Crosstalk between KCNK3-Mediated Ion Current and Adrenergic Signaling Regulates Adipose Thermogenesis and Obesity.

Adrenergic stimulation promotes lipid mobilization and oxidation in brown and beige adipocytes, where the harnessed energy is dissipated as heat in a process known as adaptive thermogenesis. The signaling cascades and energy-dissipating pathways that facilitate thermogenesis have been extensively described, yet little is known about the counterbalancing negative regulatory mechanisms. Here, we identify a two-pore-domain potassium channel, KCNK3, as a built-in rheostat negatively regulating thermogenesis. Kcnk3 is transcriptionally wired into the thermogenic program by PRDM16, a master regulator of thermogenesis. KCNK3 antagonizes norepinephrine-induced membrane depolarization by promoting potassium efflux in brown adipocytes. This limits calcium influx through voltage-dependent calcium channels and dampens adrenergic signaling, thereby attenuating lipolysis and thermogenic respiration. Adipose-specific Kcnk3 knockout mice display increased energy expenditure and are resistant to hypothermia and obesity. These findings uncover a critical K+-Ca2+-adrenergic signaling axis that acts to dampen thermogenesis, maintain tissue homeostasis, and reveal an electrophysiological regulatory mechanism of adipocyte function.

A Non-canonical Voltage-Sensing Mechanism Controls Gating in K2P K(+) Channels.

Two-pore domain (K2P) K(+) channels are major regulators of excitability that endow cells with an outwardly rectifying background "leak" conductance. In some K2P channels, strong voltage-dependent activation has been observed, but the mechanism remains unresolved because they lack a canonical voltage-sensing domain. Here, we show voltage-dependent gating is common to most K2P channels and that this voltage sensitivity originates from the movement of three to four ions into the high electric field of an inactive selectivity filter. Overall, this ion-flux gating mechanism generates a one-way "check valve" within the filter because outward movement of K(+) induces filter opening, whereas inward movement promotes inactivation. Furthermore, many physiological stimuli switch off this flux gating mode to convert K2P channels into a leak conductance. These findings provide insight into the functional plasticity of a K(+)-selective filter and also refine our understanding of K2P channels and the mechanisms by which ion channels can sense voltage.

The TWIK2 Potassium Efflux Channel in Macrophages Mediates NLRP3 Inflammasome-Induced Inflammation.

Potassium (K+) efflux across the plasma membrane is thought to be an essential mechanism for ATP-induced NLRP3 inflammasome activation, yet the identity of the efflux channel has remained elusive. Here we identified the two-pore domain K+ channel (K2P) TWIK2 as the K+ efflux channel triggering NLRP3 inflammasome activation. Deletion of Kcnk6 (encoding TWIK2) prevented NLRP3 activation in macrophages and suppressed sepsis-induced lung inflammation. Adoptive transfer of Kcnk6-/- macrophages into mouse airways after macrophage depletion also prevented inflammatory lung injury. The K+ efflux channel TWIK2 in macrophages has a fundamental role in activating the NLRP3 inflammasome and consequently mediates inflammation, pointing to TWIK2 as a potential target for anti-inflammatory therapies.

KCNK1 promotes proliferation and metastasis of breast cancer cells by activating lactate dehydrogenase A (LDHA) and up-regulating H3K18 lactylation.

Breast cancer is the most prevalent malignancy and the most significant contributor to mortality in female oncology patients. Potassium Two Pore Domain Channel Subfamily K Member 1 (KCNK1) is differentially expressed in a variety of tumors, but the mechanism of its function in breast cancer is unknown. In this study, we found for the first time that KCNK1 was significantly up-regulated in human breast cancer and was correlated with poor prognosis in breast cancer patients. KCNK1 promoted breast cancer proliferation, invasion, and metastasis in vitro and vivo. Further studies unexpectedly revealed that KCNK1 increased the glycolysis and lactate production in breast cancer cells by binding to and activating lactate dehydrogenase A (LDHA), which promoted histones lysine lactylation to induce the expression of a series of downstream genes and LDHA itself. Notably, increased expression of LDHA served as a vicious positive feedback to reduce tumor cell stiffness and adhesion, which eventually resulted in the proliferation, invasion, and metastasis of breast cancer. In conclusion, our results suggest that KCNK1 may serve as a potential breast cancer biomarker, and deeper insight into the cancer-promoting mechanism of KCNK1 may uncover a novel therapeutic target for breast cancer treatment.

TREK-1 and Best1 channels mediate fast and slow glutamate release in astrocytes upon GPCR activation.

Astrocytes release glutamate upon activation of various GPCRs to exert important roles in synaptic functions. However, the molecular mechanism of release has been controversial. Here, we report two kinetically distinct modes of nonvesicular, channel-mediated glutamate release. The fast mode requires activation of G(αi), dissociation of G(ßγ), and subsequent opening of glutamate-permeable, two-pore domain potassium channel TREK-1 through direct interaction between G(ßγ) and N terminus of TREK-1. The slow mode is Ca(2+) dependent and requires G(αq) activation and opening of glutamate-permeable, Ca(2+)-activated anion channel Best1. Ultrastructural analyses demonstrate that TREK-1 is preferentially localized at cell body and processes, whereas Best1 is mostly found in microdomains of astrocytes near synapses. Diffusion modeling predicts that the fast mode can target neuronal mGluR with peak glutamate concentration of 100 µM, whereas slow mode targets neuronal NMDA receptors at around 1 µM. Our results reveal two distinct sources of astrocytic glutamate that can differentially influence neighboring neurons.

C-type inactivation and proton modulation mechanisms of the TASK3 channel.

The TWIK-related acid-sensitive K+ channel 3 (TASK3) belongs to the two-pore domain (K2P) potassium channel family, which regulates cell excitability by mediating a constitutive "leak" potassium efflux in the nervous system. Extracellular acidification inhibits TASK3 channel, but the molecular mechanism by which channel inactivation is coupled to pH decrease remains unclear. Here, we report the cryo-electron microscopy structures of human TASK3 at neutral and acidic pH. Structural comparison revealed selectivity filter (SF) rearrangements upon acidification, characteristic of C-type inactivation, but with a unique structural basis. The extracellular mouth of the SF was prominently dilated and simultaneously blocked by a hydrophobic gate. His98 protonation shifted the conformational equilibrium between the conductive and C-type inactivated SF toward the latter by engaging a cation-π interaction with Trp78, consistent with molecular dynamics simulations and electrophysiological experiments. Our work illustrated how TASK3 is gated in response to extracellular pH change and implies how physiological stimuli might directly modulate the C-type gating of K2P channels.

Structural basis for pH gating of the two-pore domain K+ channel TASK2.

TASK2 (also known as KCNK5) channels generate pH-gated leak-type K+ currents to control cellular electrical excitability1-3. TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem4-6 and pH homeostasis by kidney proximal tubule cells7,8. These roles depend on channel activation by intracellular and extracellular alkalization3,8,9, but the mechanistic basis for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy structures of Mus musculus TASK2 in lipid nanodiscs in open and closed conformations. We identify two gates, distinct from previously observed K+ channel gates, controlled by stimuli on either side of the membrane. Intracellular gating involves lysine protonation on inner helices and the formation of a protein seal between the cytoplasm and the channel. Extracellular gating involves arginine protonation on the channel surface and correlated conformational changes that displace the K+-selectivity filter to render it nonconductive. These results explain how internal and external protons control intracellular and selectivity filter gates to modulate TASK2 activity.

A lower X-gate in TASK channels traps inhibitors within the vestibule.

TWIK-related acid-sensitive potassium (TASK) channels-members of the two pore domain potassium (K2P) channel family-are found in neurons1, cardiomyocytes2-4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8-11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12-15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate-which we designate as an 'X-gate'-created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders.

Myometrium infection decreases TREK1 through NHE1 and increases contraction in pregnant mice.

Intrauterine infection during pregnancy can enhance uterine contractions. A two-pore K+ channel TREK1 is crucial for maintaining uterine quiescence and reducing contractility, with its properties regulated by pH changes in cell microenvironment. Meanwhile, the sodium hydrogen exchanger 1 (NHE1) plays a pivotal role in modulating cellular pH homeostasis, and its activation increases smooth muscle tension. By establishing an infected mouse model of Escherichia coli (E. coli) and lipopolysaccharide (LPS), we used Western blotting, real-time quantitative polymerase chain reaction, and immunofluorescence to detect changes of TREK1 and NHE1 expression in the myometrium, and isometric recording measured the uterus contraction. The NHE1 inhibitor cariporide was used to explore the effect of NHE1 on TREK1. Finally, cell contraction assay and siRNA transfection were performed to clarify the relationship between NHE1 and TREK1 in vitro. We found that the uterine contraction was notably enhanced in infected mice with E. coli and LPS administration. Meanwhile, TREK1 expression was reduced, whereas NHE1 expression was upregulated in infected mice. Cariporide alleviated the increased uterine contraction and promoted myometrium TREK1 expression in LPS-injected mice. Furthermore, suppression of NHE1 with siRNA transfection inhibited the contractility of uterine smooth muscle cells and activated the TREK1. Altogether, our findings indicate that infection increases the uterine contraction by downregulating myometrium TREK1 in mice, and the inhibition of TREK1 is attributed to the activation of NHE1.NEW & NOTEWORTHY Present work found that infection during pregnancy will increase myometrium contraction. Infection downregulated NHE1 and followed TREK1 expression and activation decrease in myometrium, resulting in increased myometrium contraction.

Critical contribution of the intracellular C-terminal region to TRESK channel activity is revealed by the epithelial Na+ current ratio method.

TRESK (K2P18.1) possesses unique structural proportions within the K2P background potassium channel family. The previously described TRESK regulatory mechanisms are based on the long intracellular loop between the second and the third transmembrane segments (TMS). However, the functional significance of the exceptionally short intracellular C-terminal region (iCtr) following the fourth TMS has not yet been examined. In the present study, we investigated TRESK constructs modified at the iCtr by two-electrode voltage clamp and the newly developed epithelial sodium current ratio (ENaR) method in Xenopus oocytes. The ENaR method allowed the evaluation of channel activity by exclusively using electrophysiology and provided data that are otherwise not readily available under whole-cell conditions. TRESK homodimer was connected with two ENaC (epithelial Na+ channel) heterotrimers, and the Na+ current was measured as an internal reference, proportional to the number of channels in the plasma membrane. Modifications of TRESK iCtr resulted in diverse functional effects, indicating a complex contribution of this region to K+ channel activity. Mutations of positive residues in proximal iCtr locked TRESK in low activity, calcineurin-insensitive state, although this phosphatase binds to distant motifs in the loop region. Accordingly, mutations in proximal iCtr may prevent the transmission of modulation to the gating machinery. Replacing distal iCtr with a sequence designed to interact with the inner surface of the plasma membrane increased the activity of the channel to unprecedented levels, as indicated by ENaR and single channel measurements. In conclusion, the distal iCtr is a major positive determinant of TRESK function.

Long-term follow-up of TREK-1 KO mice reveals the development of bladder hypertrophy and impaired bladder smooth muscle contractility with age.

Stretch-activated two-pore domain K+ (K2P) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction.NEW & NOTEWORTHY This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology.

Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Two-Pore Domain Potassium Channels as Drug Targets: Anesthesia and Beyond.

Two-pore domain potassium (K2P) channels stabilize the resting membrane potential of both excitable and nonexcitable cells and, as such, are important regulators of cell activity. There are many conditions where pharmacological regulation of K2P channel activity would be of therapeutic benefit, including, but not limited to, atrial fibrillation, respiratory depression, pulmonary hypertension, neuropathic pain, migraine, depression, and some forms of cancer. Up until now, few if any selective pharmacological regulators of K2P channels have been available. However, recent publications of solved structures with small-molecule activators and inhibitors bound to TREK-1, TREK-2, and TASK-1 K2P channels have given insight into the pharmacophore requirements for compound binding to these sites. Together with the increasing availability of a number of novel, active, small-molecule compounds from K2P channel screening programs, these advances have opened up the possibility of rational activator and inhibitor design to selectively target K2P channels.

Synapse development is regulated by microglial THIK-1 K+ channels.

Microglia are the resident immune cells of the central nervous system. They constantly survey the brain parenchyma for redundant synapses, debris, or dying cells, which they remove through phagocytosis. Microglial ramification, motility, and cytokine release are regulated by tonically active THIK-1 K+ channels on the microglial plasma membrane. Here, we examined whether these channels also play a role in phagocytosis. Using pharmacological blockers and THIK-1 knockout (KO) mice, we found that a lack of THIK-1 activity approximately halved both microglial phagocytosis and marker levels for the lysosomes that degrade phagocytically removed material. These changes may reflect a decrease of intracellular [Ca2+]i activity, which was observed when THIK-1 activity was reduced, since buffering [Ca2+]i reduced phagocytosis. Less phagocytosis is expected to result in impaired pruning of synapses. In the hippocampus, mice lacking THIK-1 expression had an increased number of anatomically and electrophysiologically defined glutamatergic synapses during development. This resulted from an increased number of presynaptic terminals, caused by impaired removal by THIK-1 KO microglia. The dependence of synapse number on THIK-1 K+ channels, which control microglial surveillance and phagocytic ability, implies that changes in the THIK-1 expression level in disease states may contribute to altering neural circuit function.

Ultrasound activates mechanosensitive TRAAK K+ channels through the lipid membrane.

Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.

The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

KCNT1 Channel Blockers: A Medicinal Chemistry Perspective.

Potassium channels have recently emerged as suitable target for the treatment of epileptic diseases. Among potassium channels, KCNT1 channels are the most widely characterized as responsible for several epileptic and developmental encephalopathies. Nevertheless, the medicinal chemistry of KCNT1 blockers is underdeveloped so far. In the present review, we describe and analyse the papers addressing the issue of KCNT1 blockers' development and identification, also evidencing the pros and the cons of the scientific approaches therein described. After a short introduction describing the epileptic diseases and the structure-function of potassium channels, we provide an extensive overview of the chemotypes described so far as KCNT1 blockers, and the scientific approaches used for their identification.

Activation of neurokinin-III receptors modulates human atrial TASK-1 currents.

RATIONALE: The neurokinin-III receptor was recently shown to regulate atrial cardiomyocyte excitability by inhibiting atrial background potassium currents. TASK-1 (hK2P3.1) two-pore-domain potassium channels, which are expressed atrial-specifically in the human heart, contribute significantly to atrial background potassium currents. As TASK-1 channels are regulated by a variety of intracellular signalling cascades, they represent a promising candidate for mediating the electrophysiological effects of the Gq-coupled neurokinin-III receptor. OBJECTIVE: To investigate whether TASK-1 channels mediate the neurokinin-III receptor activation induced effects on atrial electrophysiology. METHODS AND RESULTS: In Xenopus laevis oocytes, heterologously expressing neurokinin-III receptor and TASK-1, administration of the endogenous neurokinin-III receptor ligands substance P or neurokinin B resulted in a strong TASK-1 current inhibition. This could be reproduced by application of the high affinity neurokinin-III receptor agonist senktide. Moreover, preincubation with the neurokinin-III receptor antagonist osanetant blunted the effect of senktide. Mutagenesis studies employing TASK-1 channel constructs which lack either protein kinase C (PKC) phosphorylation sites or the domain which is regulating the diacyl glycerol (DAG) sensitivity domain of TASK-1 revealed a protein kinase C independent mechanism of TASK-1 current inhibition: upon neurokinin-III receptor activation TASK-1 channels are blocked in a DAG-dependent fashion. Finally, effects of senktide on atrial TASK-1 currents could be reproduced in patch-clamp measurements, performed on isolated human atrial cardiomyocytes. CONCLUSIONS: Heterologously expressed human TASK-1 channels are inhibited by neurokinin-III receptor activation in a DAG dependent fashion. Patch-clamp measurements, performed on human atrial cardiomyocytes suggest that the atrial-specific effects of neurokinin-III receptor activation on cardiac excitability are predominantly mediated via TASK-1 currents.

Physiological and pathophysiological roles of the KCNK3 potassium channel in the pulmonary circulation and the heart.

Potassium channel subfamily K member 3 (KCNK3), encoded by the KCNK3 gene, is part of the two-pore domain potassium channel family, constitutively active at resting membrane potentials in excitable cells, including smooth muscle and cardiac cells. Several physiological and pharmacological mediators, such as intracellular signalling pathways, extracellular pH, hypoxia and anaesthetics, regulate KCNK3 channel function. Recent studies show that modulation of KCNK3 channel expression and function strongly influences pulmonary vascular cell and cardiomyocyte function. The altered activity of KCNK3 in pathological situations such as atrial fibrillation, pulmonary arterial hypertension and right ventricular dysfunction demonstrates the crucial role of KCNK3 in cardiovascular homeostasis. Furthermore, loss of function variants of KCNK3 have been identified in patients suffering from pulmonary arterial hypertension and atrial fibrillation. This review focuses on current knowledge of the role of the KCNK3 channel in pulmonary circulation and the heart, in healthy and pathological conditions.

Allosteric coupling between transmembrane segment 4 and the selectivity filter of TALK1 potassium channels regulates their gating by extracellular pH.

Opening of two-pore domain K+ channels (K2Ps) is regulated by various external cues, such as pH, membrane tension, or temperature, which allosterically modulate the selectivity filter (SF) gate. However, how these cues cause conformational changes in the SF of some K2P channels remains unclear. Herein, we investigate the mechanisms by which extracellular pH affects gating in an alkaline-activated K2P channel, TALK1, using electrophysiology and molecular dynamics (MD) simulations. We show that R233, located at the N-terminal end of transmembrane segment 4, is the primary pHo sensor. This residue distally regulates the orientation of the carbonyl group at the S1 potassium-binding site through an interacting network composed of residues on transmembrane segment 4, the pore helix domain 1, and the SF. Moreover, in the presence of divalent cations, we found the acidic pH-activated R233E mutant recapitulates the network interactions of protonated R233. Intriguingly, our data further suggested stochastic coupling between R233 and the SF gate, which can be described by an allosteric gating model. We propose that this allosteric model could predict the hybrid pH sensitivity in heterodimeric channels with alkaline-activated and acidic-activated K2P subunits.