Modulation of Kv1.5 Currents by Protein Kinase A, Tyrosine Kinase, and Protein Tyrosine Phosphatase Requires an Intact Cytoskeleton

Abstract

The regulation of cardiac delayed rectifier potassium (Kv) currents by cAMP-dependent protein kinase (PKA) contributes to the control of blood pressure and heart rate. We investigated the modulation by PKA and protein phosphatases of cloned Kv1.5 channels expressed in Xenopus laevis oocytes. Exposure of oocytes to activators of PKA (100 nM forskolin, 1 mM 8-bromo-cAMP, or 1 mM 3-isobutyl-1-methylxanthine) had no effect on the amplitude of Kv1.5 currents. Inhibition of PKA by injection of protein kinase A inhibitor peptide or exposure to myristoylated protein kinase A inhibitor peptide (M-PKI; 100 nM) reduced currents mediated by Kv1.5. M-PKI also reduced the amplitude of currents mediated by mutated Kv1.5 channels in which the COOH terminal PKA phosphorylation sites and PSD-95, Disc-large, and ZO-1-binding domain were removed. The reduction of Kv1.5 currents by M-PKI was attenuated by inhibition of actin polymerization by 1 M cytochalasins B and D, but was not affected by 10 M phalloidin (stabilizes actin filaments) or 50 M colchicine (disrupts microtubules). Treatment of oocytes with antisense oligonucleotides against ␣-actinin-2 abolished the reduction in Kv1.5 current by M-PKI. These observations suggest that Kv1.5 currents are activated by endogenous PKA in "resting" oocytes and that inhibition of PKA activity reveals the action of endogenous phosphatases. Indeed, injection of alkaline phosphatase reduced currents mediated by Kv1.5. Further preincubation of oocytes with 1 mM sodium orthovanadate (a protein tyrosine phosphatase inhibitor) abolished the reduction in Kv1.5 currents by M-PKI. We conclude that currents encoded by Kv1.5 are regulated by PKA and protein tyrosine phosphatase and that this regulation requires an intact actin cytoskeleton and ␣-actinin-2.

Redox modulation of basal and β‐adrenergically stimulated cardiac L‐type Ca2+ channel activity by phenylarsine oxide

“…It has been established that this type of regulation involves the production of cAMP and subsequent phosphorylation by protein kinase A (PKA). Tyrosine kinases have also been reported to modulate the activity of cardiac ion channels (Shuba & McDonald, 1997;Zhou et al, 1997;Wang & Lipsius, 1998;Hool et al, 1998;Maier et al, 1999;Mason et al, 2002;Tiran et al, 2003). We previously demonstrated that genistein, a tyrosine kinase inhibitor, can increase the sensitivity of cardiac ion channels to b 1 -AR stimulation, suggesting that basal tyrosine kinase activity inhibits badrenergic responsiveness in cardiac myocytes (Hool et al, 1998).…”

Mouse models of altered protein kinase A signaling

“…A myriad of studies over the past 40 years have identified hundreds (if not thousands) of PKA substrates, both in the nucleus and in the cytoplasm (Budovskaya et al 2005, Huang et al 2007, Neuberger et al 2007, Gao et al 2008. Like other post-translational protein modifications, PKAmediated phosphorylation can affect any aspect of target protein function, including activity, localization, and stability (Huang et al 2000, Mason et al 2002, Higuchi et al 2003, Qiao et al 2003, Hoang et al 2004, Hino et al 2005, Rolli-Derkinderen et al 2005, such that PKA exerts complex effects on most biologic systems. For this reason, pharmacologic or genetic manipulation in tissue culture cells may produce only a limited understanding of the pleomorphic role of PKA signaling.…”

Direct block of cloned hKv1.5 channel by cytochalasins, actin-disrupting agents

“…Cardiac Kv1.5 channels also couple to the actin cytoskeleton, which regulates current density and channel localization (20). Recently, it has been shown that modulation of Kv1.5 currents by protein kinase A requires an intact cytoskeleton (21). Specific disruptors for cytoskeletons have been used in analyzing the functional roles of the cytoskeleton, including modulation of ion channel activities.…”

“…There are several previous reports (20,21,33,37) showing the regulation of ion channels by cytoskeleton. Maruoka et al (20) have reported that disruption of the actin cytoskeleton with cytochalasin B or cytochalasin D significantly increases Kv1.5-dependent K ϩ currents in Kv1.5-expressing human embryonic kidney cells.…”