Professor Stephen Tucker

Abstract:

The TREK subfamily of 2-pore domain potassium channels act as polymodal sensors of multiple physical and chemical stimuli, coupling electrical excitability to the cellular environment. A nuanced understanding of TREK channel pharmacology is necessary for the development of novel experimental tools, for potential pharmacotherapies, and for understanding potential beneficial or adverse effects of clinically used drugs. Here, we sought to determine the mechanism of action of 2 TREK channel inhibitors: the β-blocker carvedilol and the antipsychotic fluphenazine. Using patch and 2-electrode voltage-clamp electrophysiology, we show that both carvedilol and fluphenazine exhibit an activation-mode dependence, with mechanical- and temperature-activation of TREK channels reducing drug sensitivity approximately 10-fold. Mutagenesis at a key intramembrane fenestration site leucine, modulator competition experiments, and in silico docking studies are all consistent with carvedilol and fluphenazine binding at a shared, overlapping binding site. These findings establish a conserved conformation-dependent mechanism of action for structurally diverse TREK inhibitors and identify the fenestration as a promiscuous binding site for a range of clinically used drugs. SIGNIFICANCE STATEMENT: TREK potassium channels play key roles in regulating membrane excitability in a wide range of cells. Modulation of these channels has been proposed for potential antiarrhythmic and antidepressant effects. This study defined the off-target molecular mechanism of action of the clinically used drugs carvedilol and fluphenazine on TREK channels and identify the TREK channel fenestration as a promiscuous binding site for inhibitory drugs.

Abstract:

Gain-of-function (GoF) missense variants in the two-pore domain (K2P) K+ channel TASK-1 (KCNK3) result in developmental delay with sleep apnea (DDSA), a neurodevelopmental channelopathy, while loss-of-function (LoF) variants cause pulmonary arterial hypertension. However, for the related TASK-3 channel (KCNK9), both LoF and GoF variants underlie a distinct neurodevelopmental disorder, KCNK9 imprinting syndrome (KIS). The relationship between genotype and phenotype in these disorders is further complicated because TASK-1 and TASK-3 can co-assemble into heteromeric channels with distinct functional properties. Here, we report additional patients with missense variants in KCNK3 and KCNK9 and investigate the effect of four novel genetic variants on the functional properties of homomeric and heteromeric TASK channels. Interestingly, two of these new pathogenic GoF variants (R131H and L122V) are found in both TASK-1 and TASK-3 and have equivalent functional effects on heteromeric TASK-1/TASK-3 channel activity, yet result in different clinical phenotypes. We have also determined a cryo-EM structure for the pathogenic L122V mutant TASK-3 channel, which suggests that subtle changes in gating and permeation within the inner cavity are responsible for its activatory effect. Overall, these results highlight the dominant role that homomeric TASK channels likely play in defining their associated channelopathies as well as the complexity of interpreting K+ channel dysfunction in pathophysiology.

Abstract:

Two-Pore Domain K+ (K2P) channels are crucial determinants of the resting membrane potential and of cellular electrical excitability in many different cell types. TASK-1 and TASK-3 K2P channel activity is also coupled to GPCR signalling pathways via Gαq and their subsequent inhibition is via direct interaction with diacylglycerol (DAG) generated from phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis. This regulation is defective in two different neurodevelopmental disorders, but the molecular mechanisms underlying this inhibitory process and the reasons for the GPCR-insensitivity of these disease-causing mutations remain unclear. Here we show that GqPCR inhibition inversely correlates with channel open probability, and results from a state-dependent destabilisation of the open state by DAG promoting channel closure. We also identify a DAG interaction-site within a groove between the M2, M3 and M4 domains, and show the crucial role of residues within this site in mediating the inhibitory effect and defining channel sensitivity. These results not only reveal the structural and molecular mechanisms underlying GqPCR regulation of TASK channels, but also explain the pathogenic effect of a common regulatory defect linked to different K2P channelopathies.

Abstract:

Pain management in animal experimentation is crucial for both ethical and scientific reasons, as unmanaged pain can distort physiological responses compromising data reliability. Current strategies are often invasive and pharmacology-based, introducing variability and confounding effects. Here, we present Light-Induced Analgesia, a drug-free, non-invasive method for pain relief in animals. We show that 365 nm illumination activates the pain-inhibitory TRAAK two-pore domain potassium (K2P) channel. This activation is driven by the oxidation of a native methionine at TRAAK’s regulatory fenestration site, triggering a conformational switch from its inactive (down) to active (up) state. We further demonstrate that this mechanism can be transferred to other related K2Ps via a single-point mutation, rendering them light-sensitive. In rodents, gentle skin exposure to 365 nm is sufficient to activate endogenous TRAAK, silence nociceptors, and produce potent, long-lasting analgesia that outperforms standard treatments. Light-Induced Analgesia thus offers an effective, drug-free alternative that can enhance animal welfare and experimental reliability in preclinical research.