Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR’s pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR’s role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.

The tandem of pore domain in a weak inwardly rectifying K + channel (Twik)-related acid-arachidonic activated K + channel (TRAAK) and Twikrelated K + channels (TREK) 1 and TREK2 are active as homodimers gated by stretch, fatty acids, pH, and G protein-coupled receptors. These two-pore domain potassium (K 2P ) channels are broadly expressed in the nervous system where they control excitability. TREK/TRAAK KO mice display altered phenotypes related to nociception, neuroprotection afforded by polyunsaturated fatty acids, learning and memory, mood control, and sensitivity to general anesthetics. These channels have emerged as promising targets for the development of new classes of anesthetics, analgesics, antidepressants, neuroprotective agents, and drugs against addiction. Here, we show that the TREK1, TREK2, and TRAAK subunits assemble and form active heterodimeric channels with electrophysiological, regulatory, and pharmacological properties different from those of homodimeric channels. Heteromerization occurs between all TREK variants produced by alternative splicing and alternative translation initiation. These results unveil a previously unexpected diversity of K 2P channels that will be challenging to analyze in vivo, but which opens new perspectives for the development of clinically relevant drugs. + channels (TREK) 1, TREK2, and Twik-related acid-arachidonic activated K + channel (TRAAK) channels produce inhibitory background K + currents (1-3). These channels respond to neuroprotective fatty acids, mechanical stretch, temperature, pH, and neurotransmitters through G proteincoupled receptors (GPCRs) (for a recent review, see ref. 4). TREK1 is activated by volatile anesthetics and plays a role in general anesthesia. These channels have emerged as promising targets for the development of other classes of clinical compounds. TREK1 is activated by opioid receptors and contributes to morphine-induced analgesia, but is not involved in morphine-induced constipation, respiratory depression, and dependence (5). TREK1 openers, acting downstream from opioid receptors, might have strong analgesic effects without adverse effects (6). TRAAK may also be a good target for analgesia: Its activation by angiotensin II receptors is responsible for the painless nature of the early lesions of the necrotizing tropical disease Buruli ulcer (7). Also, activation of TREK1 by GABA B receptors in the hippocampus (8), and inhibition of TREK2 by neurotensin receptors in the entorhinal cortex (9), suggests that specific modulators of these channels might also have beneficial actions against drug abuse, and against learning slow down and memory deficits in Alzheimer's disease. Finally, inhibition of TREK1 by spadin (10), an endogenous peptide, or by the antidepressant fluoxetine (Prozac) (11), pinpoints it as a valuable target for the treatment of depression. TREK/TRAAK channels are broadly expressed in the nervous system (12-15). TREK1 is more specifically expressed in the striatum, TREK2 in the cerebellum, and TRAAK in the thalamus. All...

Background: THIK1 and THIK2 are related leak K ϩ channel subunits.Results: Assembly of THIK1 with THIK2 was shown by dominant negative effects of pore-mutated subunits, in situ proximity ligation assay, FRET, and electrophysiology of covalent THIK1/THIK2 dimers. Conclusion: THIK1 and THIK2 assemble and form active channels. Significance: In cell and tissues co-expressing THIK1 and THIK2, heterodimeric channels may contribute to cell excitability.Despite a high level of sequence homology, tandem pore domain halothane-inhibited K ؉ channel 1 (THIK1) produces background K ؉ currents, whereas THIK2 is silent. This lack of

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.

Intracellular organelles change their size during trafficking and maturation. This requires the transport of ions and water across their membranes. Macropinocytosis, a ubiquitous form of endocytosis of particular importance for immune and cancer cells, generates large vacuoles that can be followed optically. Shrinkage of macrophage macropinosomes depends on TPC-mediated Na+ efflux and Cl− exit through unknown channels. Relieving osmotic pressure facilitates vesicle budding, positioning osmotic shrinkage upstream of vesicular sorting and trafficking. Here we identify the missing macrophage Cl− channel as the proton-activated Cl− channel ASOR/TMEM206. ASOR activation requires Na+-mediated depolarization and luminal acidification by redundant transporters including H+-ATPases and CLC 2Cl−/H+ exchangers. As corroborated by mathematical modelling, feedback loops requiring the steep voltage and pH dependencies of ASOR and CLCs render vacuole resolution resilient towards transporter copy numbers. TMEM206 disruption increased albumin-dependent survival of cancer cells. Our work suggests a function for the voltage and pH dependence of ASOR and CLCs, provides a comprehensive model for ion-transport-dependent vacuole maturation and reveals biological roles of ASOR.

Among K2P 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 K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated anion channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying ASOR chloride channel. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.

No abstract