Potassium channels are tetrameric membrane proteins that provide a highly selective conduit for potassium ions to diffuse across the hydrophobic barrier of cell membranes. As such, their formation and biophysical properties are critical for processes like neuronal excitability, secretion of hormones, and muscle contraction. The formation of ion channels includes biogenesis of monomeric channel subunits, assembly of subunits into the tetrameric channel, and trafficking of the channel to the appropriate cellular membrane where it performs its functional role. Although the structure and function of mature potassium channels have been studied extensively, little is known about the early folding events in channel biogenesis. Defects in translation and folding have downstream consequences for assembly, trafficking, and function of potassium channels, and underlie pathology. The long-term goal of our research program is to elucidate basic principles of translation and protein folding in the biogenesis of voltage-gated potassium (Kv) channels, including folding events in the ribosome-nascent peptide complex. This proposal is divided into four interrelated Projects, each having several Aims. Project I is devoted to understanding how the T1 domain, critical for assembly and targeting of Kv channels, moves through the tunnel and is persuaded to fold during biogenesis. No models of this progression exist. The expected outcomes of our studies will fill this gap. Project II maps the events and location of voltage- sensor (VS) formation. Thes findings bear on all voltage-gated channels and mechanisms for VS folding defects. Project III defines prerequisites for pore formation and the defects in pore architecture that underlie the impaired trafficking responsible for channel diseases, e.g., Long QT2 Syndrome. Project IV will generate new paradigms for allosteric communication in the ribosome-nascent peptide complex, challenge existing paradigms that refute the important role of peptide-tunnel dynamics. Our results will advance a new technology for determining rates of peptide movement in the tunnel during peptide elongation. The results of our studies bear not only on Kv channel formation and cellular levels of Kv protein, but also on broader issues in biogenesis and folding of proteins, and will provide a paradigm for rational design of therapeutics for Kv folding defects. Many of the methods and strategies used in these proposed studies (e.g., pegylation, intramolecular crosslinking assays of a nascent peptide attached to a ribosomal tunnel) were introduced and developed in my laboratory to assay secondary, tertiary, and quaternary folding events. The proposed experiments will use a range of techniques including novel biochemical micro-assays of Kv biogenic intermediates, electrophysiology of Xenopus oocytes, and computational analysis.
Formation of voltage-gated potassium channels includes synthesis of the channel protein, assembly of these proteins into multimeric channels, and delivery of these channels to the plasma membrane. Defects in any of these steps will result in altered expression of the channels at the cell surface, with pathological and often lethal consequences. Our studies of ion channel folding bear on basic principles of protein folding and pave the way for understanding the molecular basis of protein misfolding, a fundamental cause of many diseases.
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