Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells

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.

Cyclic AMP-Dependent Regulation of Kv7 Voltage-Gated Potassium Channels

“…β2-adrenergic receptor agonists are commonly used for the treatment of asthma to relieve hyperconstriction of the airway (Cazzola et al, 2013), and are able to enhance Kv7 currents in ASMCs thereby inducing relaxation of rat airways (Brueggemann et al, 2014a). A later study by Brueggemann et al (2018) revealed that β adrenoceptor stimulation activated the Kv7.5 channels in cultured human ASMCs, a response which was mediated by the PKA pathway (Brueggemann et al, 2018). This study identified serine 53 on the Kv7.5 N-terminus to be responsible for the increased Kv7.5 currents in response to cAMP elevation, which could be a common mechanism for β adrenoceptor/cAMP activation of Kv7.5 channels in vascular smooth muscle cells.…”

“…Kv7.5/Kv7.3 channels show similar currents to the Kv7.2/Kv7.3 currents and are also inhibited by M1 muscarinic receptor activation (Schroeder et al, 2000a). Kv7.5 can be phosphorylated and stimulated by PKA on serine 53 on the N-terminus (Brueggemann et al, 2018); however, it is still unclear whether the heterotetrameric Kv7.3/Kv7.5 channel found in neurones are regulated in the same way by cAMP.…”

Kv7 Channels in Lung Diseases

“…The specific expression of certain Kv7 channel in different types of cells is of great pharmacological interest, since their selective modulation could provide beneficial therapeutic effects while avoiding the adverse effects associated with other isoforms (Stott et al, 2014). A clear example comes from the previous experience with retigabine and flupirtine, which have been used as analgesic and anticonvulsant, respectively, for many years but were withdrawn from the marked due to their adverse effects including the risk of serious liver injury, pigment changes in the retina and urinary retention (Herrmann et al, 1987;Brickel et al, 2012). While, as mentioned, both drugs activate Kv7.2-Kv7.5, they appear to display greater efficacy for Kv7.2 and Kv7.3 channels over Kv7.4 and Kv7.5 channels, which predominate in smooth muscle.…”

“…On the other hand, it is well recognized that stimulation of β-adrenergic receptor increases the activity of different Kv7 channels in different tissues such as cardiac (Kv7.1), neuronal (Kv7.2/Kv7.3), and vascular (Kv7.4/Kv7.5 and Kv7.5) channels ( Jentsch, 2000 ; Marx et al, 2002 ; Chadha et al, 2014 ; Mani et al, 2016 ; Barrese et al, 2018 ). Likewise, in human airway smooth muscle cells, Kv7.5 channels are robustly enhanced by activation of β-adrenoceptors via a mechanism involving cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-dependent phosphorylation ( Brueggemann et al, 2018 ). This effect might be shared with other GPCRs but does not seem to account for Kv7.4 activation.…”

“…Studies in human tracheal muscle revealed abundant expression of KCNQ1 relative to guinea pig, a modest expression of KCNQ4 and KCNQ5 and no detectable expression of KCNQ2 or KCNQ3 ( Brueggemann et al, 2011 ). A further study showed that cultured human ASMCs express all KCNQ genes with a predominant relative abundance of KCNQ5 mRNA followed by KCNQ3 or KCNQ4 , while KCNQ2 and KCNQ1 mRNA are expressed to a lesser extent ( Brueggemann et al, 2018 ). These differences in the expression profile of Kv7 channels in human tissue could be due to phenotypic changes associated with cell culture.…”