ABSTRACT
P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X(4) receptors was 5.6 nm. Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.
Subject(s)
Fluorescence Resonance Energy Transfer/methods , Microscopy, Electron/methods , Receptors, Purinergic P2/chemistry , Carrier Proteins/chemistry , Cell Line , Green Fluorescent Proteins/metabolism , Humans , Ions , Lectins/chemistry , Lipid Bilayers/chemistry , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X4 , Time FactorsABSTRACT
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 alpha4beta2 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 alpha4beta2 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 alpha4beta2 channels may form a dimer, with the channels approximately 80 A apart. The measurements also show that P2X2 subunits interact specifically and robustly with the beta2 subunits in alpha4beta2 channels. The data provide direct evidence for the close spatial apposition of full-length P2X2 and alpha4beta2 channels within 100 nm of the plasma membrane of living cells.
Subject(s)
Fluorescence Resonance Energy Transfer , Microscopy, Fluorescence , Neurons/physiology , Receptors, Nicotinic/physiology , Receptors, Purinergic P2/physiology , Adenosine Triphosphate/physiology , Animals , Cell Membrane/physiology , Cell Membrane/ultrastructure , Cells, Cultured , Electrophysiology , Fluorescence Resonance Energy Transfer/methods , Hippocampus/cytology , Ion Channel Gating/physiology , Kidney/cytology , Mesencephalon/cytology , Mice , Microscopy, Fluorescence/methods , Neurons/ultrastructure , Rats , Receptors, Nicotinic/genetics , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X2 , TransfectionABSTRACT
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.
