An angstrom scale interaction between plasma membrane ATP-gated P2X2 and alpha4beta2 nicotinic channels measured with fluorescence resonance energy transfer and total internal reflection fluorescence microscopy.
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.
Khakh
Baljit S
BS
Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom. bsk@mrc-lmb.cam.ac.uk
Fisher
James A
JA
Nashmi
Raad
R
Bowser
David N
DN
Lester
Henry A
HA
D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002478:Cells, Cultured; D004594:Electrophysiology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006624:Hippocampus / Q000166:cytology; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000502:physiology* / Q000648:ultrastructure; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000502:physiology*; D058476:Receptors, Purinergic P2X2; D014162:Transfection
C546401:P2RX2 protein, human; C546402:P2rx2 protein, mouse; D011978:Receptors, Nicotinic; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; C403531:nicotinic receptor alpha4beta2; D000255:Adenosine Triphosphate