Differential Activation of Vascular Smooth Muscle Kv7.4, Kv7.5, and Kv7.4/7.5 Channels by ML213 and ICA-069673

Abstract

Recent research suggests that smooth muscle cells express Kv7.4 and Kv7.5 voltage-activated potassium channels, which contribute to maintenance of their resting membrane voltage. New pharmacologic activators of Kv7 channels, ML213 (N-mesitybicyclo[2.2.1]heptane-2-carboxamide) and ICA-069673 N-(6-chloropyridin-3-yl)-3,4-difluorobenzamide), have been reported to discriminate among channels formed from different Kv7 subtypes. We compared the effects of ML213 and ICA-069673 on homomeric human Kv7.4, Kv7.5, and heteromeric Kv7.4/7.5 channels exogenously expressed in A7r5 vascular smooth muscle cells. We found that, despite its previous description as a selective activator of Kv7.2 and Kv7.4, ML213 significantly increased the maximum conductance of homomeric Kv7.4 and Kv7.5, as well as heteromeric Kv7.4/7.5 channels, and induced a negative shift of their activation curves. Current deactivation rates decreased in the presence of the ML213 (10 mM) for all three channel combinations. Mutants of Kv7.4 (W242L) and Kv7.5 (W235L), previously found to be insensitive to another Kv7 channel activator, retigabine, were also insensitive to ML213 (10 mM). In contrast to ML213, ICA-069673 robustly activated Kv7.4 channels but was significantly less effective on homomeric Kv7.5 channels. Heteromeric Kv7.4/7.5 channels displayed intermediate responses to ICA-069673. In each case, ICA-069673 induced a negative shift of the activation curves without significantly increasing maximal conductance. Current deactivation rates decreased in the presence of ICA-069673 in a subunit-specific manner. Kv7.4 W242L responded to ICA-069673-like wild-type Kv7.4, but a Kv7.4 F143A mutant was much less sensitive to ICA-069673. Based on these results, ML213 and ICA-069673 likely bind to different sites and are differentially selective among Kv7.4, Kv7.5, and Kv7.4/7.5 channel subtypes.

Kv7.5 Potassium Channel Subunits Are the Primary Targets for PKA-Dependent Enhancement of Vascular Smooth Muscle Kv7 Currents

“…4B); a slight suppression of the current observed in the presence of rolipram was not statistically significant. Subsequent application of the nonselective Kv7.2-Kv7.5 activator ML213 (10 mM) (Yu et al, 2011;Brueggemann et al, 2014a) robustly enhanced the MASMC Kv7 currents and this effect was reversed by the selective Kv7 channel blocker XE991 (10 mM) (Fig. 4B), demonstrating the expected pharmacological characteristics of the Kv7 currents and confirming our ability to detect an increase in current amplitude in these cells.…”

“…4). A7r5 cells natively express only Kv7.5 channels (Brueggemann et al, 2007, whereas MASMCs predominantly express Kv7.4/Kv7.5 channels (Brueggemann et al, , 2014a. Insensitivity of MASMC Kv7.4/7.5 channels to rolipram was replicated when this same subunit combination was expressed in A7r5 cells, suggesting that the difference in regulation of native Kv7 currents between A7r5 cells and MASMCs relates primarily to the Kv7 channel subunit stoichiometry rather than to differences in PDE isoforms (Polson and Strada, 1996;Dunkerley et al, 2002;Maurice et al, 2003).…”

The Novel KV7.2/KV7.3 Channel Opener ICA-069673 Reveals Subtype-Specific Functional Roles in Guinea Pig Detrusor Smooth Muscle Excitability and Contractility

“…Although it is less potent than its related analog ICA-27243, ICA-069673 displays greater efficacy and improved selectivity for heteromeric K V 7.2/K V 7.3 over K V 7.1 channels (Amato et al, 2011). In contrast with retigabine, which binds to the pore region (S5 to S6) of K V 7.2-K V 7.5 channels (Gunthorpe et al, 2012), evidence suggests that ICA-069673 is among an emerging novel class of compounds known as "gating modifiers," named so for their ability to bind to the S1-S4 voltage-sensing domain of K V 7 channels Peretz et al, 2010;Brueggemann et al, 2014a).…”

“…ICA-069673 did not affect K V 7.1 channels, which are predominately expressed in the heart (Amato et al, 2011). A recent study on A7r5 smooth muscle cells demonstrated that ICA-069673 was effective in activating recombinantly expressed K V 7.4 channels (Brueggemann et al, 2014a). However, K V 7.4 channel protein expression was not observed in guinea pig DSM (Afeli et al, 2013).…”

Sequence determinants of subtype‐specific actions of KCNQ channel openers

“…; Brueggemann et al . ). Several studies have suggested the possibility that certain KCNQ activators may act on a distinct site present in certain KCNQ subtypes.…”