The Kawate Lab

Research

Overview

Integral membrane proteins (IMPs) constitute about 25% of the animal proteome and orchestrate essential life processes like body formation and brain function. While many IMPs emerge as potential therapeutic targets for devastating diseases such as cancer and chronic pain, drug development has not been able to meet the desperate need. Major impediments in finding novel drugs targeting IMPs stem from the lack of mechanistic understanding of how those important proteins function. Importantly, the lack of structural information has severely hampered rational drug design.

Our research team aims to unveil structures of eukaryotic IMPs and elucidate the mechanisms underlying their function, modulation, and regulation. We are particularly interested in IMPs that regulate extracellular ATP mediated signaling, an important mode of cell-cell communication that mediates immune responses and neurotransmission. We use multidisciplinary techniques including cryo-EM, X-ray crystallography, electrophysiology, and functional reconstitution in a lipidic environment.

Crystal structures of the P2X7 receptor

The P2X7 receptor is a non-selective cation channel activated by extracellular ATP. Chronic activation of P2X7 underlies many health problems such as pathologic pain, yet we lack effective antagonists due to poorly understood mechanisms of inhibition. In this study, we obtained the first crystal structures of a mammalian P2X7 receptor in the presence of five structurally different drugs. We discovered a new drug-binding pocket and a unique turret-like structure in the P2X7 receptor, both of which seem to narrow after ATP-binding. We propose that these unique structural rearrangements account for the subtype-specific action of the P2X7 drugs.

Functional reconstitution of P2X7

Since the early 70's, a number of labs have reported that activation of the P2X7 receptor leads to the opening of a peculiar dye-permeable pore. While the physiological significance of such a pore remains unclear, it was controversial whether P2X7 itself forms a pore or recruits other large-conductance channels. In either case, the consensus of the field was that its unique C-terminal domain (CTD) is absolutely necessary for the formation of the pore. In this study, we performed the first functional reconstitution of the P2X7 receptor using liposomes composed of various synthetic lipids. We demonstrated that 1) P2X7 alone forms a dye-permeable large pore in the absence of other cellular components, 2) its activation heavily depends on lipid compositions, and 3) the large pore forms immediately after stimulation, which disagrees with the pore dilation mechanism. To our surprise, we discovered that the CTD is not necessary for the large pore per se, but for counteracting the inhibitory action of cholesterol in the membrane.