Structure Function Studies on the Mechanisms of P2X3 Receptor Desensitization

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DESCRIPTION (provided by applicant): Purinergic receptors (P2X) are proteins that are activated by the binding of ATP and adenosine. Because they are actively expressed in the cardiovascular, nervous, and immune systems, the structural mechanisms by which these receptors function are important to understand how they modulate processes such as smooth muscle contraction, platelet activation, synaptic transmission and inflammation. As such, they have become a key potential pharmacologic target for cardiovascular, neuronal and inflammatory diseases. Despite being an actively pursued pharmacologic target, only two high-resolution structures exist for any of the purinergic receptor subtypes and neither is on a human homolog. Structures of an apo-closed state as well as an ATP-bound state of zebra fish P2X4 receptor have provided some structural insight into receptor activation; however, the mechanisms surrounding desensitization (rapid vs. slow) remain unanswered. Because P2X3 receptor displays rapid desensitization kinetics, as opposed to P2X4, which displays slow desensitization kinetics, structural differences between the two receptors will give insight into mechanisms of desensitization. This proposal outlines the use of proven strategies to determine a high-resolution structure of human P2X3 receptor in the desensitized state using X-ray crystallography. First, I will identify a "minimal functional construct" of human P2X3 which displays activation and desensitization properties identical to wild type yet is optimal for expression and purification in a mammalian expression system. Through this process, I will identify key residues in the N- and C-terminal domains of human P2X3 that are important for activation and desensitization. Crystallization trials will not be limited to classical methods usig detergent/protein micelles but will also include new techniques that allow crystallization in a lipid-rich environment, such as bicelle and lipidic cubic phase crystallization. To increase the likelihood of obtaining a structure of the desensitized state, co-crystallization will be performed in the presence of two toxins that are known to stabilize the desensitized state as well as with Fab fragments from conformation-specific antibodies. The results of this work will substantially contribute to the understanding of the mechanisms for purinergic receptor activation and subsequent desensitization.