William Rudolf Kobertz, PhD - US grants

The long-term goal of this research is to elucidate the molecular and cellular mechanisms that ensure potassium (K+) channels assemble with the appropriate membrane-embedded regulatory subunits for proper physiological function. N-glycosylation is a vital co- and post-translational modification that facilitates the assembly and trafficking of K+ channel subunits. Mutations that block glycosylation of KCNE K+ regulatory subunits have been directly linked to the genetic and drug-induced forms of cardiac arrhythmias (Long QT Syndrome). Accordingly, the two aims of this proposal investigate K+ channel subunit glycosylation in the ER, Golgi and plasma membrane: (1) We will determine the molecular and cellular bases of K+ channel subunit co- and post-translational N-glycosylation. (2) We will reengineer the cell's glycocalyx to fluorescently visualize K+ efflux from living cells.

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular and cellular mechanisms that ensure potassium channel protein-protein complexes properly assembly and maintain K+ homeostasis in the cochlear duct. The KCNQ1-KCNE1 K+ channel complex is the exclusive mechanism for endolymphatic K+ secretion into the cochlear duct. Genetic mutations in either KCNQ1 or KCNE that disrupt the assembly, trafficking or function of the complex give rise to Jervell and Lange-Nielsen Syndrome, a recessive form of congenital hearing loss accompanied with syncopal episodes. This application is directed at determining the basic mechanisms of KCNQ1-KCNE1 assembly and trafficking in the inner ear. There are three aims to this application: (1) we will identify the residues that line the protein-protein interface of the KCNQ1-KCNE1 complex utilizing a combination of biochemical and electrophysiological experiments;(2) we will determine the cellular mechanisms that ensure KCNE1 assembles with KCNQ1 by examining the cellular trafficking patterns of the proteins using enzymatic deglycosylation, membrane fractionation, cell surface labeling methods and immunofluorescence;(3) we will investigate a Jervell and Lange-Nielsen Syndrome (JLNS) mutation that disrupts assembly and trafficking of the complex via N-linked glycosylation. For this aim, we will examine the role of N-linked glycosylation in KCNE1 biogenesis, complex assembly and cellular trafficking. The results from these aims will provide a molecular and cellular framework, which will aid in the understanding of JLNS and other KCNQ1-KCNE-linked diseases.

DESCRIPTION (provided by applicant): DESCRIPTION (provided by applicant): Abstract: This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic, 06-GM-102: Chemist/Biologist collaborations facilitating tool development. The goal of this project is to develop a new set of molecular reagents that deliver chemical probes to specific ion channel complexes functioning in living cells. The development of this novel technology will enable the facile determination of the subunit composition and membrane localization of ion channel complexes in native cells. For this project, we will target the N-linked glycans on ion- conducting and regulatory subunits of two K+ channels found in cardiomyocytes: Kv11 K+ channels and KCNE type I transmembrane peptides. There are three aims of this project. In Aim 1, we will derivatize two peptide toxins such that when they bind to their cognate K+ channel complex they will covalently label the complex with either biotin or a fluorescent probe. The labeling efficiency and specificity of the derivatized peptide toxins will be demonstrated in electrical recordings, biochemical assays and fluorescence imaging experiments. Aims 2 and 3 will demonstrate the utility of the panel of derivatized toxins by identifying the native regulatory KCNE subunits co-assembled with cardiac K+ channels and determining the cell surface localization and turnover of K+ channels in living cardiomyocytes. Ion channels function as macromolecular protein complexes composed of membrane-embedded ion-conducting and regulatory subunits. Complex assembly is vital for proper cellular function, as mutations that prevent co-assembly give rise to neurological, cardiac, muscular and respiratory diseases. This project describes the development of a novel set of reagents to probe the structure, function and cellular localization of healthy and diseased ion channel complexes.

DESCRIPTION (provided by applicant): Calmodulin proteins are vital for both the assembly and regulation of auditory KCNQ channels. Although there are many informative high resolution structures of calmodulin bound to peptide fragments derived from ion channels, the location of calmodulin on functioning channels and the calcium-induced structural changes responsible for physiological function have remained elusive for all ion channels. This proposal outlines an innovative new approach that detects KCNQ-bound calmodulin and its distance from the ion conduction pathway. Using these experimentally determined distance restraints and the many high resolution structures of potassium channels and calmodulin bound to peptides, we will generate quaternary structures of the differently calcified KCNQ4- and KCNQ1/KCNE1-calmodulin complexes. These quaternary structures combined with the proposed functional studies will provide unprecedented molecular insight into calcium-regulated potassium recycling in the ear.

? DESCRIPTION (provided by applicant): The goal of this project is fluorescently visualize potassium (K+) egress and extracellular accumulation at the cell surface. The development of this innovative technology has the potential to enable the large-scale spatiotemporal resolution of neuronal and glial cell activity. For this project, we are exploiting the presence of the cell's glycocalyx to attach potassium-sensitive fluorophores exactly where K+ accumulates and is reabsorbed by glia. There is one aim to this project: To synthesize a novel, near-IR K+ sensor that covalently modifies the glycocalyces of living cells to fluorescently detect and measure K+ efflux and accumulation. The completion of this aim will yield a transformative set of chemical-biological tools and methodologies to investigate the physiology and pathophysiology of K+ activity in neurons, glia, and potentially in living animals.

Abstract: The goal of this Focused Technology and Development grant is to repurpose intracellular fluorescent ion and metabolite sensors to detect extracellular fluxes. The three orthogonal approaches directly target the cell?s glycocalyx: the nanometer thick, sugar?coating where ion and metabolite concentrations vary greatly during ion channel and membrane transporter activity. Aim 1 describes an approach to attach any fluorescent protein sensor to cells metabolically-labeled with an unnatural azidosugar. Aim 2 is a non-genetic approach that labels all mammalian cells?including human cells?with both small molecule and protein fluorescent sensors. Aim 3 is a chemical genetic approach to achieve cell-type-specific labeling with small molecule and protein fluorescent sensors. Completion of the aims will convert these working prototypes that utilize three major classes of fluorescent sensors (small molecule, protein, and FRET) into technologies that enable the visualization of ions and metabolites at the surface of a primary cells, tissues, and potentially in living animals.