Andreas Karschin

GABAB-receptor subtypes assemble into functional heteromeric complexes

B-type receptors for the neurotransmitter GABA (γ-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABAB receptors, so it is expected that GABAB-receptor (GABABR) subtypes exist. Two GABAB-receptor splice variants have been cloned (GABABR1a and GABABR1b), but native GABAB receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult,. Here we describe a new GABAB receptor subtype, GABABR2, which does not bind available GABAB antagonists with measurable potency. GABABR1a, GABABR1b and GABABR2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABAB receptors in vivo and show that GABABR2 and GABABR1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABAB receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

IRK(1–3) and GIRK(1–4) Inwardly Rectifying K+Channel mRNAs Are Differentially Expressed in the Adult Rat Brain

Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K+ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1–3 and GIRK1–4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantia nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K+ channels in neurons and glial cells.

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

International Union of Pharmacology. LIV. Nomenclature and Molecular Relationships of Inwardly Rectifying Potassium Channels

Since the initial cDNA cloning of the first inward rectifiers Kir1.1 (ROMK1) and Kir2.1 (IRK1) in 1993, a succession of new members of this family have been identified, including the G protein-coupled Kir3 and the ATP-sensitive Kir6. These channels play an important physiological role in the

Overlapping distribution of K(ATP) channel-forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain.

ATP‐sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP‐binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.

Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels

Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABAB receptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABAB receptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABAB receptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABAB receptors in vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.

The role of conserved aspartate and serine residues in ligand binding and in function of the 5‐HT1A receptor: A site‐directed mutation study

Wild‐type and mutant serotonin 1A receptors were transiently expressed in COS‐7 cells using the infection‐transfection variant of the vaccinia virus/ T7 polymerase vector system. The amino acid substitutions in the transmembrane regions, Asp82→Asn82, Asp116 →Asn116, and Ser198→Ala198 all resulted in a decrease in affinity for 5‐HT by 60–100‐fold, without affecting the affinity for the antagonist, pindolol. The binding of agonist to the additional mutant, Thr199→Ala199, was too weak to be measured, 5‐HT induced GTPase activities for all receptors studied. These findings indicate that the residues mutated play an important role in the binding of the agonist and less critical roles in the binding of the antagonist pindolol.

THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels.

Two cDNAs encoding novel K+channels, THIK-1 and THIK-2 (tandem pore domainhalothane inhibited K +channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K+ channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1–14q24.3 (human (h) THIK-1) and 2p22–2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K+channel displaying weak inward rectification in symmetrical K+ solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation of the C‐terminus of TASK‐1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP‐tagged channel proteins. Two‐hybrid analysis showed that the C‐terminal domain of TASK‐1, TASK‐3 and TASK‐5, but not TASK‐4, interacts with isoforms of the adapter protein 14‐3‐3. A pentapeptide motif at the extreme C‐terminus of TASK‐1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14‐3‐3, whereas the last 40 amino acids of TASK‐1 were required for strong binding. Deletion of a single amino acid at the C‐terminal end of TASK‐1 or TASK‐3 abolished binding of 14‐3‐3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK‐1 mutants that failed to interact with 14‐3‐3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK‐1 S409A, which interacts with 14‐3‐3‐like wild‐type channels, displayed normal macroscopic currents. Co‐injection of 14‐3‐3ζ cRNA increased TASK‐1 current in Xenopus oocytes by about 70 %. After co‐transfection in HEK293 cells, TASK‐1 and 14‐3‐3ζ (but not TASK‐1ΔC5 and 14‐3‐3ζ) could be co‐immunoprecipitated. Furthermore, TASK‐1 and 14‐3‐3 could be co‐immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14‐3‐3 with TASK‐1 or TASK‐3 may promote the trafficking of the channels to the surface membrane.

The Metabotropic GABAB Receptor Directly Interacts with the Activating Transcription Factor 4

G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic γ-aminobutyric acid type B (GABAB) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABAB receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABAB receptor (GABABR) 1 and GABABR2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABABR and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABABRs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivoreporter assays. Thus, inhibitory metabotropic GABABRs may regulate activity-dependent gene expression via a direct interaction with ATF-4.