The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine

Abstract

KCNQ (voltage-gated K؉ channel family 7 (Kv7)) channels control cellular excitability and underlie the K ؉ current sensi- Members of the voltage-gated K ϩ (Kv) 5 channel family 7 (Kv7, otherwise known as KCNQ) are key determinants of cellular excitability in the heart (Kv7.1) and brain (Kv7.2-7.5) (1, 2). There is no crystal structure for any member of Kv7 family, although they constitute prominent targets of clinically relevant drugs, and their dysfunction is implicated in a number of human diseases (1-3). In their physiological context, Kv7 channels open at subthreshold voltages and are modulated by G protein-coupled receptor signaling pathways via secondary messengers (1, 2). In neurons, Kv7.2/3 heteromers presumably underlie the K ϩ current sensitive to muscarinic receptor signaling, the M current (4 -7). Mutations in the Kv7.2 (8 -11) and Kv7.3 (11,12) subunits underlie a form of inherited human epilepsy: benign familial neonatal convulsions. Retigabine (RTG,methylamino]phenyl]carbamate), an anticonvulsant drug approved for the adjunctive treatment of partial-onset seizures in adults, is thought to act primarily by increasing the propensity to reside in the open conformation of neuronal Kv7.2/3 channels (13-15), leading to hyperpolarization of the membrane potential toward the K ϩ equilibrium potential and, therefore, attenuating the repetitive firing pattern that underlies seizures (16 -19). However, the molecular mechanism by which RTG opens the Kv7.2/3 channels is not well understood. Remarkably, cardiac Kv7.1 channels are insensitive to RTG (15,20,21). This exquisite specificity affords an opportunity to dissect the target site of action on Kv7.2/3 channels.We selected the PM of the Kv7 (KCNQ) channel family because the amino acid sequence of its pore and voltage sensor modules is similar to that of KvLm, a bacterial Kv for which we solved the structure of the pore-only module in a lipid environment at atomic resolution (22). We exploit the structural independence of the pore and sensor modules of to produce homomers (6, 7, 13-15, 20, 21, 25-27) and heteromers (6, 7, 13-15, 20, 25-27) of Kv7.2 and Kv7.3 PMs by in vitro transcription and translation. Single-channel activity of the purified pore-only module (PM) was characterized after reconstitution in lipid bilayers. Single-channel currents through purified Kv7.2, Kv7.3, and Kv7.2/Kv7.3 PMs are K ϩ -selective and blocked by specific Kv7 channel blockers (28). Interestingly, RTG potentiates channel function by increasing single channel open probability in all functional assemblies of Kv7 PMs, validating that the site of action of RTG is embodied in the sensorless PM. Furthermore, a key mutation in Kv7.3 PM W265L renders the channel insensitive to RTG, further confirming the PM of Kv7 channels as the interaction site for the drug. Experimental Procedures

Retigabine holds KV7 channels open and stabilizes the resting potential

“…Here, for the first time, we provide an example of hysteresis in the activity of a K V channel that directly affects the channel’s pharmacology under physiological conditions. In line with recent findings on the action of Retigabine on the isolated pore of K V 7 channels ( Syeda et al, 2015 ), our observations clearly show that the stabilized open state of the K V 7.2/K V 7.3 channel is the target for the action of the anticonvulsant Retigabine. These findings have several implications.…”

Modulation of KV7 Channel Deactivation by PI(4,5)P2

“…Likewise, such drugs can partially compensate for the decrease in M-currents caused by PI(4,5)P 2 depletion (Zhou et al, 2013). Retigabine stabilizes the channels' open states as it increases the dwell time of open events (Tatulian and Brown, 2003;Syeda et al, 2015). Consistently, Retigabine also decreases the deactivation rate of K V 7.2/K V 7.3 channels (Main et al, 2000;Linley et al, 2012;Corbin-Leftwich et al, 2016;Yau et al, 2018).…”

“…Although the mechanism underlying this action remains unclear, single channel recordings show that Retigabine increases the lifetime of open state events of K V 7.2/K V 7.3 channels, while decreasing the dwelling-time of closed channel events (Tatulian and Brown, 2003). Further, the mean-open-time of the isolated pore of K V 7.3 channel increases in the presence of Retigabine (Syeda et al, 2015). Furthermore, the deactivation rate of both K V 7.2/K V 7.3 and K V 7.3 channels decreases in the presence of Retigabine (Wickenden et al, 2000;Corbin-Leftwich et al, 2016;Kim et al, 2017).…”

Remembering Mechanosensitivity of NMDA Receptors

“…Indeed, a recent study showed that NMDAR channels were mechanically responsive to fluid shear stress in the absence of agonists (Maneshi et al, 2017), which seems also to indicate a physiological role for NMDAR in the glymphatic clearance pathway in the brain (Jessen and Kotz, 2015; Cox et al, 2019; Martinac and Bilston, 2019). Moreover, like mechanosensitive MscS, MscL, TREK-1, TREK-2, TRAAK, Piezo1, and OSCA channels (Kloda et al, 2007a; Brohawn et al, 2014; Cox et al, 2016; Syeda et al, 2016; Murthy et al, 2018), the NMDAR channel protein could be reconstituted into liposomes and activated by stretching liposome patches (Kloda et al, 2007a), which indicates that NMDAR is inherently mechanosensitive meaning that lipid-protein interactions are sufficient for the channel activation by mechanical force.…”

“…What is the consequence of these findings for the mechanosensitivity of NMDA receptors? The bonus of these findings is that local curvature on the scale of tens of nanometers can be influenced by a host of proteins, lipid types and unilateral insertion of physiologically active amphipathic molecules, such as arachidonic or lysophosphatidic acid (Figure 1; Zimmerberg and Kozlov, 2006; Kloda et al, 2007b; Frisca et al, 2012; Syeda et al, 2016). Moreover, the list of amphipathic molecules produced and released in physiological settings that could modify NMDAR channel function is potentially very long.…”