Christian Derst

Glutamatergic Afferents of the Ventral Tegmental Area in the Rat

Glutamatergic inputs to the ventral tegmental area (VTA), thought crucial to the capacity of the VTA to detect and signal stimulus salience, have been reported to arise in but a few structures. However, the afferent system of the VTA comprises very abundant neurons within a large formation extending from the prefrontal cortex to the caudal brainstem. Neurons in nearly all parts of this continuum may be glutamatergic and equivalently important to VTA function. Thus, we sought to identify the full range of glutamatergic inputs to the VTA by combining retrograde transport of wheat germ agglutinin-bound gold after injections into the VTA with nonisotopic in situ hybridization of the vesicular glutamate transporters (VGLUTs) 1, 2, and 3. We found glutamatergic neurons innervating the VTA in almost all structures projecting there and that a majority of these are subcortical and VGLUT2 mRNA positive. The tremendous convergence of glutamatergic afferents from many brain areas in the VTA suggests that (1) the function of the VTA requires integration of manifold and diverse bits of information and (2) the activity of the VTA reflects the ongoing activities of various combinations of its afferents.

Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome.

Andersens syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersens syndrome. The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x–Kir2.y concatemers expressed inwardly rectifying K+ channels with a conductance of 28–30 pS. (ii) Expression of Kir2.x–Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba2+ sensitive currents. (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC50 for Ba2+ block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersens syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersens syndrome.

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

1 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. 2 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. 3 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. 4 Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 5 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. 6 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. 7 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 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. 8 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. 9 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.

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.

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.

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 μs, 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.

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

1 The full‐length cDNAs of two different α‐subunits (Kir6.1 and Kir6.2) and partial cDNAs of three different β‐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. 2 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. 3 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. 4 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 μm 2,4‐dinitrophenol plus 10 mM deoxyglucose) induced a current that reversed near the calculated K+ equilibrium potential. 5 Rilmakalim (1 μm), diazoxide (300 μm) 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 μm). 6 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.

A covalently bound catalytic intermediate in Escherichia coli asparaginase : Crystal structure of a Thr‐89‐Val mutant

Escherichia coli asparaginase II catalyzes the hydrolysis of l‐asparagine to l‐aspartate via a threonine‐bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threomines, Thr‐89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 Å resolution, shows high overall similarity to the wild‐type enzyme, but an aspartyl moiety is covalently bound to Thr‐12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.

Effects of divalent cations and spermine on the K+ channel TASK-3 and on the outward current in thalamic neurons

The potassium channels TASK‐1 and TASK‐3 show high sequence homology but differ in their sensitivity to extracellular divalent cations. Heterologous expression in HEK293 cells showed that the single‐channel conductance of TASK‐3 increased approximately four‐fold after removal of external divalent cations, whereas the conductance of TASK‐1 was unaffected. Replacing the glutamate at position 70 of TASK‐3 by a lysine or arginine residue abolished the sensitivity to divalent cations. The reverse mutation in TASK‐1 (K70E) induced sensitivity to divalent cations. The organic polycations spermine and ruthenium red modulated the conductance of TASK‐3 in a similar way as Ca2+ or Mg2+. Our data suggest that these effects were mediated by shielding of the negative charges in the extracellular loops of TASK‐3. Whole‐cell currents carried by TASK‐3 channels were inhibited by spermine and ruthenium red even in the presence of external divalent cations. These data suggest that, in addition to their effect on single‐channel conductance, spermine and ruthenium red decreased the open probability of TASK‐3 channels, probably by binding to residue E70. The standing outward current in thalamocortical relay neurons, which is largely carried by TASK channels, was also inhibited by divalent cations and spermine. Using the differential sensitivity of TASK‐1 and TASK‐3 to divalent cations and spermine we found that about 20% of the standing outward current in thalamocortical relay neurons flows through TASK‐3 channels. We conclude from our results that inhibition of TASK‐3 channels may contribute to the neuromodulatory effect of spermine released from neurons during repetitive activity or during hypoxia.

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.