Abstract
The Kv7 family (Kv7.1-7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels.
Highlights
Kv7 potassium channels are regulated by protein kinase C (PKC) and may assemble as Kv7.4/Kv7.5-heteromers
We visualized TRPC6 non-selective cation channels, which are known to be expressed in mesenteric artery smooth muscle cells (MASMCs) [33], but, as an ion channel of a different class, would not be expected to physically interact with Kv7 channel subunits
To detect an interaction between Kv7.4 and Kv7.5, MASMCs were incubated with both mouse anti-KCNQ4 and rabbit anti-KCNQ5 antibodies
Summary
Kv7 potassium channels are regulated by protein kinase C (PKC) and may assemble as Kv7.4/Kv7.5-heteromers. We provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Expression of an inducible protein kinase C␣ (PKC␣) translocation system revealed that PKC␣ activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Biochemical evidence for the formation and regulation of endogenous Kv7.4/7.5 heteromers is lacking It remains to be determined whether the Kv7.4 and Kv7.5 subunits in vascular smooth muscle cells are phosphorylated or if they are sensitive to PKC-dependent regulation by vasoconstrictor agonists. Vascular Kv7.4/7.5 Heteromers Regulated by PKC vasoconstrictor hormone arginine vasopressin (AVP) induces translocation of PKC␣ from the cytosol to the plasma membrane in A7r5 rat aortic smooth muscle cells [23]. The ability to be phosphorylated corresponds to the sensitivity of the Kv7 channels to AVP in the rank order: Kv7.5 Ͼ Kv7.4/7.5 ϾϾ Kv7.4
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