Date on Master's Thesis/Doctoral Dissertation

5-2016

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Degree Program

Biochemistry and Molecular Biology, PhD

Committee Chair

Bhatnagar, Aruni

Committee Co-Chair (if applicable)

Gregg, Ronald

Committee Member

Gregg, Ronald

Committee Member

Conklin, Daniel

Committee Member

Clark, Barbara

Committee Member

Cheng, Alan

Author's Keywords

electrophysiology; action potential; potassium; cardiomyocyte; repolarization

Abstract

Myocardial voltage-gated potassium (Kv) channels regulate the resting membrane potential and the repolarization phase of the action potential. Members of the Kv1 and Kv4 family associate with ancillary subunits, such as the Kvβ proteins, that modify channel kinetics, gating and trafficking. Previous investigation into the function of cardiac β subunits demonstrated that Kvβ1 regulates Ito and IK,slow currents in the heart, but the role of Kvβ2 in the myocardium remains unknown. In heterologous expression systems, Kvβ2 increases surface expression of Kv1 channels, shifts the activation potential of Kv1 channels to more polarized voltages, and increases the inactivation of Kv1 channels. Accordingly, the electrophysiological phenotype in Kvβ2-/- mice was examined to uncover its role. To investigate the effects of the loss of Kvβ2 on cardiac repolarization, we performed whole-cell electrophysiology on primary cardiac myocytes. We found Kv current density was reduced and action potential duration prolonged in myocytes lacking Kvβ2. To isolate the molecular interactions by which Kvβ2 was affecting Kv currents, we show that Kvβ2 co-immunoprecipitates with Kv1.4 and Kv1.5 in heart lysates. To measure if surface expression of these Kv channels was reduced with the loss of Kvβ2, we performed immunofluorescent confocal microscopy of isolated cardiac myocytes. We found that the surface expression of Kv1.5 was reduced in Kvβ2-/- myocytes. We also performed a membrane fractionation technique to demonstrate that the proportion of total cellular Kv1.5 at the membrane was reduced in Kvβ2-/-. Together, these findings support our hypothesis that Kvβ2 plays a role in the generation of functional Kv currents in the myocardium by interacting with members of the Kv family. The pyridine nucleotides, NAD[P](H), are ubiquitous cofactors utilized as electron donors and acceptors by over 250 cellular oxidoreductases. Work out of our laboratory has shown that the Kvβ proteins are functional enzymes of the aldo-keto reductase family, that utilize NAD[P]H to catalyze the reduction of substrates. Furthermore, follow up work has shown that the redox status of bound pyridine nucleotide (PN) modifies the gating of Kvα-Kvβ channel complexes in heterologous expression systems. To examine a physiological role for PN in cardiac repolarization, whole-cell and single channel cardiac myocyte currents were recorded under the exposure to various PN redox states. We found that the inactivation rates and open probabilities of Kv currents in isolated myocytes are sensitive to the redox status of PN, and that surface action potentials in an isolated heart model are prolonged by treatment with factors that increase intracellular NADH concentration.

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