The acrylamide (S)-1 differentially affects Kv7 (KCNQ) potassium channels
Introduction
Kv7 (KCNQ) channels belong to the family of K+ selective, voltage-gated channels composed homomeric or heteromeric α-subunit tetramers. Five members of the Kv7 family have been identified to date including the primarily cardiac and epithelial Kv7.1 and the neuronal Kv7.2–5 channels (for review see Jentsch, 2000). Kv7 channels are known to co-assemble with auxiliary subunits belonging to the KCNE family, which results in changes in expression levels and biophysical and pharmacological properties of the channel (for review see McCrossan and Abbott, 2004).
Noteworthy, mutations in four of the five members are associated with human diseases. The slowly activating repolarizing cardiac IKs current consists of co-assembled Kv7.1 and KCNE1 subunits (Barhanin et al., 1996, Sanguinetti et al., 1996). Mutations in either Kv7.1 or KCNE1 can cause long QT syndrome (Splawski et al., 1997, Wang et al., 1996). Mutations in Kv7.2 or Kv7.3 are the genetic basis of benign familial neonatal convulsion, which is a rare form of neonatal epilepsy (Biervert et al., 1998, Charlier et al., 1998, Singh et al., 1998). Kv7.4 is mainly expressed in the auditory system and mutations in Kv7.4 are linked to hearing loss (Kubisch et al., 1999). The close association between Kv7 channels and disease illustrates the significance of this channel family in controlling cellular excitability.
Several types of neurons express an endogenous potassium current called the M-current governing their excitability. The M-current is voltage-gated and serves to stabilize the membrane potential. Because the M-current activates at potentials around −60 mV, it plays a central role in the sub-threshold control of firing. In addition, the M-current is modulated by a number of neurotransmitters including acetylcholine acting on muscarinic receptors (for review see Brown and Yu, 2000). Kv7.2 and Kv7.3 heterotetramers have been shown to be the molecular correlate of the M-current (Wang et al., 1998). However, both Kv7.4 and Kv7.5 are expressed in the brain and display M-current characteristics (Kubisch et al., 1999, Lerche et al., 2000).
Modulators of the M-current have been under clinical investigation. These include blockers (e.g., linopirdine) for cognition enhancement and openers (e.g., retigabine) for treatment of diseases characterized by excessive neuronal activity such as epilepsy, neuropathic pain and migraine. Dampening neuronal excitability by enhancing M-currents might represent a novel pharmaceutical treatment for several diseases related to neuronal hyperexcitability.
In the present study we have thoroughly investigated the effect of Bristol–Myers Squibb compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1] on cloned human Kv7.1–5 potassium channels. The compound has previously been reported to be a Kv7.2 channel opener and found to have an effect in a rat model of migraine (Wu et al., 2003).
We demonstrate that (S)-1 is an activator of all neuronal Kv7 channels and blocks Kv7.1 as well as Kv7.1/KCNE1. The tryptophan residue 242 in the 5th transmembrane region, also known to be crucial for the effect of retigabine, is found to be critical for the enhancing effect of (S)-1 on Kv7.4. Furthermore, no additive effect on Kv7.4 current amplitude was observed when (S)-1 was applied in the presence of saturating concentrations of retigabine.
Section snippets
Molecular biology
cDNAs encoding human Kv7.1–5 and KCNE1 were subcloned into the dual-function expression vector pXOOM, containing the 5′- and 3′-untranslated regions for Xenopus laevis β-globin as well as a poly-A segment, as described previously (Jespersen et al., 2002).
The point mutation W242L of human Kv7.4 was constructed using standard overlap PCR techniques. The PCR-fragment was subsequently cloned into the pGEM-HE vector for expression in Xenopus laevis oocytes. Constructs were sequenced using an ABI 377
(S)-1 affects Kv7.2–5 current amplitude differently
The effect of (S)-1 on Kv7 channels expressed in Xenopus laevis oocytes was studied using two-electrode voltage-clamp. The oocytes were held at −80 mV for 3 s to release any form of inactivation, and channels were activated by depolarizing steps from −100 mV to +60 mV (20 mV increments, 2 or 5 s duration) in the presence and absence of (S)-1 (Fig. 1). Current traces from oocytes injected with 10 ng Kv7.2 cRNA (Fig. 1A) revealed a slowly activating and deactivating current activating around −60 mV.
Discussion
(S)-1 has previously been shown to increase currents through mKv7.2 channels expressed in Xenopus laevis oocytes and HEK cells and demonstrated to have a positive effect in a rat model of migraine (Wu et al., 2003). Here we show that (S)-1 enhances currents through all human neuronal Kv7 channels including homomeric Kv7.2, Kv7.4 and Kv7.5 and heteromeric Kv7.2/Kv7.3 channels expressed in Xenopus laevis oocytes. (S)-1 shifts the voltage dependence of activation to more hyperpolarized potentials
Acknowledgments
The work was supported by The Danish National Research Foundation to S.P.O. and by a NovoNordisk scholarship to B.H.B.
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