Project Summary The focus of this proposal is to investigate the mechanism by which the conserved molecular chaperone, GRP170/Lhs1, regulates the degradation, assembly, and trafficking of the epithelial sodium channel, ENaC. ENaC is responsible for salt reabsorption across epithelia of the kidney and lung, and controls both blood pressure and ion and fluid homeostasis. Gain- and loss-of-function mutations in ENaC lead to disease, and ENaC activity is also associated with other diseases associated with epithelial malfunction. ENaC is a heterotrimeric channel composed of an α, β, and γ subunit. Each subunit contains two transmembrane domains, a large extracellular loop, and short cytosolic N- and C-termini. Soon after synthesis, ENaC is subject to Endoplasmic Reticulum Associated Degradation (ERAD), which targets misfolded proteins and orphaned subunits of multimeric complexes for destruction by the cytosolic 26S proteasome. Not surprisingly, ENaC subunits individually are targeted for ERAD, but a significant percent of ENaC is degraded even when all three ENaC subunits are present. How sufficient subunit assembly occurs in order to escape ERAD is mysterious. However, data from this team of investigators uncovered a new role for the Lhs1 chaperone (GRP170 in mammalian cells) during ENaC biogenesis. Specifically, Lhs1 facilitated the degradation of the α subunit but had no effect on β or γ subunit degradation, yet when all three ENaC subunits were expressed, intersubunit interactions between the transmembrane domains blocked Lhs1-dependent ERAD. Consistent with these data, GRP170 also targeted the α subunit for ERAD in mammalian cells but promoted trafficking of the assembled heterotrimeric channel. Three model systems will be used to further understand these events: 1) An established, genetically facile yeast system will be used to define the structural elements required to differentiate between an orphaned ENaC subunit and the assembled heterotrimeric channel; 2) A Fischer rat thyroid (FRT) cell system will be used to confirm results from the yeast system and define amino acid motifs required for GRP170-mediated channel assembly and trafficking; 3) A conditional GRP170 knock out mouse, which lacks GRP170 in kidney tubules, will be used to determine how ENaC regulation by the GRP170 chaperone affects renal physiology. Overall, this proposal will use a multi-system approach to define how a single molecular chaperone regulates ENaC and—for the first time—indicate how chaperones can select an orphaned subunit for degradation as well as facilitate assembly of an oligomeric protein. Together, understanding the mechanism of action of GRP170 will provide novel insights into ENaC function and associated disease states. More generally, this work will help decipher how membrane assembly of a multimeric protein in the ER results in stabilization and trafficking, which is vital for the function of numerous other ion transporters in the kidney. The experiments ...