The proposed project continues the original specific goal of understanding potassium (K) permeation and gating through the renal, inward rectifying, K channel: ROMK (Kir1.1). However, the project now encompasses a more general theme of understanding the structural mechanics of gating (opening &closing) in the inward rectifier K channel family (Kir). Recent crystallographic data on the closed and partially open structures of the bacterial channels: KirBac1.1 and KirBac3.1 have allowed us to construct detailed homology models of ROMK in the closed state and partial open-state. The ROMK channel is uniquely suited for combined structure and function studies to elucidate gating in a mammalian channel because we already have a large collection of physiological data on both ligand (pH) gating and permeant-ion gating in ROMK. This, together with our homology modeling, should allow us to clarify the molecular processes of ROMK gating as well as provide new insight into the gating dynamics of other inward rectifier channels. Our experiments would address 4 aspects of conformational change during gating. (1) Is the pH sensor formed by C-terminal salt bridging? (2) Do the 2 conserved Gly in the inner TM2 helix function as hinge points or are they more important for helix packing? (3) Are there other gates in the permeation path besides the principal ligand gate at the bundle crossing? If so, how are these two gates linked together at a structural level? Is external K gating of ROMK dependent on the molecular structure of the pH gate at the bundle crossing? Do changes in selectivity filter conformation constitute a second (C-type inactivation) gate in series with the bundle-crossing gate? (4) We also propose to directly measure conformational changes during gating, using lanthanide resonance energy transfer (LRET) methods. This technique, which involves genetically encoding donor and acceptor tags into the channel, would permit us to look at state-dependent changes in distance and address several important structural questions about ROMK gating: (a) Do the Kir C-termini move toward each other during opening of the bundle-crossing gate? (b) What are the helix motions at the level of the bundle crossing gate during ROMK (Kir1.1) pH gating? (c) What is the angular motion of the slide helix during ROMK (Kir1.1) pH gating? These experiments would be done in collaboration with Prof. Francisco Bezanilla, nearby at the Univ. of Chicago, using a novel technique for isolating large segments of (inside-up) oocyte membrane suitable for the proposed LRET experiments. Results of this project would do much to further our understanding of the renal ROMK potassium channel that is essential for K balance in the human kidney. This would not only help patients with the antenatal variant of Bartter's disease, caused by a congenital defect in ROMK, but would also pave the way for a molecular characterization of gating in other inward rectifier potassium channels. These channels play essential roles in heart cells, pancreatic beta cells (diabetes and hypoglycemia), disorders of acid-base balance, modulation of neuronal activity as well as potassium buffering in brain glial cells. A thorough characterization of their gating is essential for understanding the molecular basis of a variety of channelopathies.
The proposed project would characterize the underlying molecular basis for ion channel gating (opening &closing) in the renal inward rectifier (ROMK) family of potassium (K) channels. Results of this study would not only be relevant for renal diseases like Bartter's syndrome but would also have profound implications for G-protein regulated inward rectifier channels in the heart and nervous system, as well as K channels in pancreatic beta cells that are implicated in diabetes and hypoglycemia. This could ultimately be used for targeted drug design to correct a variety of congenital ion channelopathies affecting the kidney, heart, and pancreas.
Showing the most recent 10 out of 24 publications
