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Updated: May 11, 2026
Proteomics to Identify Proteins Interacting with P2X2 Ligand-Gated Cation Channels
Published on: May 19, 2009
Direct observation of ATP-induced conformational changes in single P2X(4) receptors
Youichi Shinozaki1, Koji Sumitomo, Makoto Tsuda
1NTT Basic Research Laboratories, NTT Corporation, Kanagawa, Japan. shinozak@will.brl.ntt.co.jp
Abstract:
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.
Insights
The P2X(4) receptor
Area of Science:
- Molecular biology
- Biophysics
- Cell biology
Background:
- The ATP-gated P2X(4) receptor is a cation channel implicated in numerous pathophysiological processes.
- The precise architecture and ATP-induced structural dynamics of the P2X(4) receptor remain incompletely understood.
Purpose of the Study:
- To investigate the architecture and ATP-induced structural modifications of P2X(4) receptors.
- To elucidate the functional consequences of these structural changes on ion channel activity.
Main Methods:
- Utilized fast-scanning atomic force microscopy (AFM) to image P2X(4) receptors in both membrane-dissociated and membrane-inserted states.
- Performed time-lapse imaging to capture dynamic structural changes upon ATP binding.
- Conducted dye uptake assays to assess ion channel pore function under varying calcium (Ca2+) conditions.
Main Results:
- AFM revealed an upward orientation of P2X(4) receptors, leaning to one side.
- Two distinct activated structures were observed under Ca2+-free conditions: a trimer and a pore dilation-like tripartite structure.
- ATP-activated P2X(4) receptors exhibited pore dilation in the absence of Ca2+, confirmed by dye uptake.
- In the presence of Ca2+, P2X(4) receptors formed a disengaged trimer with no observed dye uptake.
Conclusions:
- The study provides novel insights into the structural rearrangements of P2X(4) receptors upon ATP binding.
- Observed structural changes, particularly pore dilation, directly correlate with the receptor's ion transport dynamics.
- Calcium ions play a critical role in modulating P2X(4) receptor conformation and function.
