Molecular Cloning of Epithelial K Channels

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ABSTRACT 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, allows us to design new experiments that should hopefully 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 5 aspects of conformational change during gating. (1) Is the pH sensor formed by C-terminal salt bridging? (2) How is the ligand (pH) signal transmitted from the C-terminus to the transmembrane helices and to the principal gate at the inner helix bundle crossing? (3) Is gating produced by bending all along the inner (TM2) helix or only at 2 conserved glycines? (4) 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? (5) We also propose to directly measure conformational changes during gating, using lanthanide resonance energy transfer (LRET) methods. This would specifically address two hypotheses. Do the Kir C-termini move toward each other during opening of the bundle-crossing gate? Do the slide, outer and inner helices rotate relative to each other during gating? We plan to use a variety of techniques to answer these questions: (1) computer based molecular modeling, (2) lanthanide resonance energy transfer to measure changes in molecular distance between labelled residues during channel opening and closure, and (3) site-directed mutagenesis to determine the locus of putative salt-bridge sensors in the cytoplasmic domain. 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.