Two-pore-domain potassium (K(+)) channels are substrates for resting K(+) currents in neurons. They are major targets for endogenous modulators, as well as for clinically important compounds such as volatile anesthetics. In the current study, we report on the CNS distribution in the rat and mouse of mRNA encoding seven two-pore-domain K(+) channel family members: TASK-1 (KCNK3), TASK-2 (KCNK5), TASK-3 (KCNK9), TREK-1 (KCNK2), TREK-2 (KCNK10), TRAAK (KCNK4), and TWIK-1 (KCNK1). All of these genes were expressed in dorsal root ganglia, and for all of the genes except TASK-2, there was a differential distribution in the CNS. For TASK-1, highest mRNA accumulation was seen in the cerebellum and somatic motoneurons. TASK-3 was much more widely distributed, with robust expression in all brain regions, with particularly high expression in somatic motoneurons, cerebellar granule neurons, the locus ceruleus, and raphe nuclei and in various nuclei of the hypothalamus. TREK-1 was highest in the striatum and in parts of the cortex (layer IV) and hippocampus (CA2 pyramidal neurons). mRNA for TRAAK also was highest in the cortex, whereas expression of TREK-2 was primarily restricted to the cerebellar granule cell layer. There was widespread distribution of TWIK-1, with highest levels in the cerebellar granule cell layer, thalamic reticular nucleus, and piriform cortex. The differential expression of each of these genes likely contributes to characteristic excitability properties in distinct populations of neurons, as well as to diversity in their susceptibility to modulation.

Inhibition of "leak" potassium (K+) channels is a widespread CNS mechanism by which transmitters induce slow excitation. We show that TASK-1, a two pore domain K+ channel, provides a prominent leak K+ current and target for neurotransmitter modulation in hypoglossal motoneurons (HMs). TASK-1 mRNA is present at high levels in motoneurons, including HMs, which express a K+ current with pH- and voltage-dependent properties virtually identical to those of the cloned channel. This pH-sensitive K+ channel was fully inhibited by serotonin, norepinephrine, substance P, thyrotropin-releasing hormone, and 3,5-dihydroxyphenylglycine, a group I metabotropic glutamate receptor agonist. The neurotransmitter effect was entirely reconstituted in HEK 293 cells coexpressing TASK-1 and the TRH-R1 receptor. Given its expression patterns and the widespread prevalence of this neuromodulatory mechanism, TASK-1 also likely supports this action in other CNS neurons.

Despite widespread use of volatile general anesthetics for well over a century, the mechanisms by which they alter specific CNS functions remain unclear. Here, we present evidence implicating the two-pore domain, pH-sensitive TASK-1 channel as a target for specific, clinically important anesthetic effects in mammalian neurons. In rat somatic motoneurons and locus coeruleus cells, two populations of neurons that express TASK-1 mRNA, inhalation anesthetics activated a neuronal K ϩ conductance, causing membrane hyperpolarization and suppressing action potential discharge. These membrane effects occurred at clinically relevant anesthetic levels, with precisely the steep concentration dependence expected for anesthetic effects of these compounds. The native neuronal K ϩ current displayed voltage-and time-dependent properties that were identical to those mediated by the open-rectifier TASK-1 channel. Moreover, the neuronal K ϩ channel and heterologously expressed TASK-1 were similarly modulated by extracellular pH. The decreased cellular excitability associated with TASK-1 activation in these cell groups probably accounts for specific CNS effects of anesthetics: in motoneurons, it likely contributes to anesthetic-induced immobilization, whereas in the locus coeruleus, it may support analgesic and hypnotic actions attributed to inhibition of those neurons.

Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K ؉ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G␣ q -coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R p )-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

The three vertebrate Gli proteins play a central role in mediating Hedgehog(Hh)-dependent cell fate specification in the developing spinal cord; however,their individual contributions to this process have not been fully characterized. In this paper, we have addressed this issue by examining patterning in the spinal cord of Gli2;Gli3 double mutant embryos, and in chick embryos transfected with dominant activator forms of Gli2 and Gli3. In double homozygotes, Gli1 is also not expressed; thus, all Gli protein activities are absent in these mice. We show that Gli3 contributes activator functions to ventral neuronal patterning, and plays a redundant role with Gli2 in the generation of V3 interneurons. We also show that motoneurons and three classes of ventral neurons are generated in the ventral spinal cord in double mutants, but develop as intermingled rather than discrete populations. Finally, we provide evidence that Gli2 and Gli3 activators control ventral neuronal patterning by regulating progenitor segregation. Thus, multiple ventral neuronal types can develop in the absence of Gli function, but require balanced Gli protein activities for their correct patterning and differentiation.

Shh-Gli signaling controls cell fates in the developing ventral neural tube by regulating the patterned expression of transcription factors in neural progenitors. However, the molecular mechanisms that limit target gene responses to specific domains are unclear. Here, we show that Wnt pathway inhibitors regulate the threshold response of a ventral Shh target gene, Nkx2.2, to establish its restricted expression in the ventral spinal cord. Identification and characterization of an Nkx2.2 enhancer reveals that expression is directly regulated by positive Shh-Gli signaling and negative Tcf repressor activity. Our data indicate that the dorsal limit of Nkx2.2 is controlled by Tcf4-mediated transcriptional repression, and not by a direct requirement for high-level Shh-Gli signaling, arguing against a simple model based on differential Gli factor affinities in target genes. These results identify a transcriptional mechanism that integrates graded Shh and Wnt signaling to define progenitor gene expression domains and cell fates in the neural tube.

Leak K+ currents contribute to the resting membrane potential and are important for modulation of neuronal excitability. Within the past few years, an entire family of genes has been described whose members form leak K+ channels, insofar as they generate potassium-selective currents with little voltage- and time-dependence. They are often referred to as "two-pore-domain" channels because of their predicted topology, which includes two pore-forming regions in each subunit. These channels are modulated by a host of different endogenous and clinical compounds such as neurotransmitters and anesthetics, and by physicochemical factors such as temperature, pH, oxygen tension, and osmolarity. They also are subject to long-term regulation by changes in gene expression. In this review, the authors describe multiple roles that modulation of leak K+ channels play in CNS function and discuss evidence that members of the two-pore-domain family are molecular substrates for these processes.

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