TRPC3の構造解析:電顕像の単粒子解析法の進歩

抄録

TRPC3 plays important roles in neuronal differentiation and immune cell maturation by mediating the cationic current in response to phospholipase C activation, Ca²⁺ depletion, and diacylglycerol stimulation. In collaboration with Dr, Yasuo Mori (Kyoto Univ.), we purified the TRPC3 channel as a glycosylated tetramer and observed the structure using electron microscopy for single particle analysis¹⁾. Negatively stained specimens demonstrate homogeneous protein particles containing an internal cavity-like structure. These particle images were selected by automated pick-up programs²⁾, aligned, and classified by the growing neural gas network method³⁾. Similarly oriented projections were averaged to decrease the signal-to-noise ratio. The averaged images progress from the top view to the side views, which are representative of their raw images. The top view confirmed the hypothesis of a four-domain structure, and the side view demonstrates a large cytoplasmic domain with a capped structure at the bottom, which is near a predicted locus of ion release. The total image of the protein is a blunt-edged trapezoid: both width and height of the molecule are over 200 angstrom. This large dimension of TRPC3 is also supported by the Stokes radius (92 angstrom) obtained from gel filtration chromatography. 1) Mio, K., Ogura, T., Hara, Y., Mori, Y. & Sato, C. The non-selective cation-permeable channel TRPC3 is a tetrahedron with a cap on the large cytoplasmic end. Biochem. Biophys. Res. Commun. 333, 768-777 (2005). 2) Ogura, T. & Sato, C. J Struct Biol 146, 344-58 (2004). 3) Ogura, T., Iwasaki, K. & Sato, C. J Struct Biol 143, 185-200 (2003). [J Physiol Sci. 2006;56 Suppl:S11]