Contribution of Kv7.4/Kv7.5 Heteromers to Intrinsic and Calcitonin Gene-Related Peptide–Induced Cerebral Reactivity

Abstract

Objective-Middle cerebral artery (MCA) diameter is regulated by inherent myogenic activity and the effect of potent vasodilators such as calcitonin gene-related peptide (CGRP). Previous studies showed that MCAs express KCNQ1, 4, and 5 potassium channel genes, and the expression products (Kv7 channels) participate in the myogenic control of MCA diameter. The present study investigated the contribution of Kv7.4 and Kv7.5 isoforms to myogenic and CGRP regulation of MCA diameter and determined whether they were affected in hypertensive animals. Approach and Results-Isometric tension recordings performed on MCA from normotensive rats produced CGRP vasodilations that were inhibited by the pan-Kv7 channel blocker linopirdine (P<0.01) and after transfection of arteries with siRNA against KCNQ4 (P<0.01) but not KCNQ5. However, isobaric myography revealed that myogenic constriction in response to increases in intravascular pressure (20-80 mm Hg) was affected by both KCNQ4 and KCNQ5 siRNA. Proximity ligation assay signals were equally abundant for Kv7.4/Kv7.4 or Kv7.4/Kv7.5 antibody combinations but minimal for Kv7.5/Kv7.5 antibodies or Kv7.4/7.1 combinations. In contrast to systemic arteries, Kv7 function and Kv7.4 abundance in MCA were not altered in hypertensive rats. Conclusions-This study reveals, for the first time to our knowledge, that in cerebral arteries, Kv7.4 and Kv7.5 proteins exist predominantly as a functional heterotetramer, which regulates intrinsic myogenicity and vasodilation attributed to CGRP. Surprisingly, unlike systemic arteries, Kv7 activity in MCAs is not affected by the development of hypertension, and CGRP-mediated vasodilation is well maintained. As such, cerebrovascular Kv7 channels could be amenable for therapeutic targeting in conditions such as cerebral vasospasm. evidence demonstrated that Kv7.4 channels in systemic arteries, such as the mesenteric and renal, are severely compromised in hypertension. 2,4 A similar situation in cerebral circulation would lead to a predilection toward vasospasm and may underlie ischemic stroke. Here, we used a combination of molecular and functional approaches to define the interaction of different Kv7 channels in MCA and to ascertain the functional impact of individual Kv7 isoforms in the intrinsic and CGRP-mediated regulation of arterial diameter. Materials and MethodsMaterials and Methods are available in the online-only Supplement. ResultsInitial studies used a pharmacological approach to tease out a functional role for KCNQ1, 4, and 5 that are dominantly expressed in the MCA. 5 The application of a selective Kv7.1 channel blocker, HMR1556 (10 μmol/L), 12 had no effect on MCA tone, whereas the pan-Kv7 channel blocker, linopirdine, evoked robust contractions of MCAs under similar conditions ( Figure 1A). In MCAs precontracted with 0.1 μmol/L U46619, the application of Kv7.2 to Kv7.5 channel activators, retigabine and S-1, caused relaxation in a concentration-dependent manner with the latter being more potent ( Figure 1B). In contrast, the Kv7.1-selective...

Complex role of Kv7 channels in cGMP and cAMP-mediated relaxations

“…Moreover, in spontaneously hypertensive animals which show decreased vascular Kv7.4 expression, these responses were all compromised. This is consistent with previous findings that endogenous vasodilation that are Kv7 dependent are compromised in hypertensive animals ( 2,4,6 summarised in Fig. 1).…”

“…Further studies have since shown that in other vascular beds Kv7 channels contribute to the cAMP-dependent relaxations. In the cerebral arteries calcitonin gene related peptide 2 and forskolin relaxations 5 are inhibited by linopirdine, while in coronary arteries adenosine relaxations are Kv7 dependent. 6 These studies have established the importance of Kv7 channels in the mediation of cAMP dependent relaxations in the vasculature, but their role in other endogenous signaling pathways was not clear.…”

“…Of the 5 Kv7 subtypes, Kv7.1 7.4 and 7.5 are consistently expressed in the vasculature where the predominant molecular architecture has been shown to be a Kv7.4/ Kv7.5 heterotetramer. 2,3 Recent research has focused on the role of Kv7 channels in vasodilations mediated by endogenous molecules, uncovering several vasodilators which act at least in part via activation of Kv7 channels. The seminal study by Chadha et al 4 established that Kv7 channels contribute to cyclic AMP (cAMP) dependent isoproterenol mediated vasorelaxations in the rat renal artery.…”

G-protein βγ subunits are positive regulators of Kv7.4 and native vascular Kv7 channel activity

“…There are five Kv7 isoforms (Kv7.1-Kv7.5) of which Kv7.1, Kv7.4, and Kv7.5 are consistently expressed within VSM, where the predominant molecular architecture is a Kv7.4/Kv7.5 heterotetramer (2,3). Activation of Kv7 channels produces relaxation of numerous arteries (4)(5)(6)(7)(8), whereas blockade of Kv7 channels results in contraction of vessels at rest (7,(9)(10)(11) or an inhibition of endogenously derived vasorelaxations (2,(11)(12)(13). In addition, molecular reduction of Kv7.4 reduces responses to various Gs-coupled vasodilators in a number of arteries (2,11).…”

“…Activation of Kv7 channels produces relaxation of numerous arteries (4)(5)(6)(7)(8), whereas blockade of Kv7 channels results in contraction of vessels at rest (7,(9)(10)(11) or an inhibition of endogenously derived vasorelaxations (2,(11)(12)(13). In addition, molecular reduction of Kv7.4 reduces responses to various Gs-coupled vasodilators in a number of arteries (2,11). Crucially, Kv7.4 abundance is reduced in various arteries from hypertensive animals (6,11,12) where relaxant responses to endogenous vasodilators are also impaired (11,12).…”