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.

Coronary Artery Disease (CAD) typically kills more people globally each year than any other single cause of death. A better understanding of genetic predisposition to CAD and the underlying mechanisms will help to identify those most at risk and contribute to improved therapeutic approaches. KCNE2 is a functionally versatile, ubiquitously expressed potassium channel β subunit associated with CAD and cardiac arrhythmia susceptibility in humans and mice. Here, to identify novel KCNE2 interaction partners, we employed yeast two-hybrid screening of adult and fetal human heart libraries using the KCNE2 intracellular C-terminal domain as bait. Testin (encoded by TES), an endothelial cell-expressed, CAD-associated, focal adhesion protein, was identified as a high-confidence interaction partner for KCNE2. We confirmed physical association between KCNE2 and Testin in vitro by co-immunoprecipitation. Whole-cell patch clamp electrophysiology revealed that KCNE2 negative-shifts the voltage dependence and increases the rate of activation of the endothelial cell and cardiomyocyte-expressed Kv channel α subunit, Kv1.5 in CHO cells, whereas Testin did not alter Kv1.5 function. However, Testin nullified KCNE2 effects on Kv1.5 voltage dependence and gating kinetics. In contrast, Testin did not prevent KCNE2 regulation of KCNQ1 gating. The data identify a novel role for Testin as a tertiary ion channel regulatory protein. Future studies will address the potential role for KCNE2-Testin interactions in arterial and myocyte physiology and CAD.

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 β-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 (EC₅₀ = 0.7 µM), 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 γ-amino-β-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 STATEMENT: Ketogenic diets are used to treat refractory epilepsy but the therapeutic mechanism is not fully understood. Here, we show that clinically relevant concentrations of β-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 β-hydroxybutyrate.

Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K⁺ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

Transmembrane channel-like protein isoform 1 (TMC1) is essential for the generation of mechano-electrical transducer currents in hair cells of the inner ear. TMC1 disruption causes hair cell degeneration and deafness in mice and humans. Although thought to be expressed at the cell surface in vivo, TMC1 remains in the endoplasmic reticulum when heterologously expressed in standard cell lines, precluding determination of its roles in mechanosensing and pore formation. Here, we report that the KCNQ1 Kv channel forms complexes with TMC1 and rescues its surface expression when coexpressed in Chinese Hamster Ovary cells. TMC1 rescue is specific for KCNQ1 within the KCNQ family, is prevented by a KCNQ1 trafficking-deficient mutation, and is influenced by KCNE β subunits and inhibition of KCNQ1 endocytosis. TMC1 lowers KCNQ1 and KCNQ1-KCNE1 K⁺ currents, and despite the surface expression, it does not detectably respond to mechanical stimulation or high salt. We conclude that TMC1 is not intrinsically mechano- or osmosensitive but has the capacity for cell surface expression, and requires partner protein(s) for surface expression and mechanosensitivity. We suggest that KCNQ1, expression of which is not thought to overlap with TMC1 in hair cells, is a proxy partner bearing structural elements or a sequence motif reminiscent of a true in vivo TMC1 hair cell partner. Discovery of the first reported strategy to rescue TMC1 surface expression should aid future studies of the TMC1 function and native partners.

Transmembrane channel-like protein isoform 1 (TMC1) is essential for the generation of mechano-electrical transducer currents in hair cells of the inner ear. TMC1 disruption causes hair cell degeneration and deafness in mice and humans. Although thought to be expressed at the cell surface in vivo, TMC1 remains in the endoplasmic reticulum when heterologously expressed in standard cell lines, precluding determination of its roles in mechanosensing and pore formation. Here, we report that the KCNQ1 Kv channel forms complexes with TMC1 and rescues its surface expression when coexpressed in Chinese Hamster Ovary cells. TMC1 rescue is specific for KCNQ1 within the KCNQ family, is prevented by a KCNQ1 trafficking-deficient mutation, and is influenced by KCNE β subunits and inhibition of KCNQ1 endocytosis. TMC1 lowers KCNQ1 and KCNQ1-KCNE1 K⁺ currents, and despite the surface expression, it does not detectably respond to mechanical stimulation or high salt. We conclude that TMC1 is not intrinsically mechano- or osmosensitive but has the capacity for cell surface expression, and requires partner protein(s) for surface expression and mechanosensitivity. We suggest that KCNQ1, expression of which is not thought to overlap with TMC1 in hair cells, is a proxy partner bearing structural elements or a sequence motif reminiscent of a true in vivo TMC1 hair cell partner. Discovery of the first reported strategy to rescue TMC1 surface expression should aid future studies of the TMC1 function and native partners.