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Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

Significance Statement

Arteries have the remarkable ability to adjust the amount and force of blood that flows through them. Such adjustments are made possible by contraction or relaxation of arterial smooth muscle cells (ASMCs) within the walls of the arteries. Many hormonal and neuronal actions serve to adjust the contraction of ASMCs, to modulate blood flow and pressure. At the cellular level, contraction of ASMCs depends on the flow of calcium ions into the cells through specialized protein pores or “channels” on the cell’s plasma membrane. Opening of voltage-sensitive calcium channels (VSCCs) involves positive changes to the voltage across the plasma membrane. Relaxation of ASMCs occurs when membrane voltage is maintained around -60 millivolts (negative inside compared to outside) by a flux of potassium ions out of the cells through channels that selectively conduct potassium; this negative voltage inhibits the opening of VSCCs. Among the many types of potassium channels on the ASMC plasma membrane, Kv7 channels, are particularly well suited as targets for hormonal and neuronal regulation of ASMC contraction to adjust blood pressure and blood flow.

Membrane voltage, and hence the flux of calcium ions via VSCCs, is very sensitive to the opening (activation) or closing (e.g. blocking) of Kv7 channels. These channels are tetrameric assemblies constituted by four Kv7 channel alpha subunits. There are five different types of Kv7 channel alpha subunits, named Kv7.1 through 7.5. In ASMCs, Kv7 channels are composed of four Kv7.4 subunits, four Kv7.5 subunits, or by some combination of Kv7.4/7.5 subunits (i.e. heterotetrameric channels). Altering the activity of the Kv7 channels with chemicals that bind directly to the channels has been shown to influence arterial contractility and diameter. However, physiological regulation of Kv7 channel activity is still poorly understood. In particular, it is unclear how the subunit composition of the channels influences the regulation of their activity.

In the article by Mani et al. (PMID: 26700561), the activity of ASMC Kv7 channels was monitored in response to activation of cell surface receptors and intracellular signaling regulators to better understand the physiological regulation of these channels. Furthermore, to study how different channel subunits respond to these stimuli, cultured rat aorta ASMCs (A7r5 cells) that naturally express only Kv7.5 subunits were used, and compared with the responses of ASMCs freshly isolated from rat arteries, which predominantly express heterotetrameric Kv7.4/Kv7.5 channels. In addition, human Kv7 channel alpha subunits were artificially introduced into A7r5 cells to compare regulation of human Kv7.4 or human Kv7.5 channels expressed individually or expressed together.

The results indicated that activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), a well-known vasodilatory stimulus, robustly increased the activity of Kv7.5 channels. Activation of cAMP/PKA using chemicals, or by engagement of beta adrenergic receptors that activate this signaling pathway, similarly increased the activity of naturally occurring Kv7.5 channels or of artificially introduced human Kv7.5 channels in A7r5 cells. In contrast, activity of freshly isolated rat artery Kv7.4/Kv7.5 channels or artificially introduced human Kv7.4/7.5 channels were only modestly enhanced, and human Kv7.4 channels were insensitive to activation of this signaling pathway. It was further demonstrated that the changes in activity of the channels by the signaling pathway are dependent on temporary addition of a phosphate group to the Kv7.5 channel subunits. No phosphate additions were detectable by activation of the signaling pathway in cells with artificially introduced Kv7.4 channels.

In summary, these results suggest that the responsiveness of arterial smooth muscle Kv7 channel subunits to intracellular cAMP/PKA signal activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4. The differences in Kv7 channel subunit response may have important implications in terms of arterial function, as the Kv7 channel subunit expression patterns may differ among vascular beds and may change during development or with disease.


Figure Legend: Phosphorylation of Kv7.5 alpha subunits enhances efflux of potassium ions from arterial smooth muscle cells. Schematic diagram illustrating the signal transduction pathway whereby activation of β-adrenergic receptors (β-Adr) leads to elevation of cytosolic concentrations of cyclic adenosine monophosphate (cAMP) and hence to activation of Protein Kinase A. Protein kinase A can catalyze the transfer of phosphate (P) to Kv7.5 alpha subunits, which increases the opening of channels containing these subunits (Kv7.5 homotetramers or Kv7.4/Kv7.5 heterotetramers; solid arrows). Opening of the channels increases efflux of potassium ions (K+), which promotes smooth muscle relaxation. Kv7.4 alpha subunits are not phosphorylated by Protein Kinase A, and the activity of Kv7.4 homotetramers is not altered by this mechanism (dashed arrow).

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About The Author

Bharath Mani received his DVM degree in 2001 and MS degree in 2003 from Tamil Nadu Veterinary and Animal Sciences University, Chennai, India, and Ph.D (Pharmacology) degree in 2012 from Loyola University Chicago, USA. His doctoral research examined the role of arterial smooth muscle Kv7 channel function in influencing arterial contraction status.

He is now a postdoctoral fellow at the University of Texas Southwestern Medical Center at Dallas, Texas, USA. His current research investigates neuroendocrine regulation of metabolic and cardiovascular function.

Journal Reference

Mol Pharmacol. 2016; 89(3):323-34.

Mani BK, Robakowski C, Brueggemann LI, Cribbs LL, Tripathi A, Majetschak M, and Byron KL.

Loyola University Chicago, Dept. of Molecular Pharmacology & Therapeutics, Maywood, IL 60153, USA.


Kv7 (KCNQ) channels, formed as homo- or heterotetramers of Kv7.4 and Kv7.5 α-subunits, are important regulators of vascular smooth musclecell (VSMC) membrane voltage. Recent studies demonstrate that direct pharmacological modulation of VSMC Kv7 channel activity can influence blood vessel contractility and diameter. However, the physiologic regulation of Kv7 channel activity is still poorly understood. Here, we study the effect of cAMP/protein kinase A (PKA) activation on whole cell K(+) currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth musclecells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific α-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo- or heterotetrameric channels in A7r5 cells. Stimulation of Gαs-coupled β-adrenergic receptors with isoproterenol induced PKA-dependent activation of endogenous Kv7.5 currents in A7r5 cells. The receptor-mediated enhancement of Kv7.5 currents was mimicked by pharmacological agents that increase [cAMP] (forskolin, rolipram, 3-isobutyl-1-methylxanthine, and papaverine) or mimic cAMP (8-bromo-cAMP); the 2- to 4-fold PKA-dependent enhancement of currents was also observed with exogenously expressed Kv7.5 channels. In contrast, exogenously-expressed heterotetrameric Kv7.4/7.5 channels in A7r5 cells or native mesenteric artery smooth muscle Kv7.4/7.5 channels were only modestly enhanced, and homo-tetrameric Kv7.4 channels were insensitive to this regulatory pathway. Correspondingly, proximity ligation assays indicated that isoproterenol induced PKA-dependent phosphorylation of exogenously expressed Kv7.5 channel subunits, but not of Kv7.4 subunits. These results suggest that signal transduction-mediated responsiveness of vascular smooth muscle Kv7 channel subunits to cAMP/PKA activation follows the order of Kv7.5 >> Kv7.4/Kv7.5 > Kv7.4.

Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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