Direct observation of ATP-induced conformational changes in single P2X(4) receptors.
The ATP-gated P2X(4) receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X(4) receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X(4) receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X(4) receptors and a functional analysis revealed that P2X(4) receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X(4) receptors revealed two different forms of activated structures under 0 Ca(2+) conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X(4) receptors display pore dilation in the absence of Ca(2+). With Ca(2+), the P2X(4) receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X(4) receptors that correlate with pore dynamics.
NTT Basic Research Laboratories, NTT Corporation, Kanagawa, Japan. shinozak@will.brl.ntt.co.jp
Youichi
Y
Shinozaki
Koji
K
Sumitomo
Makoto
M
Tsuda
Schuichi
S
Koizumi
Kazuhide
K
Inoue
Keiichi
K
Torimitsu
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000538:Aluminum Silicates / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport; D018625:Microscopy, Atomic Force; D008958:Models, Molecular; D011107:Polylysine / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059008:Time-Lapse Imaging
D000538:Aluminum Silicates; D005456:Fluorescent Dyes; D055767:Immobilized Proteins; D058848:Receptors, Purinergic P2X4; D011994:Recombinant Proteins; D011107:Polylysine; D000255:Adenosine Triphosphate; D002118:Calcium; C011934:mica