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A Karschin

Publications and source records attributed to A Karschin.

At least 19 recordsLinked to original sources
PubMed2001

Dynamic activation of K(ATP) channels in rhythmically active neurons.

1. The respiratory centre within the brainstem is one of the most active neuronal networks that generates ongoing rhythmic activity. Stabilization of such vital activity requires efficient processes for activity-correlated adjustment of neuronal excitability. Recent investigations have shown that a regulatory factor coupling electrical activity with cell metabolism comprises ATP-dependent K(+) channels (K(ATP) channels), which continuously adjust the excitability of respiratory neurons during normoxia and increasingly during hypoxia. 2. We used the single-cell antisense RNA amplification-polymerase chain reaction (PCR) technique to demonstrate that respiratory neurons co-express the sulphonylurea receptor SUR1 with the Kir6.2 potassium channel protein. 3. Single channel measurements on rhythmically active inspiratory neurons of the brainstem slice preparation of newborn mice revealed that K(ATP) channels are periodically activated in synchrony with each respiratory cycle. 4. The Na(+)-K(+)-ATPase was inhibited with ouabain to demonstrate that oscillations of the channel open probability disappear, although respiratory activity persists for a longer time. Such findings indicate that K(ATP) channel open probability reflects activity-dependent fluctuations in the ATP concentration within submembrane domains. 5. We also examined the effects of extracellular [K(+)] and hypoxia. All changes in the respiratory rhythm (i.e. changes in cycle length and burst durations) affected the periodic fluctuations of K(ATP) channel activity. 6. The data indicate that K(ATP) channels continuously modulate central respiratory neurons and contribute to periodic adjustment of neuronal excitability. Such dynamic adjustment of channel activity operates over a high range of metabolic demands, starting below physiological conditions and extending into pathological situations of energy depletion.

ATP-Binding Cassette Transporters↗
PubMed2001

Genetic and functional linkage of Kir5.1 and Kir2.1 channel subunits.

We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K(+) channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1-24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.

Alternative Splicing↗
PubMed2001

Expression pattern in brain of TASK-1, TASK-3, and a tandem pore domain K(+) channel subunit, TASK-5, associated with the central auditory nervous system.

TWIK-related acid-sensitive K(+) (TASK) channels contribute to setting the resting potential of mammalian neurons and have recently been defined as molecular targets for extracellular protons and volatile anesthetics. We have isolated a novel member of this subfamily, hTASK-5, from a human genomic library and mapped it to chromosomal region 20q12-20q13. hTASK-5 did not functionally express in Xenopus oocytes, whereas chimeric TASK-5/TASK-3 constructs containing the region between M1 and M3 of TASK-3 produced K(+) selective currents. To better correlate TASK subunits with native K(+) currents in neurons the precise cellular distribution of all TASK family members was elucidated in rat brain. A comprehensive in situ hybridization analysis revealed that both TASK-1 and TASK-3 transcripts are most strongly expressed in many neurons likely to be cholinergic, serotonergic, or noradrenergic. In contrast, TASK-5 expression is found in olfactory bulb mitral cells and Purkinje cells, but predominantly associated with the central auditory pathway. Thus, TASK-5 K(+) channels, possibly in conjunction with auxiliary proteins, may play a role in the transmission of temporal information in the auditory system.

Amino Acid Sequence↗
PubMed2001

Kir6.1 is the principal pore-forming subunit of astrocyte but not neuronal plasma membrane K-ATP channels.

ATP-sensitive potassium channels (K-ATP channels) directly couple the energy state of a cell to its excitability, are activated by hypoxia, and have been suggested to protect neurons during disturbances of energy metabolism such as transient ischemic attacks or stroke. Molecular studies have demonstrated that functional K-ATP channels are octameric protein complexes, consisting of four sulfonylurea receptor proteins and four pore-forming subunits which are members of the Kir6 family of inwardly rectifying potassium channels. Here we show, using specific antibodies against the two known pore-forming subunits (Kir6.1 and Kir6.2) of K-ATP channels, that only Kir6.1 and not Kir6.2 subunits are expressed in astrocytes. In addition to a minority of neurons, Kir6.1 protein is present on hippocampal, cortical, and cerebellar astrocytes, tanycytes, and Bergmann glial cells. We also provide ultrastructural evidence that Kir6.1 immunoreactivity is primarily localized to distal perisynaptic and peridendritic astrocyte plasma membrane processes, and we confirm the presence of functional K-ATP channels in Bergmann glial cells by slice-patch-clamp experiments. The identification of Kir6.1 as the principal pore-forming subunit of plasma membrane K-ATP channels in astrocytes suggests that these glial K-ATP channels act in synergy with neuronal Kir6.2-mediated K-ATP channels during metabolic challenges in the brain.

Adenosine Triphosphate↗
PubMed2001

Cellular localization of the potassium channel Kir7.1 in guinea pig and human kidney.

BACKGROUND: K(+) channels have important functions in the kidney, such as maintenance of the membrane potential, volume regulation, recirculation, and secretion of potassium ions. The aim of this study was to obtain more information on the localization and possible functional role of the inwardly rectifying K(+) channel, Kir7.1. METHODS: Kir7.1 cDNA (1114 bp) was isolated from guinea pig kidney (gpKir7.1), and its tissue distribution was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, a genomic DNA fragment (6153 bp) was isolated from a genomic library. cRNA was expressed in Xenopus laevis oocytes for functional studies. Immunohistochemistry and RT-PCR were used to localize Kir7.1 in guinea pig and human kidney. RESULTS: The expression of gpKir7.1 in Xenopus laevis oocytes revealed inwardly rectifying K(+) currents. The reversal potential was strongly dependent on the extracellular K(+) concentration, shifting from -14 mV at 96 mmol/L K(+) to -90 mV at 1 mmol/L K(+). gpKir7.1 showed a low affinity for Ba(2+). Significant expression of gpKir7.1 was found in brain, kidney, and lung, but not in heart, skeletal muscle, liver, or spleen. Immunocytochemical detection in guinea pig identified the gpKir7.1 protein in the basolateral membrane of epithelial cells of the proximal tubule. RT-PCR analysis identified strong gpKir7.1 expression in the proximal tubule and weak expression in glomeruli and thick ascending limb. In isolated human tubule fragments, RT-PCR showed expression in proximal tubule and thick ascending limb. CONCLUSION: Our results suggest that Kir7.1 may contribute to basolateral K(+) recycling in the proximal tubule and in the thick ascending limb.

Amino Acid Sequence↗
PubMed2001

Functional heterogeneity of ROMK mutations linked to hyperprostaglandin E syndrome.

BACKGROUND: The renal K(+) channel ROMK (Kir1.1) controls salt reabsorption in the kidney. Loss-of-function mutations in this channel cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), which is characterized by severe renal salt and fluid wasting. METHODS: We investigated 10 HPS/aBS patients for mutations in the ROMK gene by single-strand conformation polymorphism analysis (SSCA) and direct sequencing. To assess the functional consequences, Ba(2+)-sensitive K(+) currents were measured in five mutants of the core region as well as one mutant with truncated C-terminus, using the two-electrode voltage-clamp technique after an injection of mutant cRNA into Xenopus oocytes. RESULTS: Three novel ROMK mutations were identified together with six mutations described previously. The mutations were categorized into three groups: (1) amino acid exchanges in the core region (M1-H5-M2), (2) truncation at the cytosolic C-terminus, and (3) deletions of putative promoter elements. While the core mutations W99C, N124K, and I142T led to significantly reduced macroscopic K(+) currents (1 to 8% of wild-type currents), the A103V and P110L variants retained substantial K(+) conductivity (23 and 35% of wild-type currents, respectively). Coexpression of A103V and P110L, resembling the compound heterozygous state of the affected individual, further reduced macroscopic currents to 9% of the wild-type currents. All mutants in the core region exerted a dominant-negative effect on wild-type ROMK1. The C-terminal frameshift (fs) mutation (H354fs) did not change current amplitudes compared with ROMK1 wild type, suggesting that a mechanism other than alteration of the electrophysiological properties may responsible for loss of channel activity. CONCLUSIONS: Analysis of ROMK mutants linked to HPS/aBS revealed a spectrum of mechanisms accounting for loss of channel function. Further characterization of the molecular defects might be helpful for the development of new therapeutic approaches.

Animals↗
PubMed2001

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel.

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.

Animals↗
PubMed2000

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 gamma-aminobutyric acid type B (GABA(B)) 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 GABA(B) 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 GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R 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 GABA(B)Rs 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 vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.

Activating Transcription Factor 4↗
PubMed2000

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 domain halothane 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.

Amino Acid Sequence↗
PubMed2000

Genomic structure and promoter analysis of the rat kir7.1 potassium channel gene (Kcnj13).

In the brain inwardly rectifying potassium channel Kir7.1 subunits are predominantly expressed in the choroid plexus and meninges. To investigate this tissue-specific expression pattern, we characterized the genomic organization and the 5' proximal promoter of the rat Kir7.1 gene (Kcnj13). Starting from the major transcriptional initiation site, three exons in Kcnj13 give rise to the dominant approximately 1.45 kb transcript in brain. Adjacent to the transcriptional start the minimal promoter which, uncommon for ion channels, contains a TATA- and CAAT-box is controlled by AP-1 factors and accounts for high gene expression levels. Luciferase reporter gene responses driven by the first 2.1 kb of the 5' flanking region were similarly high in epithelial FRTL-5 and neuronal N2A cells, suggesting that neuron-specific repressor elements are located remote from the non-selective minimal promoter.

Animals↗
PubMed2000

Brain localization and behavioral impact of the G-protein-gated K+ channel subunit GIRK4.

Neuronal G-protein-gated potassium (K(G)) channels are activated by several neurotransmitters and constitute an important mode of synaptic inhibition in the mammalian nervous system. K(G) channels are composed of combinations of four subunits termed G protein-gated inwardly rectifying K(+) channels (GIRK). All four GIRK subunits are expressed in the brain, and there is a general consensus concerning the expression patterns of GIRK1, GIRK2, and GIRK3. The localization pattern of GIRK4, however, remains controversial. In this study, we exploit the negative background of mice lacking a functional GIRK4 gene to identify neuronal populations that contain GIRK4 mRNA. GIRK4 mRNA was detected in only a few regions of the mouse brain, including the deep cortical pyramidal neurons, the endopiriform nucleus and claustrum of the insular cortex, the globus pallidus, the ventromedial hypothalamic nucleus, parafascicular and paraventricular thalamic nuclei, and a few brainstem nuclei (e.g., the inferior olive and vestibular nuclei). Mice lacking GIRK4 were viable and appeared normal and did not display gross deficiencies in locomotor activity, visual tasks, and pain perception. Furthermore, GIRK4-deficient mice performed similarly to wild-type controls in the passive avoidance paradigm, a test of aversive learning. GIRK4 knock-out mice did, however, exhibit impaired performance in the Morris water maze, a test of spatial learning and memory.

Animals↗
PubMed2000

TASK-3, a novel tandem pore domain acid-sensitive K+ channel. An extracellular histiding as pH sensor.

Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.

Amino Acid Sequence↗
PubMed2000

Stable cation coordination at a single outer pore residue defines permeation properties in Kir channels.

In epithelial Kir7.1 channels a non-conserved methionine in the outer pore region adjacent to the G-Y-G selectivity filter (position +2) was found to determine unique properties for permeant and blocking ions characteristic of a K(+) channel in a single-occupancy state. The monovalent cation permeability sequence of Kir7.1 channels expressed in Xenopus oocytes was Tl(+)>K(+)>Rb(+)NH(4)(+)>Cs(+)>Na(+)>Li(+), but the macroscopic conductance for Rb(+) was approximately 8-fold larger than for the smaller K(+) ions, and decreased approximately 40-fold with the conserved arginine at the +2 position (Kir7.1M125R). Moreover, in Kir7.1 Rb(+) restored the typical permeation properties of other multi-ion channels indicating that a stable coordination of permeant ions at the +2 position defines the initial step in the conduction pathway of Kir channels.

Animals↗
PubMed2000

Neuronal inwardly rectifying K(+) channels differentially couple to PDZ proteins of the PSD-95/SAP90 family.

Several signaling proteins clustered at the postsynaptic density specialization in neurons harbor a conserved C-terminal PDZ domain recognition sequence (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 protein family. This motif is also present in the C termini of some inwardly rectifying K(+) (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitability, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2(+), Kir3.3(+) and Kir3.4(-) subunits (+, motif present; -, motif absent) were used as baits in the yeast two-hybrid assay to screen for in vivo interaction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2.3, all Kir3 fragments failed to bind PSD-95 in this assay, which was supported by the lack of coimmunoprecipitation and colocalization of the entire proteins in mammalian cells. A detailed analysis of interaction domains demonstrated that the C-terminal motif in Kir3 channels is insufficient for binding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitate with PSD-95. When coexpressed in a bicistronic internal ribosome entry site expression vector in HEK-293 cells macroscopic and elementary current analysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by >50%. This inhibitory action of PSD-95, which predominantly affects the single-channel conductance, is likely attributable to a molecular association with additional internal interaction sites in the Kir2.3 protein.

Amino Acid Sequence↗
PubMed2000

Interaction of the C-terminal tail region of the metabotropic glutamate receptor 7 with the protein kinase C substrate PICK1.

Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release. Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1. This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs. Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1. These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs.

Amino Acid Sequence↗
PubMed2000

Gain of function mutants: ion channels and G protein-coupled receptors.

Many ion channels and receptors display striking phenotypes for gain-of-function mutations but milder phenotypes for null mutations. Gain of molecular function can have several mechanistic bases: selectivity changes, gating changes including constitutive activation and slowed inactivation, elimination of a subunit that enhances inactivation, decreased drug sensitivity, changes in regulation or trafficking of the channel, or induction of apoptosis. Decreased firing frequency can occur via increased function of K+ or Cl- channels. Channel mutants also cause gain-of-function syndromes at the cellular and circuit level; of these syndromes, the cardiac long-QT syndromes are explained in a more straightforward way than are the epilepsies. G protein-coupled receptors are also affected by activating mutations.

Animals↗
PubMed1999

Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

Amino Acid Sequence↗