Franck C. Chatelain

Birk-Barel syndrome (BBS), also known as KCNK9 imprinting syndrome, is a neurodevelopmental disorder caused by mutations in the maternally expressed paternally imprinted KCNK9 gene that encodes the TASK3 potassium channel. The first mutation identified in patients with BBS was a loss-of-function variant that reduces the potassium current mediated by TASK3. Subsequent studies have uncovered additional pathogenic variants, including gain-of-function mutations, that increase TASK3 activity. Here, we show that both mutations resulted in the expression of the hyperactive Task3M159I variant or the hypoactive Task3G236R variant, which caused early postnatal lethality in mice when maternally inherited, confirming the monogenic nature of BBS. These results indicate that the pathogenesis of BBS involves mechanisms other than alterations in channel activity. Consequently, the proposed therapeutic strategies, such as pharmacological modulation of the TASK3 channel function or epigenetic reactivation of the paternal KCNK9 allele, do not seem to be viable options. These findings also demonstrate that TASK3 knockout mice are not suitable models for studying KCNK9 imprinting syndrome or for developing therapies for affected patients. Therefore, there is an urgent need for animal models that allow conditional expression of BBS-associated mutations.

Introduction: Two-pore domain potassium channels regulate neuronal excitability by generating background potassium currents that stabilize the resting membrane potential. Although several two-pore domain potassium channels have been implicated in pain signaling, the physiological role of tandem pore domain halotane-inhibited K+ channel (THIK2) remains largely unknown despite its high expression in human and mouse nociceptive dorsal root ganglion (DRG) neurons. Objective: This study aimed to determine the cellular distribution of THIK2 in sensory neurons and to investigate its contribution to neuronal excitability and nociceptive processing under physiological and inflammatory conditions. Methods: We combined molecular analyses, electrophysiological recordings, and behavioral approaches. THIK1 and THIK2 expression patterns were mapped in mouse DRGs. Whole-cell electrophysiology was used to assess membrane excitability in sensory neurons from wild-type and THIK2 ^−/− knockout mice. Behavioral tests evaluated thermal sensitivity under naive and inflammatory conditions. Results: We provide the first comprehensive characterization of THIK1 and THIK2 expression in mouse DRG. THIK2 deletion increased neuronal firing during sustained stimulation, indicating a loss of tonic inhibitory control of membrane excitability, particularly in nonpeptidergic IB4-positive C-fiber neurons. Behaviorally, THIK2 ^−/− mice displayed marked thermal hypersensitivity at baseline and during inflammation, consistent with enhanced sensory neuron hyperexcitability. Conclusion: THIK2 channels act as key regulators preventing pathological hyperexcitability in nociceptive sensory neurons. Their loss leads to increased neuronal firing and enhanced thermal sensitivity, identifying THIK2 as a promising therapeutic target for chronic inflammatory pain.

Inherited mutations in the KCNH2 gene, which encodes the cardiac hERG potassium channel, are major contributors to arrhythmogenic syndromes such as long QT and short QT syndromes. However, clinical interpretation of the growing number of missense variants – many of which are classified as variants of uncertain significance (VUS) – remains a pressing challenge. Here, we present a semi-automated in silico pipeline for predicting hERG variant pathogenicity, acting as a binary classifier and integrating five structural metrics – residue volume, hydrophobicity, charge, steric clashes, and proximity to pathogenic hotspots – into a composite structural pathogenicity score (SPS) scaled from 1 to 5. Applied to 1727 hERG variants from ClinVar and from a French nationwide cohort, this binary classifier, termed SPARC, identified 260 variants as high risk of pathogenicity with SPS ≥3.25, of which a representative subset from the French cohort was functionally validated using high-throughput automated patch-clamp. Functional phenotyping confirmed the structural predictions, including for several VUS, demonstrating that comprehensive structural scoring can reliably stratify variant pathogenicity. This approach, benchmarked with Alpha Missense and Revel, offers a superior scalable, cost-effective pre-screening tool to guide clinical variant interpretation and prioritization for experimental validation.

Two-pore domain potassium channels (K _2P ) regulate neuronal excitability by acting as hyperpolarizing leak channels. Among them, THIK2 remain poorly characterized. Although no study has yet clearly linked them to excitability or pain, their selective expression in nociceptive neurons of the Dorsal Root Ganglia (DRG) suggests a role in nociception and pain regulation. This project investigates THIK2 channels in pain pathophysiology through molecular, electrophysiological, and behavioral approaches. We mapped THIK expression patterns and in THIK2 knock-out mice, we examined DRG neuron excitability and pain sensitivity. Results reveal thermal hypersensitivity under both naive and inflammatory conditions, indicating that THIK2 normally limits neuronal hyperexcitability. These findings position THIK2 as a potential therapeutic target in chronic inflammatory pain, with peripheral inhibition potentially offering analgesia without central opioid side effects.

Highly selective for K⁺ at neutral pH, the TWIK1 channel becomes permeable to Na⁺ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5–6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K⁺-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na⁺-permeable conformation of the channel.

At neutral pH, the TWIK1 channel is highly selective for K ⁺ . When exposed to acidification, it becomes permeable to Na ⁺ . This change occurs within minutes and is reversible. By combining pKa calculations, molecular dynamics (MD) simulations, mutagenesis and electrophysiology, we identified a network of residues involved in this unique property. MD simulations captured crucial features associated with channel gating and previously observed by cryogenic electron microscopy (cryo-EM) at pH7.4 and pH5, such as the pH-dependent orientation of the lateral side chain of the proton sensor His122 and the elongation of the entire pore structure upon acidification. Between the closed and open states of TWIK1 observed by cryo-EM, MD simulations show that the channel undergoes additional conformational changes between pH 7.5 to 6 that involves the His122, Glu235, Lys246 and Phe109 residues. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K ⁺ -selective state at high pH, disappear upon acidification. This causes dissociation of the Phe109 aromatic side chain from this network, ultimately leading to the Na ⁺ -permeable conformation of the channel.

Two-pore domain (K _2P ) potassium channels are active as dimers. They produce inhibitory currents regulated by a variety of stimuli. Among them, TALK1, TALK2 and TASK2 form a subfamily of structurally related K _2P channels stimulated by extracellular alkalosis. The human genes encoding them are clustered on chromosomal region 6p21. They are expressed in different tissues including the pancreas. By analyzing single cell transcriptomic data, we show that these channels are co-expressed in insulin-secreting pancreatic β cells. By different approaches we show that they form functional heterodimers. Heteromerization of TALK2 with TALK1 or with TASK2 endorses TALK2 with sensitivity to extracellular alkalosis in the physiological range. The association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K _2P channel family expanding the range of their potential physiological and pathophysiological roles.

Among K_2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined “silent subunits”. Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K_2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.