Observation of Ligand-Induced Conformational Changes in Single ATP Receptors with Fast-Scanning Atomic Force Microscopy


Youichi Shinozaki1, Koji Sumitomo1, Makoto Tsuda2, Schuichi Koizumi3,
Kazuhide Inoue2, and Keiichi Torimitsu1
1Materials Science Laboratory, 2Kyushu University, 3Yamanashi University

 Proteins, known as "receptors" that exist in cells, exhibit their functions via binding with small compounds such as hormones. When the receptor is a type of ion channel, it opens its pore after binding with compounds thereby flowing ions through it and resulting in exhibiting its function. We observed the topology and stimulation-induced conformational changes in adenosine triphosphate (ATP) receptor [1], an important receptor in pain sensation, with atomic force microscopy (AFM).
 ATP receptor gene was over-expressed in immortalized cells. ATP receptor proteins were purified from their cell membranes and receptors for AFM observation were adsorbed on fleshly cleaved mica. Non-stimulated ATP receptor exhibited circular feature and that after ATP stimulation was in tripartite morphology (Fig. 1). To study whether structural difference between these two state is derived from the stimulation-induced conformational change in ATP receptor, we performed time-lapse imaging of conformational changes in ATP receptor with fast-scanning AFM. Before stimulus, ATP receptor exhibited circular feature (Fig. 2, -2.5〜0.0 s). After stimulus, ATP receptor immediately changed its structure into trimeric topology (Fig. 2, 0.5 s). Detailed analysis of trimeric ATP receptor revealed that it exhibited the further disengagement of three subunits and large pore-like structure in its center (Fig. 2, 2.0〜5.0 s). To confirm whether these structural changes are related to the physiological functions, we measured permeability of ATP receptor channel using fluorescent molecules. ATP receptor exhibited permeability to calcium ions and appeared to be functional. When imaging buffer contains no calcium, ATP receptor exhibited permeability to larger molecules (ethidium bromide) but not when imaging buffer contains calcium. These results indicate that structural changes in ATP receptor appeared to correspond to the physiological function via flowing ions through its pore. Thus, we succeeded in observation of topology and structural changes related to physiological functions of ATP receptor. We will reconstitute receptors into artificial lipid bilayer on a flat substrate [2] and analyze the relationship of receptor-lipid interaction and receptor topology/function.

[1] Y. Shinozaki et al., PLoS Biol. (accepted).
[2] Y. Shinozaki et al., Jpn. J. Appl. Phys. 47 (2008) 6164.

Fig..1. Schematics of ATP receptors before (left) and after (right) stimulation.
Fig..2. Time-lapse imaging of stimulation-induced conformational changes in ATP receptor.

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