Background potassium currents carried by the KCNK family of two-pore-domain K ϩ channels are important determinants of resting membrane potential and cellular excitability. TWIK-related acid-sensitive K ϩ 1 (TASK-1, KCNK3) and TASK-3 (KCNK9) are pHsensitive subunits of the KCNK family that are closely related and coexpressed in many brain regions. There is accumulating evidence that these two subunits can form heterodimeric channels, but this evidence remains controversial. In addition, a substantial contribution of heterodimeric TASK channels to native currents has not been unequivocally established. In a heterologous expression system, we verified formation of heterodimeric TASK channels and characterized their properties; TASK-1 and TASK-3 were coimmunoprecipitated from membranes of mammalian cells transfected with the channel subunits, and a dominant negative TASK-1(Y191F) construct strongly diminished TASK-3 currents. Tandem-linked heterodimeric TASK channel constructs displayed a pH sensitivity (pK ϳ7.3) in the physiological range closer to that of TASK-1 (pK ϳ7.5) than TASK-3 (pK ϳ6.8). On the other hand, heteromeric TASK channels were like TASK-3 insofar as they were activated by high concentrations of isoflurane (0.8 mM), whereas TASK-1 channels were inhibited. The pH and isoflurane sensitivities of native TASK-like currents in hypoglossal motoneurons, which strongly express TASK-1 and TASK-3 mRNA, were best represented by TASK heterodimeric channels. Moreover, after blocking homomeric TASK-3 channels with ruthenium red, we found a major component of motoneuronal isoflurane-sensitive TASK-like current that could be attributed to heteromeric TASK channels. Together, these data indicate that TASK-1 and TASK-3 subunits coassociate in functional channels, and heteromeric TASK channels provide a substantial component of background K ϩ current in motoneurons with distinct modulatory properties.

Large aspiny cholinergic interneurons provide the sole source of striatal acetylcholine, a neurotransmitter critical for basal ganglia function; these tonically active interneurons receive excitatory inputs from corticostriatal glutamatergic afferents that act, in part, via metabotropic glutamate receptors (mGluRs). We combined electrophysiological recordings in brain slices with molecular neuroanatomy to identify distinct ion channel targets for mGluR1/5 receptors in striatal cholinergic interneurons: transient receptor potential channel 3/7 (TrpC3/C7) and Slo2.1. In recordings obtained with methanesulfonate-based internal solutions, we found an mGluR-activated current with voltage-dependent and pharmacological properties reminiscent of TrpC3 and TrpC7; expression of these TrpC subunits in cholinergic interneurons was verified by combined immunohistochemistry and in situ hybridization, and modulation of both TrpC channels was reconstituted in HEK293 (human embryonic kidney 293) cells cotransfected with mGluR1 or mGluR5. With a chloridebased internal solution, mGluR agonists did not activate interneuron TrpC-like currents. Instead, a time-dependent, outwardly rectifying K ϩ current developed after whole-cell access, and this Cl Ϫ -activated K ϩ current was strongly inhibited by volatile anesthetics and mGluR activation. This modulation was recapitulated in cells transfected with Slo2.1, a Na ϩ -and Cl Ϫ -activated K ϩ channel, and Slo2.1 expression was confirmed histochemically in striatal cholinergic interneurons. By using gramicidin perforated-patch recordings, we established that the predominant agonist-activated current was TrpC-like when ambient intracellular chloride was preserved, although a small K ϩ current contribution was observed in some cells. Together, our data indicate that mGluR1/5-mediated glutamatergic excitation of cholinergic interneurons is primarily a result of activation of TrpC3/TrpC7-like cationic channels; under conditions when intracellular NaCl is elevated, a Slo2.1 background K ϩ channel may also contribute.

General anesthetics cause sedation, hypnosis, and immobilization via CNS mechanisms that remain incompletely understood; contributions of particular anesthetic targets in specific neural pathways remain largely unexplored. Among potential molecular targets for mediating anesthetic actions, members of the TASK subgroup [TASK-1 (K2P3.1) and TASK-3 (K2P9.1)] of background K ϩ channels are appealing candidates since they are expressed in CNS sites relevant to anesthetic actions and activated by clinically relevant concentrations of inhaled anesthetics. Here, we used global and conditional TASK channel single and double subunit knock-out mice to demonstrate definitively that TASK channels account for motoneuronal, anesthetic-activated K ϩ currents and to test their contributions to sedative, hypnotic, and immobilizing anesthetic actions. In motoneurons from all knock-out mice lines, TASK-like currents were reduced and cells were less sensitive to hyperpolarizing effects of halothane and isoflurane. In an immobilization assay, higher concentrations of both halothane and isoflurane were required to render TASK knock-out animals unresponsive to a tail pinch; in assays of sedation (loss of movement) and hypnosis (loss-of-righting reflex), TASK knock-out mice showed a modest decrease in sensitivity, and only for halothane. In conditional knock-out mice, with TASK channel deletion restricted to cholinergic neurons, immobilizing actions of the inhaled anesthetics and sedative effects of halothane were reduced to the same extent as in global knock-out lines. These data indicate that TASK channels in cholinergic neurons are molecular substrates for select actions of inhaled anesthetics; for immobilization, which is spinally mediated, these data implicate motoneurons as the likely neuronal substrates.

TASK-1 (KCNK3) and TASK-3 (KCNK9) are members of the two-pore domain potassium channel family and form either homomeric or heteromeric open-rectifier (leak) channels. Recent evidence suggests that these channels contribute to the resting potential and input resistance in several neuron types, including hippocampal CA1 pyramidal cells. However, the evidence for TWIK-related acid-sensitive potassium (TASK)-like conductances in inhibitory interneurons is less clear, and mRNA expression has suggested that TASK channels are expressed in only a subpopulation of interneurons. Here we use immunocytochemistry to demonstrate prominent TASK-3 protein expression in both parvalbumin-positive-and a subpopulation of glutamic acid decarboxylase (GAD)67-positive interneurons. In addition, a TASK-like current (modulated by both pH and bupivacaine) was detected in 30 -50% of CA1 stratum oriens interneurons of various morphological classes. In most neurons, basic shifts in pH had a larger effect on the TASK-like current than acidic, suggesting that the current is mediated by TASK-1/TASK-3 heterodimers. These data suggest that TASK-like conductances are more prevalent in inhibitory interneurons than previously supposed.

Berg AP, Bayliss DA. Striatal cholinergic interneurons express a receptor-insensitive homomeric TASK-3-like background K ϩ current. J Neurophysiol 97: 1546 -1552, 2007. First published December 13, 2006; doi:10.1152/jn.01090.2006. Large aspiny cholinergic interneurons provide the sole source of striatal acetylcholine, a neurotransmitter essential for normal basal ganglia function. Cholinergic interneurons engage in multiple firing patterns that depend on interactions among various voltagedependent ion channels active at different membrane potentials. Leak conductances, particularly leak K ϩ channels, are of primary importance in establishing the prevailing membrane potential. We have combined molecular neuroanatomy with whole cell electrophysiology to demonstrate that TASK-3 (K 2P 9.1, Kcnk9) subunits contribute to leak K ϩ currents in striatal cholinergic interneurons. Immunostaining for choline acetyltransferase was combined with TASK-3 labeling, using nonradioactive cRNA probes or antisera selective for TASK-3, to demonstrate that striatal cholinergic neurons universally express TASK-3. Consistent with this, we isolated a pH-, anesthetic-, and Zn 2ϩ -sensitive current with properties expected of TASK-3 homodimeric channels. Surprisingly, activation of G␣q-linked receptors (metabotropic glutamate mGluR1/5 or histamine H1) did not appear to modulate native interneuron TASK-3-like currents. Together, our data indicate that homomeric TASK-3-like background K ϩ currents contribute to establishing membrane potential in striatal cholinergic interneurons and they suggest that receptor modulation of TASK channels is dependent on cell context. I N T R O D U C T I O NAcetylcholine (ACh) is essential for striatal function and changes in striatal cholinergic drive are implicated in the pathophysiology of movement disorders (Calabresi et al. 2000). A sparse population of large aspiny cholinergic interneurons provides the sole source of striatal ACh and regulation of membrane excitability of interneurons is therefore integral to many aspects of striatal function.Cholinergic interneurons discharge spontaneously in either a tonic or bursting pattern. The expression of these distinct firing patterns involves complex interactions among various voltagedependent ion channels-TTX-sensitive sodium channels; hyperpolarization-activated HCN channels; voltage-sensitive, inwardly rectifying, and calcium-activated K . In striatum, TREK-1 (K 2P 2.1, Kcnk2) is evenly distributed in a pattern most consistent with expression in medium spiny neurons (Heurteaux et al. 2004;Talley et al. 2001). On the other hand, TASK-3 is expressed in relatively large cells in a diffuse pattern similar to that of cholinergic interneurons (Karschin et al. 2001;Talley et al. 2001). In this study, we used histochemical and electrophysiological approaches to show that TASK-3 channels are functionally expressed in these cells, where they provide a background K ϩ conductance that contributes to the membrane potential; these predominantly homomeric, native TASK-3-...

Highlights d ALS patient-derived motor neurons are hyperexcitable d A high-throughput GCaMP screen is used to identify ALS excitability modulators d Kv7 ion channels, AMPA receptors, and the type 2 dopamine receptor are major targets d The D2 dopamine receptor modulates both neuronal excitability and survival

High-throughput screening for drug discovery is increasingly utilizing cellular systems of high physiological relevance, such as patient primary cells and organoid cultures. We used 3D-cultured cystic fibrosis patient bronchial epithelial cells to screen for new small-molecule correctors of the disease-causing F508del mutation in CFTR. Impaired mucociliary clearance due to insufficient airway hydration is a hallmark of cystic fibrosis and we used a simple measure of surface liquid levels to quantify F508del CFTR correction in cultured bronchial epithelial cells. Two robust assay formats were configured and used to screen more than 100,000 compounds as mixtures or individual compounds in 96-well format. The corrector discovery success rate, as measured by the number of hits confirmed by an electrophysiology assay on patient primary bronchial epithelial cells, was superior to screens in cell lines expressing recombinant F508del CFTR. Several novel corrector scaffolds were discovered that when combined with the clinical corrector VX-809 delivered combination responses greater than double that of VX-809 alone. This work exemplifies the advantages of a disease-relevant readout and 3D-cultured patient primary cells for the discovery of new drug candidates.

Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0–4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.