TARGETING RENAL OUTER MEDULLARY K+ CHANNEL ROMK FOR NEW CLASS OF DIURETICS

肾外髓K+通道（ROMK）的突变可引起肾纤维化

Mutations in the renal outer medullary K+ channel ROMK produce an antenatal variant of Bartter's syndrome manifested by hypokalemic alkalosis, polyuria and hypotension. We have shown that ROMK mutations reduce K+ flux and propose this as the pathogenesis for the disease. A corollary is that ROMK blockers should act as diuretics and our long-term goal is to develop such blockers. Our specific aims are: 1) characterize the ROMK mutations that produce Bartter's syndrome to identify important functional domains in the protein and to design mutation-specific therapy; 2) use phage display to generate peptide ligands which regulate ROMK channel function and which will serve as ROMK tags; and 3) determine the status of ROMK glycosylation in kidney using either antibodies or high affinity ligands that we will develop. Aim 3 derives from our finding that K+ currents in un-glycosylated ROMK are markedly reduced. The project will not only provide therapy for the ROMK variant of Bartter's but will also provide a new class of loop diuretics. Experiments are designed to test the effects of ROMK mutations on K+ currents and assembly, trafficking, phosphorylation and proteolysis of ROMK channels. We will map the functional changes to a topological model of ROMK that we have developed using glycosylation site insertion mutagenesis. To discover ROMK-specific peptide ligands we will screen phage display libraries by biopanning with cells expressing ROMK1. To provide another rationale for altering ROMK currents we will examine the glycosylation of ROMK in kidney cells using biochemical and immunocytochemical methods. The research methods include: recombinant DNA to engineer Bartter's mutant, expression of recombinant proteins in Spodoptera frugiperda (Sf9) cells; patch-clamp measurements of K+ currents; biochemical methods for analysis of protein; screening phage display libraries by biopanning; sequencing isolated clones; and immunocytochemistry for localization of ROMK protein in kidney and Sf9 cells.