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Multiple Mechanisms Underlie Accessory Subunit-Mediated Regulation of Functional Kv4-Encoded Cardiac Fast Transient Outward (Ito,f) K+ Channels
Date of Award
Doctor of Philosophy (PhD)
The fast transient K+ current (Ito,f), which determines the rapid phase of action potential repolarization in ventricular myocytes, contributes importantly to the propagation of activity through the myocardium and to the generation of normal cardiac rhythms. Down-regulation of Ito,f is evident in the hypertrophied and failing heart and is associated with increased risk of ventricular arrhythmias and sudden death. Accumulating evidence suggests that native Ito,f channels function in macromolecular protein complexes, comprising Kv4 pore–forming (α) subunits together with cytosolic and transmembrane accessory subunits, including K+ Channel Interacting Protein 2 (KChIP2) and Dipeptidyl Peptidase 6 (DPP6), although the molecular mechanisms underlying accessory subunit–mediated effects on native Ito,f channels are poorly understood. Experiments here revealed that the targeted deletion of KChIP2 results in the elimination of Kv4.2-encoded Ito,f in (KChIP2-/-) mouse ventricular myocytes. In addition, although Kcnd2 (which encodes Kv4.2) transcript expression is not decreased, Kv4.2 protein is undetectable in KChIP2-/- ventricles, suggesting that KChIP2–mediated stabilization of Kv4 α is an important determinant of Ito,f channel expression.
Biochemical and electrophysiological experiments revealed that heterologous co–expression of Kv4.2 with KChIP2 increases Kv4.2 current densities and total Kv4.2 protein expression approximately 2–3 fold; KChIP2 protein is also increased ∼5 fold. Cell surface Kv4.2 protein expression, however, is increased ∼40 fold in the presence of KChIP2. Truncation of the distal Kv4.2 N–terminus results in the loss of Kv4.2/KChIP2 complex formation, reciprocal Kv4.2–KChIP2 protein stabilization and KChIP2–mediated increases in Kv4.2 current densities. KChIP2 co–expression also decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling.
Co–expression with DPP6, in contrast, selectively increases cell surface Kv4.2 protein expression (∼10 fold) and slows Kv4.2 degradation. In addition, and in contrast with KChIP2, DPP6 is not reciprocally stabilized by Kv4.2 co–expression and truncation of the Kv4.2 N–terminus does not abolish DPP6-mediated increases in cell surface Kv4.2 protein or Kv4.2 current densities. Expression of Kv4.2 with both KChIP and DPP subunits results in massive (∼900 fold) increases in cell surface Kv4.2 protein expression suggesting that KChIP2 and DPP6 function independently to increase Ito,f densities through stabilization and retention of channel complexes at the cell surface.
Chair and Committee
Jeanne M Nerbonne
Kendall J Blumer, Michael Crowder, Phyllis I Hanson, Douglas L Mann, Colin G Nichols
Foeger, Nicholas Christian, "Multiple Mechanisms Underlie Accessory Subunit-Mediated Regulation of Functional Kv4-Encoded Cardiac Fast Transient Outward (Ito,f) K+ Channels" (2013). Arts & Sciences Electronic Theses and Dissertations. 83.