Voltage-gated potassium channels KCNQ2–5 generate the M-current, which controls neuronal excitability. KCNQ2–5 subunits each harbor a high-affinity anticonvulsant drug-binding pocket containing an essential tryptophan (W265 in human KCNQ3) conserved for >500 million years, yet lacking a known physiological function. Here, phylogenetic analysis, electrostatic potential mapping, in silico docking, electrophysiology, and radioligand binding assays reveal that the anticonvulsant binding pocket evolved to accommodate endogenous neurotransmitters including γ-aminobutyric acid (GABA), which directly activates KCNQ5 and KCNQ3 via W265. GABA, and endogenous metabolites β-hydroxybutyric acid (BHB) and γ-amino-β-hydroxybutyric acid (GABOB), competitively and differentially shift the voltage dependence of KCNQ3 activation. Our results uncover a novel paradigm: direct neurotransmitter activation of voltage-gated ion channels, enabling chemosensing of the neurotransmitter/metabolite landscape to regulate channel activity and cellular excitability.

Epilepsy has been treated for centuries with herbal remedies, including leaves of the African shrub Mallotus oppositifolius, yet the underlying molecular mechanisms have remained unclear. Voltage-gated potassium channel isoforms KCNQ2–5, predominantly KCNQ2/3 heteromers, underlie the neuronal M-current, which suppresses neuronal excitability, protecting against seizures. Here, in silico docking, mutagenesis and cellular electrophysiology reveal that two components of M. oppositifolius leaf extract, mallotoxin (MTX) and isovaleric acid (IVA), act synergistically to open neuronal KCNQs, including KCNQ2/3 channels. Correspondingly, MTX and IVA combine to suppress pentylene tetrazole-induced tonic seizures in mice, whereas individually they are ineffective. Co-administering MTX and IVA with the modern, synthetic anticonvulsant retigabine creates a further synergy that voltage independently locks KCNQ2/3 open. Leveraging this synergy, which harnesses ancient and modern medicines to exploit differential KCNQ isoform preferences, presents an approach to developing safe yet effective anticonvulsants.

Botanical folk medicines have been used throughout human history to treat common disorders such as hypertension, often with unknown underlying mechanisms. Here, we discovered that hypotensive folk medicines from a genetically diverse range of plant species each selectively activated the vascular-expressed KCNQ5 potassium channel, a feature lacking in the modern synthetic pharmacopeia, whereas nonhypotensive plant extracts did not. Analyzing constituents of the hypotensive Sophora flavescens root, we found that the quinolizidine alkaloid aloperine is a KCNQ-dependent vasorelaxant that potently and isoform-selectively activates KCNQ5 by binding near the foot of the channel voltage sensor. Our findings reveal that KCNQ5-selective activation is a defining molecular mechanistic signature of genetically diverse traditional botanical hypotensives, transcending plant genus and human cultural boundaries. Discovery of botanical KCNQ5-selective potassium channel openers may enable future targeted therapies for diseases including hypertension and KCNQ5 loss-of-function encephalopathy.

Voltage-gated potassium channels of the KCNQ (Kv7) subfamily are essential for control of cellular excitability and repolarization in a wide range of cell types. Recently, we and others found that some KCNQ channels functionally and physically interact with sodium-dependent solute transporters, including myo-inositol transporters SMIT1 and SMIT2, potentially facilitating various modes of channel-transporter signal integration. In contrast to indirect effects such as channel regulation by SMIT-transported, myo-inositol-derived phosphatidylinositol 4,5-bisphosphate (PIP), the mechanisms and functional consequences of the physical interaction of channels with transporters have been little studied. Here, using co-immunoprecipitation with different channel domains, we found that SMIT1 binds to the KCNQ2 pore module. We next tested the effects of SMIT1 co-expression, in the absence of extracellular myo-inositol or other SMIT1 substrates, on fundamental functional attributes of KCNQ2, KCNQ2/3, KCNQ1, and KCNQ1-KCNE1 channels. Without exception, SMIT1 altered KCNQ ion selectivity, sensitivity to extracellular K, and pharmacology, consistent with an impact on conformation of the KCNQ pore. SMIT1 also altered the gating kinetics and/or voltage dependence of KCNQ2, KCNQ2/3, and KCNQ1-KCNE1. In contrast, SMIT1 had no effect on Kv1.1 (KCNA1) gating, ion selectivity, or pharmacology. We conclude that, independent of its transport activity and indirect regulatory mechanisms involving inositol-derived increases in PIP, SMIT1, and likely other related sodium-dependent solute transporters, regulates KCNQ channel ion selectivity, gating, and pharmacology by direct physical interaction with the pore module.

IntroductionGabapentin (Neurontin) and pregabalin (Lyrica) are synthetic antiepileptic and antinociceptive gabapentinoid compounds originally designed as analogues of the neurotransmitter γ-aminobutyric acid (GABA), and both are in widespread clinical use (Calandre et al., 2016). However, the mechanisms of action of gabapentinoids, exemplified by gabapentin and pregabalin, are incompletely understood. [ 3 H]-gabapentin binding was first described in membrane fractions from rat brain homogenates, and the target protein identified as the α2-δ subunit of voltage-gated calcium (CaV) channels. The findings were later recapitulated using porcine brain tissue, heterologously expressed α2-δ, and also with pregabalin; binding was found to be exclusive to α2-δ1 and 2 isoforms (Brown and Gee, 1998; Field et al., 2006; Fuller-Bicer et al., 2009; Gee et al., This article has not been copyedited and formatted. The final version may differ from this version.Molecular Pharmacology Fast Forward. Published on as DOI: 10.1124 at ASPET Journals on April 27, 2019 molpharm.aspetjournals.org Downloaded from MOL #112953 4 1996). Gabapentin and pregabalin are generally considered inactive against canonical GABAA and GABAB receptors, despite their structural similarity to GABA (Ben-Menachem, 2004; Jensen et al., 2002; Lanneau et al., 2001;Stringer and Lorenzo, 1999;Taylor, 1997), although some investigators contend that there are some subtype-specific effects on GABAB receptors (Bertrand et al., 2003a;Ng et al., 2001;Parker et al., 2004). Binding of gabapentin and pregabalin to α2-δ is suggested to act therapeutically via impairment of Cav channel activity, thus reducing neuronal calcium currents (Stefani et al., 1998;Stefani et al., 2001), although others observed no evidence for gabapentin-induced changes in neuronal Cav activity (Rock et al., 1993;Schumacher et al., 1998).We recently made the unexpected discovery that GABA can activate voltage-gated potassium (Kv) channels composed of heteromeric assemblies of KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) poreforming α subunits (Manville et al., 2018). KCNQ (Kv7) channels comprise tetramers of α subunits, each containing six transmembrane (S) segments, organized into the voltage-sensing domain (VSD, S1-4) and the pore module (S5-6) ( Fig. 1 A, B). In vertebrate nervous systems, KCNQ2/3 (Kv7.2/3) heteromers are the primary molecular correlate of the M-current, a muscarinicinhibited Kv current essential for regulating excitability of a wide range of neurons throughout the nervous system (Brown and Adams, 1980;Marrion et al., 1989;Wang et al., 1998). We found that, like the anticonvulsant retigabine (Kim et al., 2015;Schenzer et al., 2005), GABA binds to a conserved tryptophan (W265) on KCNQ3 to activate KCNQ3 homomers and KCNQ2/3 heteromers (Manville et al., 2018) (Fig. 1B-D).Because of the structural similarities between gabapentinoids and GABA, and the known influence of the M-current in many of the disease states responsive to gabapentinoids (epilepsy, pain, anxiety, alcohol withdrawal) (Bla...

Analysis of a leaf extract used in African folk medicine reveals a high-affinity K+ channel agonist and its binding site.

Ketogenic diets are effective therapies for refractory epilepsy, yet the underlying mechanisms are incompletely understood. The anticonvulsant efficacy of ketogenic diets correlates positively to the serum concentration of b-hydroxybutyrate (BHB), the primary ketone body generated by ketosis. Voltage-gated potassium channels generated by KCNQ2-5 subunits, especially KCNQ2/3 heteromers, generate the M-current, a therapeutic target for synthetic anticonvulsants. Here, we report that BHB directly activates KCNQ2/3 channels (EC 50 5 0.7 mM), via a highly conserved S5 tryptophan (W265) on KCNQ3. BHB was also acutely effective as an anticonvulsant in the pentylene tetrazole (PTZ) seizure assay in mice. Strikingly, coadministration of g-amino-b-hydroxybutyric acid, a high-affinity KCNQ2/3 partial agonist that also acts via KCNQ3-W265, similarly reduced the efficacy of BHB in KCNQ2/3 channel activation in vitro and in the PTZ seizure assay in vivo. Our results uncover a novel, unexpected molecular basis for anticonvulsant effects of the major ketone body induced by ketosis. SIGNIFICANCE STATEMENTKetogenic diets are used to treat refractory epilepsy but the therapeutic mechanism is not fully understood. Here, we show that clinically relevant concentrations of b-hydroxybutyrate, the primary ketone body generated during ketogenesis, activates KCNQ2/3 potassium channels by binding to a specific site on KCNQ3, an effect known to reduce neuronal excitability. We provide evidence using a mouse chemoconvulsant model that KCNQ2/3 activation contributes to the antiepileptic action of b-hydroxybutyrate.

Herbs have a long history of use as folk medicine anticonvulsants, yet the underlying mechanisms often remain unknown. Neuronal voltage‐gated potassium channel subfamily Q (KCNQ) dysfunction can cause severe epileptic encephalopathies that are resistant to modern anticonvulsants. Here we report that cilantro (Coriandrum sativum), a widely used culinary herb that also exhibits antiepileptic and other therapeutic activities, is a highly potent KCNQ channel activator. Screening of cilantro leaf metabolites revealed that one, the long‐chain fatty aldehyde (E)‐2‐dodecenal, activates multiple KCNQs, including the predominant neuronal isoform, KCNQ2/ KCNQ3 [half maximal effective concentration (EC50), 60 ± 20 nM], and the predominant cardiac isoform, KCNQ1 in complexes with the type I transmembrane ancillary subunit (KCNE1) (EC50, 260 ± 100 nM). (E)‐2‐dodecenal also recapitulated the anticonvulsant action of cilantro, delaying pentylene tetrazole‐induced seizures. In silico docking and mutagenesis studies identified the (E)‐2‐dodecenal binding site, juxtaposed between residues on the KCNQ S5 transmembrane segment and S4‐5 linker. The results provide a molecular basis for the therapeutic actions of cilantro and indicate that this ubiquitous culinary herb is surprisingly influential upon clinically important KCNQ channels.—Manville, R. W., Abbott, G. W. Cilantro leaf harbors a potent potassium channel‐activating anticonvulsant. FASEB J. 33, 11349–11363 (2019). http://www.fasebj.org