Abstract
β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.
Highlights
Voltage-activated Kv7 potassium channels, encoded by KCNQ genes, mediate “M-currents”, which have been implicated in the regulation of neuronal excitability by G protein-coupled receptor agonists [1,2] and more recently identified as intermediates in smooth muscle signal transduction [3,4]
The results presented here further suggest that the ability of Gs-coupled receptor agonists to enhance native HASMC Kv7 currents is convergent with their protein kinase A (PKA)-dependent enhancement of cloned Kv7.5 currents and their well-known bronchorelaxant effects in human lungs [22]
We have provided evidence that Kv7.5 is the predominant Kv7 channel subunit in HASMCs and that the enhancement of Kv7.5 currents in response to stimulation of the β-adrenergic receptor (βAR)/Gs/cyclic adenosine monophosphate (cAMP)/PKA pathway is mediated by phosphorylation of S53 on the amino-terminal region of the Kv7.5 α-subunits
Summary
Voltage-activated Kv7 potassium channels, encoded by KCNQ genes, mediate “M-currents”, which have been implicated in the regulation of neuronal excitability by G protein-coupled receptor agonists [1,2] and more recently identified as intermediates in smooth muscle signal transduction [3,4]. WWee nnoottiicceedd ddiimmiinniisshheedd eennhhaanncceemmeenntt ooff ccuurrrreennttss tthhrroouugghh CC--tteerrmmiinnaall SS→→AAmmuuttaannttssiinnrreessppoonnssee ttooaapppplliiccaattiioonnooffffoorrsskkoolliinn//IBIBMMXXththaattddididnnoottrereaacchhssigignnifiificcaanncceeinineeaacchhininddivivididuuaallccaassee..WWeeccrreeaatteeddaa ccoommbbiinneeddmmuuttaannttKKCCNNQQ55ccoonnssttrruuccttttooeennccooddeeaacchhaannnneellwwiitthhaallllssiixxoofftthheeppuuttaattiivveeCC--tteerrmmiinnaallPPKKAA pphhoosspphhoorryyllaattiioonn ssiitteess mmuuttaatteedd ttoo aallaanniinneess ((QQ55CC--tteerrmm66xxSS→→AA).).TThheesseexxtutuppleleSS→→AAmmuuttaanntt pprroodduucceedd ffuunnccttiioonnaallcchhaannnneelslswwitihthprporpoepretiretisessimsiimlairlatro twoilwdi-ltdy-pteypKev7K.5v7(.V50.5(V=0.−551=.4−±531..24 m±V3, .n2 =m5V;,Fnig=ure5; 5FAig,Cu,rDe ).AT,hCe,DQ). We have provided evidence that Kv7.5 is the predominant Kv7 channel subunit in HASMCs and that the enhancement of Kv7.5 currents in response to stimulation of the βAR/Gs/cAMP/PKA pathway is mediated by phosphorylation of S53 on the amino-terminal region of the Kv7.5 α-subunits. The present study provides evidence for a mechanistic link between these processes: βAR receptor activation results in PKA-dependent phosphorylation of S53 on the N-terminus of Kv7.5 channel subunits, which increases channel activity, and likely contributes to βAR agonist-induced relaxation of vascular and airway smooth muscle. Understanding the physiological mechanisms by which smooth muscle Kv7 channel activity is regulated may lead to alternative strategies for modulating this activity in vascular or airway smooth muscle and thereby to development of more effective therapies for vascular and airway diseases
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