ subunits interact specifically and robustly with the β₂ subunits in α₄β₂ channels. The data provide direct evidence for the close spatial apposition of full-length P2X₂ and α₄β₂ channels within 100 nm of the plasma membrane of living cells.

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