An ankyrin G-binding motif mediates TRAAK periodic localization at axon initial segments of hippocampal pyramidal neurons.

The axon initial segment (AIS) is a critical compartment in neurons. It converts postsynaptic input into action potentials that subsequently trigger information transfer to target neurons. This process relies on the presence of several voltage-gated sodium (NaV) and potassium (KV) channels that accumulate in high densities at the AIS. TRAAK is a mechanosensitive leak potassium channel that was recently localized to the nodes of Ranvier. Here, we uncover that TRAAK is also present in AISs of hippocampal and cortical neurons in the adult rat brain as well as in AISs of cultured rat hippocampal neurons. We show that the AIS localization is driven by a C-terminal ankyrin G-binding sequence that organizes TRAAK in a 190 nm spaced periodic pattern that codistributes with periodically organized ankyrin G. We furthermore uncover that while the identified ankyrin G-binding motif is analogous to known ankyrin G-binding motifs in NaV1 and KV7.2/KV7.3 channels, it was acquired by convergent evolution. Our findings identify TRAAK as an AIS ion channel that convergently acquired an ankyrin G-binding motif and expand the role of ankyrin G to include the nanoscale organization of ion channels at the AIS.

Multicolor, Cell-Impermeable, and High Affinity BACE1 Inhibitor Probes Enable Superior Endogenous Staining and Imaging of Single Molecules.

The prevailing but not undisputed amyloid cascade hypothesis places the ß-site of APP cleaving enzyme 1 (BACE1) center stage in Alzheimer's Disease pathogenesis. Here, we investigated functional properties of BACE1 with novel tag- and antibody-free labeling tools, which are conjugates of the BACE1-inhibitor IV (also referred to as C3) linked to different impermeable Alexa Fluor dyes. We show that these fluorescent small molecules bind specifically to BACE1, with a 1:1 labeling stoichiometry at their orthosteric site. This is a crucial property especially for single-molecule and super-resolution microscopy approaches, allowing characterization of the dyes' labeling capabilities in overexpressing cell systems and in native neuronal tissue. With multiple colors at hand, we evaluated BACE1-multimerization by Förster resonance energy transfer (FRET) acceptor-photobleaching and single-particle imaging of native BACE1. In summary, our novel fluorescent inhibitors, termed Alexa-C3, offer unprecedented insights into protein-protein interactions and diffusion behavior of BACE1 down to the single molecule level.

Hijacking of internal calcium dynamics by intracellularly residing viral rhodopsins.

Rhodopsins are ubiquitous light-driven membrane proteins with diverse functions, including ion transport. Widely distributed, they are also coded in the genomes of giant viruses infecting phytoplankton where their function is not settled. Here, we examine the properties of OLPVR1 (Organic Lake Phycodnavirus Rhodopsin) and two other type 1 viral channelrhodopsins (VCR1s), and demonstrate that VCR1s accumulate exclusively intracellularly, and, upon illumination, induce calcium release from intracellular IP3-dependent stores. In vivo, this light-induced calcium release is sufficient to remote control muscle contraction in VCR1-expressing tadpoles. VCR1s natively confer light-induced Ca2+ release, suggesting a distinct mechanism for reshaping the response to light of virus-infected algae. The ability of VCR1s to photorelease calcium without altering plasma membrane electrical properties marks them as potential precursors for optogenetics tools, with potential applications in basic research and medicine.

A photoswitchable inhibitor of TREK channels controls pain in wild-type intact freely moving animals.

By endowing light control of neuronal activity, optogenetics and photopharmacology are powerful methods notably used to probe the transmission of pain signals. However, costs, animal handling and ethical issues have reduced their dissemination and routine use. Here we report LAKI (Light Activated K+ channel Inhibitor), a specific photoswitchable inhibitor of the pain-related two-pore-domain potassium TREK and TRESK channels. In the dark or ambient light, LAKI is inactive. However, alternating transdermal illumination at 365 nm and 480 nm reversibly blocks and unblocks TREK/TRESK current in nociceptors, enabling rapid control of pain and nociception in intact and freely moving mice and nematode. These results demonstrate, in vivo, the subcellular localization of TREK/TRESK at the nociceptor free nerve endings in which their acute inhibition is sufficient to induce pain, showing LAKI potential as a valuable tool for TREK/TRESK channel studies. More importantly, LAKI gives the ability to reversibly remote-control pain in a non-invasive and physiological manner in naive animals, which has utility in basic and translational pain research but also in in vivo analgesic drug screening and validation, without the need of genetic manipulations or viral infection.

Regulation of two-pore-domain potassium TREK channels and their involvement in pain perception and migraine.

The ability to sense pain signals is closely linked to the activity of ion channels expressed in nociceptors, the first neurons that transduce noxious stimuli into pain. Among these ion channels, TREK1, TREK2 and TRAAK from the TREK subfamily of the Two-Pore-Domain potassium (K2P) channels, are hyperpolarizing channels that render neurons hypoexcitable. They are regulated by diverse physical and chemical stimuli as well as neurotransmitters through G-protein coupled receptor activation. Here, we review the molecular mechanisms underlying these regulations and their functional relevance in pain and migraine induction.

Photopharmacological approaches for dissecting potassium channel physiology.

Photopharmacology allows for the remote control of ion channels and receptors by the application of light-sensitive compounds. Upon irradiation with light these molecules change their configuration, enabling channel modulation with both spatial and temporal resolution. For the control of potassium channel physiology mainly two approaches have evolved. Photoswitchable tethered ligands (PTLs) and freely diffusible photochromic ligands (PCLs), targeting K+ channels, serve to gain insights in neuronal functions of the brain and the heart, whereby the molecules have been refined in the past years with special focus on improving switching characteristics in terms of red-shifted wavelengths and temporal resolution. In this review we provide an overview about the application of these tools in studying potassium channels and neuronal circuit, highlighting recent developments towards future implementations.

TREK channel activation suppresses migraine pain phenotype.

Activation and sensitization of trigeminal ganglia (TG) sensory neurons, leading to the release of pro-inflammatory peptides such as calcitonin gene-related peptide (CGRP), are likely a key component in migraine-related headache induction. Reducing TG neuron excitability represents therefore an attractive alternative strategy to relieve migraine pain. Here by using pharmacology and genetic invalidation ex vivo and in vivo, we demonstrate that activating TREK1 and TREK2 two-pore-domain potassium (K2P) channels inhibits TG neuronal firing sufficiently to fully reverse the migraine-like phenotype induced by NO-donors in rodents. Finally, targeting TREK is as efficient as treatment with CGRP antagonists, which represents one of the most effective migraine therapies. Altogether, our results demonstrate that inhibiting TG excitability by pharmacological activation of TREK channels should be considered as an alternative to the current migraine treatment.

A fine-tuned azobenzene for enhanced photopharmacology in vivo.

Despite the power of photopharmacology for interrogating signaling proteins, many photopharmacological systems are limited by their efficiency, speed, or spectral properties. Here, we screen a library of azobenzene photoswitches and identify a urea-substituted "azobenzene-400" core that offers bistable switching between cis and trans with improved kinetics, light sensitivity, and a red-shift. We then focus on the metabotropic glutamate receptors (mGluRs), neuromodulatory receptors that are major pharmacological targets. Synthesis of "BGAG12,400," a photoswitchable orthogonal, remotely tethered ligand (PORTL), enables highly efficient, rapid optical agonism following conjugation to SNAP-tagged mGluR2 and permits robust optical control of mGluR1 and mGluR5 signaling. We then produce fluorophore-conjugated branched PORTLs to enable dual imaging and manipulation of mGluRs and highlight their power in ex vivo slice and in vivo behavioral experiments in the mouse prefrontal cortex. Finally, we demonstrate the generalizability of our strategy by developing an improved soluble, photoswitchable pore blocker for potassium channels.

KCNE1 is an auxiliary subunit of two distinct ion channel superfamilies.

Determination of what is the specificity of subunits composing a protein complex is essential when studying gene variants on human pathophysiology. The pore-forming α-subunit KCNQ1, which belongs to the voltage-gated ion channel superfamily, associates to its ß-auxiliary subunit KCNE1 to generate the slow cardiac potassium IKs current, whose dysfunction leads to cardiac arrhythmia. Using pharmacology, gene invalidation, and single-molecule fluorescence assays, we found that KCNE1 fulfils all criteria of a bona fide auxiliary subunit of the TMEM16A chloride channel, which belongs to the anoctamin superfamily. Strikingly, assembly with KCNE1 switches TMEM16A from a calcium-dependent to a voltage-dependent ion channel. Importantly, clinically relevant inherited mutations within the TMEM16A-regulating domain of KCNE1 abolish the TMEM16A modulation, suggesting that the TMEM16A-KCNE1 current may contribute to inherited pathologies. Altogether, these findings challenge the dogma of the specificity of auxiliary subunits regarding protein complexes and questions ion channel classification.

Migraine and Two-Pore-Domain Potassium Channels.

Migraine is a common, disabling neurological disorder with a genetic, environmental, and hormonal component with an annual prevalence estimated at ~15%. It is characterized by attacks of severe, usually unilateral and throbbing headache, and can be accompanied by nausea, vomiting, and photophobia. Migraine is clinically divided into two main subtypes: migraine with aura, when it is preceded by transient neurological disturbances due to cortical spreading depression (CSD), and migraine without aura. Activation and sensitization of trigeminal sensory neurons, leading to the release of pro-inflammatory peptides, is likely a key component in headache pain initiation and transmission in migraine. In the present review, we will focus on the function of two-pore-domain potassium (K2P) channels, which control trigeminal sensory neuron excitability and their potential interest for developing new drugs to treat migraine.

TREK for High-Speed and High-Frequency Conduction through the Axon.

In this issue of Neuron, Kanda et al. (2019) find that the two-pore domain potassium channels TRAAK and TREK1 drive axonal action potential repolarization for high-speed and high-frequency nervous impulses in mammalian myelinated nerves.

[Two-pore-domain potassium channels and molecular mechanisms underlying migraine]. / Canaux potassiques à deux domaines P (K2P) et migraine.

Migraine is a common, disabling neurological disorder with genetic, environmental and hormonal components and a prevalence estimated at ∼15%. Migraine episodes are notably related, among several factors, to electric hyperexcitability in sensory neurons. Their electrical activity is controlled by ion channels that generate current, specifically by the two-pore-domain potassium, K2P, channels, which inhibit electrical activity. Mutation in the gene encoding TRESK, a K2P channel, causes the formation of TRESK-MT1, the expected non-functional C-terminal truncated TRESK channel, and an additional unexpected protein, TRESK-MT2, which corresponds to a non-functional N-terminal truncated TRESK channel, through a mechanism called frameshift mutation-induced Alternative Translation Initiation (fsATI). TRESK-MT1 is inactive but TRESK-M2 targets two other ion channels, TREK1 and TREK2, inducing a great stimulation of the neuronal electrical activity that may cause migraines. These findings identify TREK1 and TREK2 as potential molecular targets for migraine treatment and suggest that fsATI should be considered as a distinct class of mutations.

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK.

It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.

Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity.

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function.

A Bacterial Toxin with Analgesic Properties: Hyperpolarization of DRG Neurons by Mycolactone.

Mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, is the etiological agent of Buruli ulcer. This lipid toxin is endowed with pleiotropic effects, presents cytotoxic effects at high doses, and notably plays a pivotal role in host response upon colonization by the bacillus. Most remarkably, mycolactone displays intriguing analgesic capabilities: the toxin suppresses or alleviates the pain of the skin lesions it inflicts. We demonstrated that the analgesic capability of mycolactone was not attributable to nerve damage, but instead resulted from the triggering of a cellular pathway targeting AT2 receptors (angiotensin II type 2 receptors; AT2R), and leading to potassium-dependent hyperpolarization. This demonstration paves the way to new nature-inspired analgesic protocols. In this direction, we assess here the hyperpolarizing properties of mycolactone on nociceptive neurons. We developed a dedicated medium-throughput assay based on membrane potential changes, and visualized by confocal microscopy of bis-oxonol-loaded Dorsal Root Ganglion (DRG) neurons. We demonstrate that mycolactone at non-cytotoxic doses triggers the hyperpolarization of DRG neurons through AT2R, with this action being not affected by known ligands of AT2R. This result points towards novel AT2R-dependent signaling pathways in DRG neurons underlying the analgesic effect of mycolactone, with the perspective for the development of new types of nature-inspired analgesics.

Heterodimerization within the TREK channel subfamily produces a diverse family of highly regulated potassium channels.

Twik-related K(+) channel 1 (TREK1), TREK2, and Twik-related arachidonic-acid stimulated K(+) channel (TRAAK) form the TREK subfamily of two-pore-domain K(+) (K2P) channels. Despite sharing up to 78% sequence homology and overlapping expression profiles in the nervous system, these channels show major differences in their regulation by physiological stimuli. For instance, TREK1 is inhibited by external acidification, whereas TREK2 is activated. Here, we investigated the ability of the members of the TREK subfamily to assemble to form functional heteromeric channels with novel properties. Using single-molecule pull-down (SiMPull) from HEK cell lysate and subunit counting in the plasma membrane of living cells, we show that TREK1, TREK2, and TRAAK readily coassemble. TREK1 and TREK2 can each heterodimerize with TRAAK, but do so less efficiently than with each other. We functionally characterized the heterodimers and found that all combinations form outwardly rectifying potassium-selective channels but with variable voltage sensitivity and pH regulation. TREK1-TREK2 heterodimers show low levels of activity at physiological external pH but, unlike their corresponding homodimers, are activated by both acidic and alkaline conditions. Modeling based on recent crystal structures, along with mutational analysis, suggests that each subunit within a TREK1-TREK2 channel is regulated independently via titratable His. Finally, TREK1/TRAAK heterodimers differ in function from TRAAK homodimers in two critical ways: they are activated by both intracellular acidification and alkalinization and are regulated by the enzyme phospholipase D2. Thus, heterodimerization provides a means for diversifying functionality through an expansion of the channel types within the K2P channels.

Area-specific development of distinct projection neuron subclasses is regulated by postnatal epigenetic modifications.

During cortical development, the identity of major classes of long-distance projection neurons is established by the expression of molecular determinants, which become gradually restricted and mutually exclusive. However, the mechanisms by which projection neurons acquire their final properties during postnatal stages are still poorly understood. In this study, we show that the number of neurons co-expressing Ctip2 and Satb2, respectively involved in the early specification of subcerebral and callosal projection neurons, progressively increases after birth in the somatosensory cortex. Ctip2/Satb2 postnatal co-localization defines two distinct neuronal subclasses projecting either to the contralateral cortex or to the brainstem suggesting that Ctip2/Satb2 co-expression may refine their properties rather than determine their identity. Gain- and loss-of-function approaches reveal that the transcriptional adaptor Lmo4 drives this maturation program through modulation of epigenetic mechanisms in a time- and area-specific manner, thereby indicating that a previously unknown genetic program postnatally promotes the acquisition of final subtype-specific features.

Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid.

Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.