scholarly journals Agonist and antagonist properties of an insect GABA-gated chloride channel (RDL) are influenced by heterologous expression conditions

PLoS ONE ◽  
2021 ◽  
Vol 16 (7) ◽  
pp. e0254251
Author(s):  
Charles L. C. Smelt ◽  
Victoria R. Sanders ◽  
Alin M. Puinean ◽  
Stuart J. Lansdell ◽  
Jim Goodchild ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Chloride Channels ◽  
Bombus Terrestris ◽  
Functional Expression ◽  
Glycine Receptors ◽  
Competitive Antagonist ◽  
Inhibitory Neurotransmitter ◽  
Apparent Affinity ◽  
Chaperone Protein ◽  
Agonist And Antagonist

Pentameric ligand-gated ion channels (pLGICs) activated by the inhibitory neurotransmitter γ-aminobutyric acid (GABA) are expressed widely in both vertebrate and invertebrate species. One of the best characterised insect GABA-gated chloride channels is RDL, an abbreviation of ‘resistance to dieldrin’, that was originally identified by genetic screening in Drosophila melanogaster. Here we have cloned the analogous gene from the bumblebee Bombus terrestris audax (BtRDL) and examined its pharmacological properties by functional expression in Xenopus oocytes. Somewhat unexpectedly, the sensitivity of BtRDL to GABA, as measured by its apparent affinity (EC50), was influenced by heterologous expression conditions. This phenomenon was observed in response to alterations in the amount of cRNA injected; the length of time that oocytes were incubated before functional analysis; and by the presence or absence of a 3’ untranslated region. In contrast, similar changes in expression conditions were not associated with changes in apparent affinity with RDL cloned from D. melanogaster (DmRDL). Changes in apparent affinity with BtRDL were also observed following co-expression of a chaperone protein (NACHO). Similar changes in apparent affinity were observed with an allosteric agonist (propofol) and a non-competitive antagonist (picrotoxinin), indicating that expression-depended changes are not restricted to the orthosteric agonist binding site. Interestingly, instances of expression-dependent changes in apparent affinity have been reported previously for vertebrate glycine receptors, which are also members of the pLGIC super-family. Our observations with BtRDL are consistent with previous data obtained with vertebrate glycine receptors and indicates that agonist and antagonist apparent affinity can be influenced by the level of functional expression in a variety of pLGICs.

scholarly journals Functional Expression of the Ectoine Hydroxylase Gene (thpD) from Streptomyces chrysomallus in Halomonas elongata

2004 ◽  
Vol 70 (5) ◽  
pp. 3130-3132 ◽  
Author(s):  
Julia Prabhu ◽  
Florian Schauwecker ◽  
Nicolas Grammel ◽  
Ullrich Keller ◽  
Michael Bernhard
Keyword(s):  
Heterologous Expression ◽  
Functional Expression ◽  
Efficient Conversion ◽  
Hydroxylase Gene ◽  
Halomonas Elongata

ABSTRACT The formation of hydroxyectoine in the industrial ectoine producer Halomonas elongata was improved by the heterologous expression of the ectoine hydroxylase gene, thpD, from Streptomyces chrysomallus. The efficient conversion of ectoine to hydroxyectoine was achieved by the concerted regulation of thpD by the H. elongata ectA promoter.

scholarly journals Functional Expression of a Bacterial Xylose Isomerase in Saccharomyces cerevisiae

2009 ◽  
Vol 75 (8) ◽  
pp. 2304-2311 ◽  
Author(s):  
Dawid Brat ◽  
Eckhard Boles ◽  
Beate Wiedemann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Xylose Fermentation ◽  
Thermus Thermophilus ◽  
Xylose Isomerase ◽  
Functional Expression ◽  
Yeast Cells ◽  
Highly Active ◽  
Excellent Starting Point ◽  
Starting Point

ABSTRACT In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

Functional expression of glycine receptors in DRG neurons of mice

2021 ◽  
Vol 899 ◽  
pp. 174034
Author(s):  
Lin Yao ◽  
Tian-Yu Zhang ◽  
Xin-Tong Diao ◽  
Juan-Juan Ma ◽  
Hu-Hu Bai ◽  
...  
Keyword(s):  
Functional Expression ◽  
Glycine Receptors ◽  
Drg Neurons

Identification of a nucleoside/nucleobase transporter from Plasmodium falciparum, a novel target for anti-malarial chemotherapy

2000 ◽  
Vol 349 (1) ◽  
pp. 67-75 ◽  
Author(s):  
Marie D. PARKER ◽  
Ralph J. HYDE ◽  
Sylvia Y. M. YAO ◽  
Louisa MCROBERT ◽  
Carol E. CASS ◽  
...  
Keyword(s):  
Xenopus Oocytes ◽  
De Novo ◽  
Functional Expression ◽  
Nucleoside Transporters ◽  
Pyrimidine Nucleoside ◽  
Parasite Protein ◽  
Apparent Affinity ◽  
Equilibrative Nucleoside Transporters ◽  
Novel Target ◽  
Membrane Spanning

Plasmodium, the aetiologic agent of malaria, cannot synthesize purines de novo, and hence depends upon salvage from the host. Here we describe the molecular cloning and functional expression in Xenopus oocytes of the first purine transporter to be identified in this parasite. This 422-residue protein, which we designate PfENT1, is predicted to contain 11 membrane-spanning segments and is a distantly related member of the widely distributed eukaryotic protein family the equilibrative nucleoside transporters (ENTs). However, it differs profoundly at the sequence and functional levels from its homologous counterparts in the human host. The parasite protein exhibits a broad substrate specificity for natural nucleosides, but transports the purine nucleoside adenosine with a considerably higher apparent affinity (Km 0.32±0.05 mM) than the pyrimidine nucleoside uridine (Km 3.5±1.1 mM). It also efficiently transports nucleobases such as adenine (Km 0.32±0.10 mM) and hypoxanthine (Km 0.41±0.1 mM), and anti-viral 3ʹ-deoxynucleoside analogues. Moreover, it is not sensitive to classical inhibitors of mammalian ENTs, including NBMPR {6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine, or nitrobenzylthioinosine} and the coronary vasoactive drugs, dipyridamole, dilazep and draflazine. These unique properties suggest that PfENT1 might be a viable target for the development of novel anti-malarial drugs.

Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex

1988 ◽  
Vol 234 (1275) ◽  
pp. 159-170 ◽  
Keyword(s):  
Cerebral Cortex ◽  
Chloride Channels ◽  
Cerebral Hemispheres ◽  
Induced Response ◽  
Messenger Rnas ◽  
Induced Current ◽  
Glycine Receptors ◽  
Adult Rats ◽  
Developing Rat ◽  
Newborn Animals

The ontogenesis of mRNAs coding for GABA and glycine receptors in the cerebral cortex of the rat was examined by extracting poly(A) + mRNA from the brains of embryonic, postnatal or adult rats and injecting it into Xenopus oocytes. The ability of a messenger to express functional receptors was then assayed by measuring the membrane currents elicited by the agonists. The size of the GABA-induced current increased progressively with age, being undetectable in oocytes injected with mRNA from embryonic day 15 and reaching a maximum in oocytes injected with mRNA from postnatal day 30. In contrast, the glycine-induced response was negligible in oocytes injected with mRNA from the cerebral hemispheres of embryos 15 days old; it increased sharply to a maximum with newborn animals and then decreased with age to become very small with mRNA from adult cortex. GABA and glycine receptors induced by mRNA from the cerebral cortex of all ages are associated with chloride channels.

scholarly journals ClC-K chloride channels: emerging pathophysiology of Bartter syndrome type 3

2015 ◽  
Vol 308 (12) ◽  
pp. F1324-F1334 ◽  
Author(s):  
Olga Andrini ◽  
Mathilde Keck ◽  
Rodolfo Briones ◽  
Stéphane Lourdel ◽  
Rosa Vargas-Poussou ◽  
...  
Keyword(s):  
Chloride Channel ◽  
Chloride Channels ◽  
Large Family ◽  
Functional Expression ◽  
Bartter Syndrome ◽  
Channel Structure ◽  
Syndrome Type ◽  
Missense Mutations ◽  
Membrane Expression ◽  
Type 3

The mutations in the CLCNKB gene encoding the ClC-Kb chloride channel are responsible for Bartter syndrome type 3, one of the four variants of Bartter syndrome in the genetically based nomenclature. All forms of Bartter syndrome are characterized by hypokalemia, metabolic alkalosis, and secondary hyperaldosteronism, but Bartter syndrome type 3 has the most heterogeneous presentation, extending from severe to very mild. A relatively large number of CLCNKB mutations have been reported, including gene deletions and nonsense or missense mutations. However, only 20 CLCNKB mutations have been functionally analyzed, due to technical difficulties regarding ClC-Kb functional expression in heterologous systems. This review provides an overview of recent progress in the functional consequences of CLCNKB mutations on ClC-Kb chloride channel activity. It has been observed that 1) all ClC-Kb mutants have an impaired expression at the membrane; and 2) a minority of the mutants combines reduced membrane expression with altered pH-dependent channel gating. Although further investigation is needed to fully characterize disease pathogenesis, Bartter syndrome type 3 probably belongs to the large family of conformational diseases, in which the mutations destabilize channel structure, inducing ClC-Kb retention in the endoplasmic reticulum and accelerated channel degradation.

scholarly journals Heterologous Expression ofMycobacteriumAlkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

2018 ◽  
Vol 84 (15) ◽  
Author(s):  
Victoria McCarl ◽  
Mark V. Somerville ◽  
Mai-Anh Ly ◽  
Rebecca Henry ◽  
Elissa F. Liew ◽  
...  
Keyword(s):  
Heterologous Expression ◽  
Stereoselective Synthesis ◽  
Cell Activity ◽  
Functional Expression ◽  
Soluble Form ◽  
Gram Negative ◽  
Content Type ◽  
Alkene Oxidation ◽  
Peptide Mass ◽  
Heterologous Expression Systems

ABSTRACTAlkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) fromMycobacterium chubuensestrain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved inMycobacterium smegmatismc2155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C2to C4), and their activity was much lower on liquid alkenes (C5to C8). Despite intensive efforts to express the complete EtnABCD enzyme inEscherichia coli, this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochromecreduced/min/mg protein). Cloning the EtnABCD gene cluster intoPseudomonas putidaKT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression ofcpn60chaperonins from eitherMycobacteriumspp. orE. coli. Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels.IMPORTANCEAlkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

Sign in / Sign up

Export Citation Format

Share Document