Develop enabling biochemical and structural tools for dissecting the roles of PKD2L2 in metabolism

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Project Summary The polycystin family falls into two classes of integral membrane proteins: 1) the PKD1-type clade comprises five 11-transmembrane (TM) spanning receptor-like proteins (PKD1, PKD1L1, PKD1L2, PKD1L3, and PKD1REJ) characterized by a large N-terminal ectodomain that likely recognizes as-yet unknown ligand(s); and 2) the PKD2-type clade consists of three 6-TM spanning transient receptor potential (TRP) ion channels (PKD2, PKD2L1, and PKD2L2). PKD1 and PKD2 are the two most extensively studied polycystin proteins largely because inactivating mutations in either PKD1 or PKD2 cause a common, life-threatening, multisystem, and incurable autosomal dominant polycystic kidney disease (ADPKD). We and others showed that PKD2 itself functions as a non-selective, domain swapped cation channel. PKD2 additionally associates with PKD1 to form a heteromeric receptor/ion channel complex at sensory cilia of renal epithelia where they may respond to as-yet unknown chemical ligand(s) and/or mechanical force, contributing to the establishment and maintenance of the exquisite tubular architecture of nephrons in the kidneys. The physiological functions and disease relevance of other polycystin family members are poorly understood, although they also assemble into various complexes that possibly serve as cellular sensors for detecting and responding to a diverse range of physiological and environmental stimuli. PKD2L2 may participate in male reproduction as it is expressed in the testis and contributes to Ca2+ signaling in sperms. The PKD2L2-/- mice generated by the Illuminating the Druggable Genome (IDG) consortium exhibit altered glucose tolerance, which is an exciting discovery that suggests that PKD2L2 may function as a Ca2+ channel that regulates Ca2+ signaling and insulin secretion in the pancreas. Built on our successful biochemical, structural, and functional studies on PKD1 and PKD2 and a recent breakthrough in determining a 3.0 Å PKD2L2 cryo-EM structure, here we will continue to develop enabling biochemical reagents and structural models to lower the barriers to entry for other researchers who share the same enthusiasm for the understudies PKD2L2 channel. Specifically, we will leverage our expertise in membrane protein biochemistry and structural biology to: 1) determine additional cryo-EM structures for PKD2L2 that capture the channel in new functional states along its gating cycle; and 2) develop specific and sensitive antibodies and nanobodies for PKD2L2 that can be used to localize the channel in native tissues and cells, which will provide insights into PKD2L2 functions by illuminating the cell types and tissues where the channel operates.