Dynamic structural rearrangements of ion channels and receptors dependent on expression density.

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Our aim of research is to elucidate the mechanisms of the environment dependent changes of ion channels and receptor function and to know their dynamic structural rearrangements. During the period of this grant, we have conducted four specific researches in the following. [1]We, for the first time, observed that the pore properties and the ligand sensitivity of ATP receptor channel P2X2 change depending on "the open channel density on the membrane" probably by mutual interaction between them. We also uncovered by systematic mutagenesis study that Ile328 in the second transmembrane region is critical for the expression density dependent changes. [2]We purified recombinant protein of P2X2 by baculo virus - Sf9 cell expression system, and carried out stoichiometry analyses by anchoring the recombinant proteins with glutaraldehyde. We observed that P2X2 is composed of three subunits. We also performed single particle structure analysis using the recombinant proteins, and confirmed that P2X2 is a trimeric protein which shows an inverted pyramid like structure. [3]As a possible mechanism of the density dependent changes of P2X2, we analyzed phospholipids dependency. We observed that the P2X2 function depends not on PIP2 but on PIP3, and that the dilated pore state is directly linked to the desensitized state. [4]We analyzed the structural rearrangements of the metabotropic glutamate receptor by FRET technique, and observed that the relative allocation of the dimeric subunits changes upon glutamate application. We also observed that the type of coupling G proteins of mGluR1 changes depending on the type of ligand, showing that mGluR1 is not a simple on-/off- switch but a multiple path regulator.