Novel Drosophila two-pore domain K channels: rescue of channel function by heteromeric assembly.

Ten genes with essential structural features of two-pore domain potassium channels were identified in the genome of Drosophila melanogaster. Two Drosophila two-pore domain potassium subunits displayed substantial amino acid similarity to human TWIK-related acid-sensitive K(+) (TASK) channels (38-43%), whereas all others were less than 26% similar to any human homolog. The cDNAs of Drosophila TASK (dTASK)-6 and dTASK-7 channels were isolated from adult fruit flies. In Northern blots dTASK transcripts were found predominantly in the head fraction of adult flies and whole-mount brain in situ hybridizations showed strongly overlapping expression patterns of both dTASK isoforms in the antennal lobes. When heterologously expressed in Drosophila Schneider 2 cells, dTASK-6 gave rise to rapidly activating K(+)-selective currents that steeply depended on external pH. Structural elements in the extracellular M1-P1 loop of dTASK-6 were found to be involved in proton sensation. In contrast to mammalian TASK channels, the pH sensitivity was independent of extracellular histidines adjacent to the GYG selectivity filter (His98). As revealed by mutational analysis, functional expression of dTASK-7 was prevented by two nonconserved amino acids (Ala92-Met93) in the pore domain. When these two residues were replaced by conserved Thr92-Thr93, typical K(+)-selective leak currents were generated that were insensitive to changes in external pH. Nonfunctional wildtype dTASK-7 channels appeared to form heteromeric assemblies with dTASK-6. Following cotransfection of dTASK-6 and wildtype dTASK-7 (or when engineered as concatemers), K(+) currents were observed that were smaller in amplitude, harbored slower activation kinetics and were considerably less inhibited by local anesthetics as compared with dTASK-6. Thus, pore-loop residues in dTASK-7 changed functional and pharmacological properties in heteromeric dTASK channels.

The retention factor p11 confers an endoplasmic reticulum-localization signal to the potassium channel TASK-1.

The interaction of the adaptor protein p11, also denoted S100A10, with the C-terminus of the two-pore-domain K+ channel TASK-1 was studied using yeast two-hybrid analysis, glutathione S-transferase pull-down, and co-immunoprecipitation. We found that p11 interacts with a 40 amino-acid region in the proximal C-terminus of the channel. In heterologous expression systems, deletion of the p11-interacting domain enhanced surface expression of TASK-1. Attachment of the p11-interacting domain to the cytosolic tail of the reporter protein CD8 caused retention/retrieval of the construct in the endoplasmic reticulum (ER). Attachment of the last 36 amino acids of p11 to CD8 also caused ER localization, which was abolished by removal or mutation of a putative retention motif (H/K)xKxxx, at the C-terminal end of p11. Imaging of EGFP-tagged TASK-1 channels in COS cells suggested that wild-type TASK-1 was largely retained in the ER. Knockdown of p11 with siRNA enhanced trafficking of TASK-1 to the surface membrane. Our results suggest that binding of p11 to TASK-1 retards the surface expression of the channel, most likely by virtue of a di-lysine retention signal at the C-terminus of p11. Thus, the cytosolic protein p11 may represent a 'retention factor' that causes localization of the channel to the ER.

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.

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. 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/.

Kir2 potassium channels in rat striatum are strategically localized to control basal ganglia function.

Parkinson's disease is the most frequent movement disorder caused by loss of dopaminergic neurons in the midbrain. Intentions to avoid side effects of the conventional therapy should aim to identify additional targets for potential pharmacological intervention. In principle, every step of a signal transduction cascade such as presynaptic transmitter release, type and occupation of postsynaptic receptors, G protein-mediated effector mechanisms, and the alterations of pre- or postsynaptic potentials as determined by the local ion channel composition, have to be considered. Due to their diversity and their widespread but distinct localizations, potassium channels represent interesting candidates for new therapeutic strategies. As a first step, the present report aimed to study in the striatum the cellular and subcellular distribution of the individual members of the Kir2 family, a group of proteins forming inwardly rectifying potassium channels. For this purpose polyclonal monospecific affinity-purified antibodies against the less conserved carboxyterminal sequences from the Kir2.1, Kir2.2, Kir2.3, and Kir2.4 proteins were prepared. All subunits of the Kir2 family were detected on somata and dendrites of most striatal neurons. However, the distribution of two of them was not homogeneous. Striatal patch areas were largely devoid of the Kir2.3 protein, and the Kir2.4 subunit was most prominently expressed on the tonically active, giant cholinergic interneurons of the striatum. These two structures are among the key players in regulating dopaminergic and cholinergic neurotransmission within the striatum, and therefore are of major importance for the output of the basal ganglia. The heterogeneous localization of the Kir2.3 and the Kir2.4 subunits with respect to these strategic structures pinpoints to these channel proteins as promising targets for future pharmacological efforts.

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 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) 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 neural cell adhesion molecule regulates cell-surface delivery of G-protein-activated inwardly rectifying potassium channels via lipid rafts.

Mice deficient in the neural cell adhesion molecule (NCAM) exhibit increased anxiety and anxiolytic sensitivity to serotonin 5-HT1A receptor agonists. Here, we investigate the relationship between NCAM and 5-HT1A receptor signaling pathways modulating G-protein-activated inwardly rectifying K+ (Kir3) channels. When studying this relationship in cultured hippocampal neurons, we observed that in cells from NCAM-deficient mice, inwardly rectifying K+ (Kir3) currents were increased compared with wild-type controls. Analysis of this modulatory mechanism in Xenopus oocytes and Chinese hamster ovary (CHO) cells revealed that the recombinantly expressed major transmembrane isoforms NCAM140 and NCAM180 specifically reduced inward currents generated by neuronal Kir3.1/3.2 and Kir3.1/3.3 but not by cardiac Kir3.1/3.4 channels. Using fluorescence measurements and surface biotinylation assays, we show that this effect was caused by a reduced surface localization of Kir3 channels. Furthermore, expression of flag-tagged Kir3 channels in cultured neurons of NCAM-deficient mice resulted in a higher transport of these channels into neurites and a higher cell-surface localization compared with wild-type neurons. Neuronal Kir3 channels and NCAM isoforms are associated with cholesterol-rich microdomains (lipid rafts) in CHO cells and in isolated brain membranes. Mutational and pharmacological disruption of the lipid raft association of NCAM140 normalizes surface delivery of channels. We conclude that the transmembrane isoforms of NCAM reduce the transport of Kir3 channels to the cell surface via lipid rafts. Thus, regulation of Kir3 channel activity by NCAM may represent a novel mechanism controlling long-term excitability of neurons.

Inwardly rectifying K+ (Kir) channels in Drosophila. A crucial role of cellular milieu factors Kir channel function.

Three cDNAs encoding inwardly rectifying potassium (Kir) channels were isolated from Drosophila melanogaster. The protein sequences of Drosophila KirI (dKirI) and dKirII are moderately (<44%) and dKirIII sequence is weakly (<27%) identical to human Kir channel subunits. During fly development, five dKir channel transcripts derived from three genes are differentially expressed. Whole mount in situ hybridizations revealed dKirI transcripts absent from embryos, but dKirII and dKirIII are expressed in the embryonic hind gut and in Malpighian tubules, respectively, thus covering the entire osmoregulatory system of the developing fly. In the head of adult flies, predominantly dKirII transcripts were detected. When expressed in Xenopus oocytes, dKir channel activity was only observed after amino acid substitutions in their cytosolic tails (e.g. exchange of a unique valine in the NH(2) terminus). In contrast, heterologous expression of wild type dKirI and dKirII in Drosophila S2 cells readily evoked typical inwardly rectifying K(+) currents, which were weakly sensitive to Ba(2+). Thus, the specific milieu of insect cells provides a crucial cellular environment for proper function of dKir channels.

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.

Two novel alternatively spliced isoforms of the human two-pore-domain potassium channel TREK-2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK-2b) and 2559 bp (TREK-2c) encode proteins of 508 amino acids each. RT-PCR showed that TREK-2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK-2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK-2c in rat brain which partially overlapped with that of TREK-1. Expression of TREK-2b and TREK-2c in human embryonic kidney (HEK) 293 cells showed that their single-channel characteristics were similar. The slope conductance at negative potentials was 163 +/- 5 pS for TREK-2b and 179 +/- 17 pS for TREK-2c. The mean open and closed times of TREK-2b at -84 mV were 133 +/- 16 and 109 +/- 11 micros, respectively. Application of forskolin decreased the whole-cell current carried by TREK-2b and TREK-2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK-2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol-12-myristate-13-acetate (PMA) also reduced whole-cell current. However, removal of the putative TREK-2b-specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK-2 contributes to the diversity of two-pore-domain K+ channels.

G Protein Regulation of Inwardly Rectifying K(+) Channels.

Inwardly rectifying K(+) (Kir) channels respond to receptor-stimulated signaling cascades that involve G proteins and other cytosolic messengers. Channel activity is controlled both by direct coupling of G protein subunits and by phosphorylation via protein serine/threonine and tyrosine kinases. The coincidence of both forms of Kir channel signaling may give rise to complex cellular responses.