The long-term goal is to determine the topology of a kidney potassium channel critical to body floud homeostasis and relate the topology to ion conduction and modulation.
The specific aims are to: 1)examine the validity of an alternative topological model that I have proposed to ROMK1; 2) investigate my model's implications for ion conduction; 3) determine whether the topology of IRK1 is similar to ROMK1. The health-relatedness of the project derives from ROMK1 being critical to potassium secretion in the kidney. A hereditary hypokalemia due to a mutant renal NA/K/Cl co-transporter has already been reported. ROMK1 may likewish be implicated in hereditary renal disease. The experimental design is to map the extracellular segments by N-glycosylaton substitution mutagenesis and epitope mapping, and to verify the predicted intracellular segments by protease digestion for ROMK1, as well as IRK1. Functional studies will test whether mutations or modifications of the Asn residues with or without carbohydrate alter the biophysical properties. The research methods combine: recombinant DNA to engineer N-glycosylation substitution mutants; expression of recombinant proteins in Spodoptera frugiperda (Sf9) cells and Xenopus oocytes; biochemical methods for ascertaining glycosylation; and patch clamp measurements of the hererologously expressed currents.
