Kv7 (KCNQ) channels, formed as homo-or heterotetramers of Kv7.4 and Kv7.5 a-subunits, are important regulators of vascular smooth muscle cell (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 1 currents through endogenous Kv7.5 channels in A7r5 rat aortic smooth muscle cells or through Kv7.4/Kv7.5 heteromeric channels natively expressed in rat mesenteric artery smooth muscle cells. The contributions of specific a-subunits are further dissected using exogenously expressed human Kv7.4 and Kv7.5 homo-or heterotetrameric channels in A7r5 cells. Stimulation of Ga scoupled b-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 PKAdependent 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.

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Kv7 (KCNQ) potassium channels are essential for maintaining resting membrane voltage in smooth muscle cells. Functional Kv7 channels in rat mesenteric artery myocytes are formed as heteromers of Kv7.4 and Kv7.5 α‐subunits, whereas cultured A7r5 rat aortic smooth muscle cells natively express only homomeric Kv7.5 channels. Here we provide evidence that a selective inhibitor of phosphodiesterase‐4 (PDE‐4), rolipram (100nM), increases Kv7.5 currents in A7r5 cells in a protein kinase A‐ (PKA‐) dependent manner. The increase in Kv7.5 currents in A7r5 cells was concentration‐dependent, reaching a maximum of 3.4±0.1‐fold increase at 100nM rolipram. Direct activation of adenylyl cyclase with 1µM forskolin, in combination with the PDE inhibitor isobutylmethylxanthine (IBMX, 500µM), also effectively enhanced endogenous Kv7.5 currents in A7r5 cells. Pretreatment with a PKA inhibitor (KT5720, 1µM) completely prevented the rolipram‐induced enhancement of Kv7.5 current relative to its vehicle control. In mesenteric artery myocytes, rolipram did not increase native Kv7 currents. Using A7r5 cells as an expression system for human (h) Kv7 subunits, we found that rolipram did not increase currents through exogenous homomeric hKv7.4 or heteromeric hKv7.4/h7.5 channels. In contrast, rolipram robustly increased the current amplitude through exogenous homomeric hKv7.5 channels or through retigabine‐insensitive hKv7.5 mutant channels, KCNQ5W235L. The latter result suggests an absence of direct binding of rolipram to the site implicated in the binding of many pharmacological Kv7 channel activators. Our results suggest differential regulation of homomeric Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels by PKA.