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 in the heart, muscle, and nerve. In the previous funding period, we developed an innovative approach to label the t-tubules and sarcolemma of cardiomyocytes with either small molecule or protein-based K+-sensitive fluorophores. For this proposal, we will use our fluorescent bioconjugates to test the long standing hypothesis that K+ accumulates faster in the t-tubules to protect the heart during physical activity (Aim 1); to fluorescently visualize specific ion channel activity in order to identify the K+ channel/KCNE complexes responsible for maintaining cardiac rhythmicity (Aim 2). The completion of these aims will yield a transformative set of methodologies to directly investigate extracellular K+ accumulation from heart, muscle, and nerve cells isolated from healthy and disease animal models.
The transverse (t)-tubules are a labyrinth of various-sized (nanometers in diameter) invaginations in heart muscle cells that create an intricate network of extracellular ion reservoirs to maintain human heart contractility and rhythmicity. Ventricular heart muscle cells from patients and animal models with cardiomyopathies and ischemic heart failure all have severely disrupted t-tubule networks. Although a healthy human heart requires ventricular t-tubule networks, it has been impossible to experimentally measure potassium concentrations in these nanometer-sized extracellular domains. This proposal overcomes this barrier by developing an approach that enables the fluorescent visualization of potassium in the t-tubules and at the sarcolemma (the non-invaginated cell surface). A greater understanding of the t-tubule?s role in extracellular ion accumulation in healthy and diseased hearts will suggest more effective treatments for hypertrophic cardiomyopathy, dilated cardiomyopathy, ischemic heart failure, and cardiac arrhythmias.
|Kobertz, William R (2018) Oddballs in the Shaker family: Kv2-related regulatory subunits. J Gen Physiol 150:1599-1601|
|Bandara, H M Dhammika; Hua, Zhengmao; Zhang, Mei et al. (2017) Palladium-Mediated Synthesis of a Near-Infrared Fluorescent K+ Sensor. J Org Chem 82:8199-8205|
|Kubat Öktem, Elif; Mruk, Karen; Chang, Joshua et al. (2016) Mutant SOD1 protein increases Nav1.3 channel excitability. J Biol Phys 42:351-70|
|Zhang, Lejie; Bellve, Karl; Fogarty, Kevin et al. (2016) Fluorescent Visualization of Cellular Proton Fluxes. Cell Chem Biol 23:1449-1457|
|Mruk, Karen; Kobertz, William R (2015) Bioreactive Tethers. Adv Exp Med Biol 869:77-100|
|Malaby, Heidi L H; Kobertz, William R (2014) The middle X residue influences cotranslational N-glycosylation consensus site skipping. Biochemistry 53:4884-93|
|Kobertz, William R (2014) Stoichiometry of the cardiac IKs complex. Proc Natl Acad Sci U S A 111:5065-6|
|Aromolaran, Ademuyiwa S; Subramanyam, Prakash; Chang, Donald D et al. (2014) LQT1 mutations in KCNQ1 C-terminus assembly domain suppress IKs using different mechanisms. Cardiovasc Res 104:501-11|
|Mruk, Karen; Farley, Brian M; Ritacco, Alan W et al. (2014) Calmodulation meta-analysis: predicting calmodulin binding via canonical motif clustering. J Gen Physiol 144:105-14|
|Malaby, Heidi L H; Kobertz, William R (2013) Molecular determinants of co- and post-translational N-glycosylation of type I transmembrane peptides. Biochem J 453:427-34|
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