KCNQ K+ channels have been implicated in human diseases ranging from cardiac arrhythmias to congenital deafness and epilepsy. In neurons, these channels underlie a voltage-sensitive K+ (Kv) current, which is negatively regulated by acetylcholine to regulate postsynaptic neuronal excitability. Although KCNQ channels had not previously been considered to play a role in vasoconstrictor signal transduction, we have recently shown that suggest that regulation of arterial myocyte excitability by physiological vasoconstrictor concentrations of arginine vasopressin (AVP, 10-100 pM) involves protein-kinase C-dependent suppression of KCNQ5 channel activity. We have also recently published evidence that this negative regulation of KCNQ channels underlies the vasoconstrictor actions of low [AVP] (30 pM) in rat mesenteric arteries. No previous studies have examined how KCNQ channels may be regulated by vasoactive hormones, the signal transduction mechanisms involved, whether their functions or regulation differ among vascular beds that express different channel subtypes, or whether these channels or signaling pathways may be useful targets for cardiovascular disease therapies. We therefore propose to: 1. Identify the subtypes of KCNQ family (Kv7.1- 7.5) channels expressed in arterial myocytes from rat mesenteric or basilar arteries and determine their functional roles in regulating artery diameter. Real time PCR and immunohistochemistry will be used to detect KCNQ channel expression. Channel function will be assessed by pressure myography in isolated arteries and patch clamp electrophysiology in isolated myocytes. Selective KCNQ channel blockers and activators and molecular knock-down approaches will be used to evaluate function of specific channel subtypes. 2. Identify the signal transduction mechanisms by which AVP (and potentially other vasoconstrictor hormones) regulate KCNQ channels. We will measure time- and concentration-dependent effects of AVP and other vasoconstrictor agonists (5-HT, AngII, and phenylephrine) on KCNQ currents in freshly isolated arterial myocytes. The roles of specific protein kinase C isoforms and A kinase-anchoring protein 150 (AKAP150) in KCNQ current regulation will be investigated using pharmacological activators/inhibitors or molecular reagents to disrupt their expression/function. The role of phosphorylation of specific residues on KCNQ channels (to be identified by mass spectrometry) will be evaluated by molecular targeting of the kinase or phosphorylation sites in cultured smooth muscle cells and by knocking in dysregulated KCNQ channels in transgenic mice. Molecules associated with KCNQ channels in signaling complexes will be identified using co-immunoprecipitation with native or FLAG-tagged KCNQ channel proteins. 3. Finally, the hypothesis that arterial KCNQ channels play an important role in vasoconstrictor actions and blood pressure regulation will be tested by measuring in vivo effects of selective KCNQ channel activators and blockers on mesenteric artery blood flow and systemic blood pressure measured acutely in anesthetized rats or in chronically instrumented conscious rats.
KCNQ channels have been recognized primarily for their role in neuronal excitation. Activators or blockers of KCNQ channels have been used clinically for treatment of epilepsy and Alzheimer's disease, respectively. The effects of these drugs on arterial constriction and their role as mediators of vasoconstrictor hormone action (demonstrated for the first time in our preliminary results) have not been appreciated and might have important implications for the use of KCNQ channel modulators in existing therapies as well as for their potential use in the treatment of cardiovascular diseases.
Showing the most recent 10 out of 13 publications
