(verbatim from application) This 5-year grant application. IRPG#1, is the first of three Component Grants comprising the overall IRPG (Interactive Research Project Grant) application. This application also contains the single CORE of resources .hat will be shared among the Component Grants. The major goals of IRPG#1 are 1) to define the functional-structural domains on the ROMK K+ channel that are involved in channel regulation (phosphorylation sites, nucleotide-binding domain and pore-forming domain), and 2) to determine using a ROMK knockout mouse model the functional roles of the ROMK channel in renal K+ handling. The hypothesis driving the first goal is that amino- and carboxy-terminal regions of the ROMK channel protein provide domains for regulation of channel activitv by both phosphorylation and nucleotide-binding interactions. We propose to examine nucleotide binding and phosphorylation at the protein level. This is now possible based on our recently established ability to produce and purify to homogeneity milligram amounts of ROMK protein. The first goal will provide crucial information necessary for our collaboration to obtain three-dimensional structural information on this K+ channel. The major hypothesis driving the second goal is that the ROMK channel is essential to K+ secretory processes in the thick ascending limb (TAL) and in principal cells of the cortical collecting duct (CCD). This hypothesis will be tested by using mice deficient for the ROMK gene, i.e., a ROMK """"""""knockout"""""""" mouse model. The key observation driving our choice of the ROMK """"""""knockout"""""""" is the demonstration that loss-of-function mutations of human ROMK produce neonatal Bartter's Syndrome, a renal NaCI wasting condition due to reduced NaC1 transport by the TAL. While the human ROMK """"""""knockout"""""""" demonstrates the importance of this K+ channel to TAL function, many unanswered questions remain concerning the pathophysiology of this disorder (e.g., mechanism for continued renal K+ loss and role of prostaglandin E2 in renal NaC1 and K+ wasting) and may best be addressed using an animal model.
Showing the most recent 10 out of 26 publications
