Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.
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.
Young
Mark T
MT
Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom.
Fisher
James A
JA
Fountain
Samuel J
SJ
Ford
Robert C
RC
North
R Alan
RA
Khakh
Baljit S
BS
D002352:Carrier Proteins / Q000737:chemistry; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007477:Ions; D037102:Lectins / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008854:Microscopy, Electron / Q000379:methods*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018048:Receptors, Purinergic P2 / Q000737:chemistry* / Q000378:metabolism; D058848:Receptors, Purinergic P2X4; D013997:Time Factors
D002352:Carrier Proteins; C485182:Cyan Fluorescent Protein; D007477:Ions; D037102:Lectins; D008051:Lipid Bilayers; C548226:P2RX4 protein, human; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D049452:Green Fluorescent Proteins