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C Derst

Publications and source records attributed to C Derst.

At least 19 recordsLinked to original sources
PubMed2006

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells.

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.

Calcium↗
PubMed2006

Tandem-pore domain potassium channels are functionally expressed in retinal (Müller) glial cells.

Tandem-pore domain (2P-domain) K+-channels regulate neuronal excitability, but their function in glia, particularly, in retinal glial cells, is unclear. We have previously demonstrated the immunocytochemical localization of the 2P-domain K+ channels TASK-1 and TASK-2 in retinal Müller glial cells of amphibians. The purpose of the present study was to determine whether these channels were functional, by employing whole-cell recording from frog and mammalian (guinea pig, rat and mouse) Müller cells and confocal microscopy to monitor swelling in rat Müller cells. TASK-like immunolabel was localized in these cells. The currents mediated by 2P-domain channels were studied in isolation after blocking Kir, K(A), K(D), and BK channels. The remaining cell conductance was mostly outward and was depressed by acid pH, bupivacaine, methanandamide, quinine, and clofilium, and activated by alkaline pH in a manner consistent with that described for TASK channels. Arachidonic acid (an activator of TREK channels) had no effect on this conductance. Blockade of the conductance with bupivacaine depolarized the Müller cell membrane potential by about 50%. In slices of the rat retina, adenosine inhibited osmotic glial cell swelling via activation of A1 receptors and subsequent opening of 2P-domain K+ channels. The swelling was strongly increased by clofilium and quinine (inhibitors of 2P-domain K+ channels). These data suggest that 2P-domain K+ channels are involved in homeostasis of glial cell volume, in activity-dependent spatial K+ buffering and may play a role in maintenance of a hyperpolarized membrane potential especially in conditions where Kir channels are blocked or downregulated.

Animals↗
PubMed2004

Unique accumulation of neuropeptides in an insect: FMRFamide-related peptides in the cockroach, Periplaneta americana.

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non-dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide-related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma-specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna-heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal-unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.

Action Potentials↗
PubMed2003

The large conductance Ca2+-activated potassium channel (pSlo) of the cockroach Periplaneta americana: structure, localization in neurons and electrophysiology.

Voltage-activated, Ca2+-sensitive K+ channels (BK or maxi K,Ca channels) play a major role in the control of neuronal excitability. We have cloned pSlo, the BK channel alpha subunit of the cockroach Periplaneta americana. The amino acid sequence of pSlo shows 88% identity to dSlo from Drosophila. There are five alternatively spliced positions in pSlo showing differential expression in various tissues. A pSlo-specific antibody prominently stained the octopaminergic dorsal unpaired median (DUM) neurons and peptidergic midline neurons in Periplaneta abdominal ganglia. HEK293 cells expressing pSlo exhibit K+ channels of 170 pS conductance. They have a tendency for brief closures, exhibit subconductance states and show slight inward rectification. Activation kinetics and voltage dependence are controlled by cytoplasmic [Ca2+]. In contrast to dSlo, pSlo channels are sensitive to charybdotoxin and iberiotoxin. Mutagenesis at two positions (E254 and Q285) changed blocking efficacy of charybdotoxin. In contrast to pSlo expressed in HEK293 cells, native IbTx-sensitive K,Ca currents in DUM and in peptidergic neurons, exhibited rapid, partial inactivation. The fast component of the K,Ca current partly accounts for the repolarization and the early after-hyperpolarization of the action potential. By means of Ca2+-induced repolarization, BK channels may reduce the risk of Ca2+ overload in cockroach neurons. Interestingly, the neurons expressing pSlo were also found to express taurine, a messenger that is likely to limit overexcitation by an autocrine mechanism in mammalian central neurons.

Alternative Splicing↗
PubMed2001

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea-pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea-pig cardiac ventricle. A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea-pig genomic library. The cell-specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi-cell RT-PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. After transfection in human embryonic kidney cells (HEK293) the mean single-channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. Cell-attached measurements in isolated guinea-pig cardiomyocytes (n = 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half-maximum block at -100 mV were 3.24 M for gpKir2.1, 0.51 M for gpKir2.2, 10.26 M for gpKir2.3 and 235 M for gpKir2.4. Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell-attached recordings. The concentration and voltage dependence of Ba2+ block of the large-conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. Our results suggest that the large-conductance inward rectifier channels found in guinea-pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate-conductance (23.8 pS) and low-conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Animals↗
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

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↗
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

Molecular cloning and functional expression of KCNQ5, a potassium channel subunit that may contribute to neuronal M-current diversity.

We have isolated KCNQ5, a novel human member of the KCNQ potassium channel gene family that is differentially expressed in subregions of the brain and in skeletal muscle. When expressed in Xenopus oocytes, KCNQ5 generated voltage-dependent, slowly activating K(+)-selective currents that displayed a marked inward rectification at positive membrane voltages. KCNQ5 currents were insensitive to the K(+) channel blocker tetraethylammonium but were strongly inhibited by the selective M-current blocker linopirdine. Upon coexpression with the structurally related KCNQ3 channel subunit, current amplitudes increased 4-5-fold. Compared with homomeric KCNQ5 currents, KCNQ3/KCNQ5 currents also displayed slower activation kinetics and less inward rectification, indicating that KCNQ5 combined with KCNQ3 to form functional heteromeric channel proteins. This functional interaction between KCNQ5 and KCNQ3, a component of the M-channel, suggests that KCNQ5 may contribute to a diversity of heteromeric channels underlying native neuronal M-currents.

Amino Acid Sequence↗
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

ATP-sensitive potassium channels in capillaries isolated from guinea-pig heart.

The full-length cDNAs of two different alpha-subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different beta-subunits (SUR1, SUR2A and SUR2B) of ATP-sensitive potassium (KATP) channels of the guinea-pig (gp) were obtained by screening a cDNA library from the ventricle of guinea-pig heart. Cell-specific reverse-transcriptase PCR with gene-specific intron-spanning primers showed that gpKir6.1, gpKir6.2 and gpSUR2B were expressed in a purified fraction of capillary endothelial cells. In cardiomyocytes, gpKir6.1, gpKir6.2, gpSUR1 and gpSUR2A were detected. Patch-clamp measurements were carried out in isolated capillary fragments consisting of 3-15 endothelial cells. The membrane capacitance measured in the whole-cell mode was 19.9 +/- 1.0 pF and was independent of the length of the capillary fragment, which suggests that the endothelial cells were not electrically coupled under our experimental conditions. The perforated-patch technique was used to measure the steady-state current-voltage relation of capillary endothelial cells. Application of K+ channel openers (rilmakalim or diazoxide) or metabolic inhibition (250 microM 2,4-dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. Rilmakalim (1 microM), diazoxide (300 microM) and metabolic inhibition increased the slope conductance measured at -55 mV by a factor of 9.0 (+/-1.8), 2.5 (+/-0.2) and 3.9 (+/-1.7), respectively. The effects were reversed by glibenclamide (1 microM). Our results suggest that capillary endothelial cells from guinea-pig heart express KATP channels composed of SUR2B and Kir6.1 and/or Kir6.2 subunits. The hyperpolarization elicited by the opening of KATP channels may lead to an increase in free cytosolic Ca2+, and thus modulate the synthesis of NO and the permeability of the capillary wall.

Adenosine Triphosphate↗
PubMed2000

A potassium channel protein encoded by chlorella virus PBCV-1.

The large chlorella virus PBCV-1, which contains double-stranded DNA (dsDNA), encodes a 94-codon open reading frame (ORF) that contains a motif resembling the signature sequence of the pore domain of potassium channel proteins. Phylogenetic analyses of the encoded protein, Kcv, indicate a previously unidentified type of potassium channel. The messenger RNA encoded by the ORF leads to functional expression of a potassium-selective conductance in Xenopus laevis oocytes. The channel blockers amantadine and barium, but not cesium, inhibit this conductance, in addition to virus plaque formation. Thus, PBCV-1 encodes the first known viral protein that functions as a potassium-selective channel and is essential in the virus life cycle.

Amantadine↗
PubMed2000

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248.

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.

Amino Acid Sequence↗
PubMed2000

Genomic structure and chromosome mapping of human and mouse RAMP genes.

The cDNAs for human and murine Receptor Activity Modifying Proteins and for the associated murine Calcitonin Receptor Like Receptor were isolated. The human RAMP1 and RAMP3 genes possess two introns and human RAMP2 possesses three introns. Human RAMP1 was assigned to chromosome 2q36-->q37.1, RAMP2 to 17q12-->q21.1 and RAMP3 to 7p13-->p12. Mouse Ramp1 was assigned to chromosome 1 and Ramp2 and Ramp3 were assigned to chromosome 11.

Amino Acid Sequence↗
PubMed1999

pH gating of ROMK (K(ir)1.1) channels: control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome.

Inward-rectifier K(+) channels of the ROMK (K(ir)1.1) subtype are responsible for K(+) secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K(ir) channels. Such anomalous titration of this lysine residue (Lys-80 in K(ir)1.1) is accomplished by the tertiary structure of the K(ir) protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K(ir)1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK(a) into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK(a) of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K(ir) channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

Arginine↗