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PubMed2001

Effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine by their transporters heterologously expressed in COS cells and in rat brain synaptosomes.

Although the neurotransmitter uptake system is considered a possible target for the presynaptic action of anesthetic agents, observations are inconsistent concerning effects on the transporter and their clinical relevance. The present study examined the effects of volatile and intravenous anesthetics on the uptake of GABA, glutamate and dopamine in COS cells heterologously expressing the transporters for these neurotransmitters and in the rat brain synaptosomes. Halothane and isoflurane, but not thiamylal or thiopental, significantly inhibited uptake by COS cell systems of GABA, dopamine and glutamic acid in a concentration-dependent manner within clinically relevant ranges for anesthesia induced by these agents. Similarly, in synaptosomes halothane and isoflurane but not thiopental significantly suppressed the uptake of GABA and glutamic acid, respectively. These results do not support the hypothesis that volatile and intravenous anesthetics exert their action via specific inhibition of GABA uptake to enhance inhibitory GABAergic neuronal activity. Rather, they suggest that presynaptic uptake systems for various neurotransmitters including GABA may be the molecular targets for volatile anesthetic agents.

ATP-Binding Cassette Transporters↗
PubMed1995

Heterologous expression of genes encoding bacterial light-harvesting complexes in Rhodobacter sphaeroides.

One of the major problems in structural work on membrane-spanning proteins is the identification of an expression system which will allow the production of enough pure protein for structural studies; an inadequate expression system can lead, for example, to the formation of unwanted protein inclusion bodies. In the present work we report the expression of genes encoding the light-harvesting 2 (LH2) membrane-spanning proteins from a number of species of purple bacteria in mutants of Rhodobacter sphaeroides that lack the native LH2 antenna. The LH2 structural genes (pucBA) from the photosynthetic bacteria Rhodopseudomonas acidophila and Rubrivivax gelatinosus were amplified and tailed by polymerase chain reaction, and cloned into an LH2 expression vector, which was then introduced into three LH2-minus Rb. sphaeroides mutants; DBC omega/G5 and DD13 (DD13/G1); the resulting transconjugant strains synthesized LH2 complexes that were examined using absorption and fluorescence spectroscopy, and Western blotting. Thus, we have created a heterologous expression system which supports the assembly of a functional "foreign" light-harvesting complex. This work opens up the possibility of creating site-directed LH2 mutants from bacteria for which no genetic system is available; this is particularly significant in the case of Rps. acidophila, since this bacterium has been the source of the LH2 complex that has recently been structurally resolved to atomic resolution.

Amino Acid Sequence↗
PubMed1996

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.

Aminoglycosides↗
PubMed1994

Regulation of serotonin2A receptors in heterologous expression systems.

The serotonin2A and serotonin2C receptors are unique among receptors coupled to guanine nucleotide binding proteins in that chronic treatment in vivo with agonists as well as antagonists decreases receptor density. In an attempt to uncover molecular events involved in down-regulation of the serotonin2A receptor, the ability of agonists and antagonists to alter receptor density was examined in three heterologous expression systems, i.e., transfected NIH 3T3, transfected Madin-Darby canine kidney, and transfected AtT-20 cells. All three transfected cell lines exhibited pharmacological properties consistent with that predicted for cells expressing the serotonin2A receptor. However, the three cell lines displayed different receptor regulation properties in response to drugs acting at the serotonin2A receptor. In transfected NIH 3T3 cells, neither agonist nor antagonist treatment altered receptor density. Treatment with agonist as well as antagonist led to up-regulation of the serotonin2A receptor in transfected Madin-Darby canine kidney cells. In transfected AtT-20 cells, treatment with agonist led to receptor down-regulation, whereas antagonist treatment increased receptor density. Thus, the cellular background in which the serotonin2A receptor is expressed appears to determine the regulation properties of the receptor.

DOM 2,5-Dimethoxy-4-Methylamphetamine↗
PubMed2004

Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras.

The nicotinic acetylcholine receptor (nAChR) alpha9 and alpha10 subunits are expressed primarily within hair cells of the inner ear and have been implicated in auditory processing. Although functional recombinant nAChRs generated by the coexpression of alpha9 and alpha10 in Xenopus laevis oocytes have been described previously, there have been no reports of the successful heterologous expression of alpha9alpha10 nAChRs in cultured cell lines. In this study, subunit chimeras (alpha9chi and alpha10chi) have been constructed that contain the extracellular, ligand binding domain of the alpha9 or alpha10 subunits fused to the C-terminal domain of the 5-hydroxytryptamine type 3A (5HT3A) subunit. Specific high-affinity binding of the nicotinic radioligand [3H]methyllycaconitine was detected in membrane preparations of mammalian cells transfected with alpha9chi or alpha10chi alone, but significantly higher levels of binding were detected when alpha9chi and alpha10chi were cotransfected, providing evidence of a requirement for coassembly of alpha9 and alpha10 for the efficient formation of a nicotinic binding site. The pharmacological profile of alpha9chialpha10chi receptors, determined by equilibrium radioligand binding studies, is broadly similar to that determined previously by electrophysiological studies conducted with native and recombinant alpha9alpha10 nAChRs. In agreement with evidence that alpha9alpha10 nAChRs exhibit an atypical pharmacological profile, we have identified specific high-affinity binding of several non-nicotinic ligands including strychnine (a glycine receptor antagonist), bicuculline (a GABAA receptor antagonist), and atropine (a muscarinic acetylcholine receptor antagonist). Results have also been compared with radioligand binding data conducted with a previously described alpha7/5HT3A (alpha7chi) subunit chimera.

Aconitine↗
PubMed1994

Heterologous expression of delta F508 CFTR results in decreased sialylation of membrane glycoconjugates.

The cystic fibrosis transmembrane conductance regulator (CFTR) is commonly mutated in cystic fibrosis to the delta F508 CFTR. CFTR has been shown to function as a adenosine 3',5'-cyclic monophosphate-dependent Cl- channel at the cell surface, and there is evidence to suggest that CFTR may also have a role in transmembrane Cl- conductance in intracellular membrane compartments. Studies using cells from cystic fibrosis patients or heterologous expression systems have demonstrated that defective Cl- conductance at the cell surface and defective acidification of the Golgi compartment are associated with the presence of mutant forms of CFTR. It is possible that mutation of CFTR could also result in altered Golgi function, consistent with reports of changes in the glycosylation of cell surface and secreted glycoproteins in cystic fibrosis. Glycosylation of cell surface glycoproteins, particularly levels of sialylation, may also be related to the increased binding of Pseudomonas to cystic fibrosis cells. The current study was undertaken to compare the sialylation of cell surface glycoconjugates in heterologous cells overexpressing normal CFTR or delta F508 CFTR. The presence of sialylated residues on cells was assessed by the surface binding of the specific lectins, wheat germ agglutinin and elderberry bark lectin. A fluorescent cholera toxin B subunit probe was used to measure surface binding to sialylated gangliosides in transfected cells. Our studies show that cells lacking CFTR and cells expressing normal CFTR have unaltered levels of sialylation. In contrast, cells expressing the delta F508 CFTR have significantly decreased amounts of sialylated glycoproteins and gangliosides on the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26).

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.

Amino Acid Sequence↗
PubMed2005

Mouse GPR40 heterologously expressed in Xenopus oocytes is activated by short-, medium-, and long-chain fatty acids.

Several orphan G protein-coupled receptors, including GPR40, have recently been shown to be responsive to fatty acids. Although previous reports have suggested GPR40 detects medium- and long-chain fatty acids, it has been reported to be unresponsive to short chain fatty acids. In this study, we have heterologously expressed mouse GPR40 in Xenopus laevis oocytes and measured fatty acid-induced increases in intracellular Ca(2+), via two electrode voltage clamp recordings of the endogenous Ca(2+)-activated chloride conductance. Exposure to 500 muM linoleic acid (C18:2), a long-chain fatty acid, stimulated significant currents in mGPR40-injected oocytes (P < 0.01, ANOVA), but not in water-injected control oocytes (not significant, ANOVA). These currents were confirmed as Ca(2+)-activated chloride conductances because they were biphasic, sensitive to changes in external pH, and inhibited by DIDS. Similar currents were observed with medium-chain fatty acids, such as lauric acid (C12:0) (P < 0.01, ANOVA), and more importantly, with short-chain fatty acids, such as butyric acid (C4:0) (P < 0.01, ANOVA). In contrast, no responses were observed in mGPR40-injected oocytes exposed to either acetic acid (C2:0) or propionic acid (C3:0). Therefore, GPR40 has the capacity to respond to fatty acids with chain lengths of four or greater. This finding has important implications for understanding the structure:function relationship of fatty acid sensors, and potentially for short-chain fatty acid sensing in the gastrointestinal tract.

Animals↗
PubMed2002

Identification by heterologous expression and gene disruption of VisA as L-lysine 2-aminotransferase essential for virginiamycin S biosynthesis in Streptomyces virginiae.

The visA gene of Streptomyces virginiae has been thought to be a part of the virginiamycin S (VS) biosynthetic gene cluster based on its location in the middle of genes that encode enzymes highly similar to those participating in the biosynthesis of streptogramin-type antibiotics. Heterologous expression of the visA gene was achieved in Escherichia coli by an N-terminal fusion with thioredoxin (TrxA), and the intact recombinant VisA protein (rVisA) was purified after cleavage with enterokinase to remove the TrxA moiety. The purified rVisA showed clear L-lysine 2-aminotransferase activity with an optimum pH of around 8.0 and an optimum temperature at 35 degrees C, with 2-oxohexanoate as the best amino acceptor, indicating that VisA converts L-lysine into Delta(1)-piperidine 2-carboxylic acid. A visA deletion mutant of S. virginiae was created by homologous recombination, and the in vivo function of the visA gene was studied by phenotypic comparison between the wild type and the visA deletion mutant. No differences in growth in liquid media or in morphological behavior on solid media were observed, indicating that visA is not involved in primary metabolism or morphological differentiation. However, the visA mutant failed to produce VS while maintaining the production of virginiamycin M(1) at a level comparable to that of the parental wild-type strain, demonstrating that visA is essential to VS biosynthesis. These results, together with the observed recovery of the defect in VS production by the external addition of 3-hydroxypicolinic acid (3-HPA), a starter molecule in VS biosynthesis, suggest that VisA is the first enzyme of the VS biosynthetic pathway and that it supplies 3-HPA from L-lysine.

Anti-Bacterial Agents↗
PubMed2000

Molecular cloning and heterologous expression of pea seedling copper amine oxidase.

The cDNA coding for copper amine oxidase has been cloned from etiolated pea seedlings (Pisum sativum). The deduced amino acid sequence, consisting of 674 residues including the signal peptide, agreed well with those reported for the enzymes from a different cultivar of P. sativum and other plant sources, except for several evolutionary replacements located mostly on the molecular surface. A heterologous expression system for the cloned pea enzyme was constructed with the yeast Pichia pastoris, using the AOX1 promoter and the yeast alpha-factor secretion signal. Adding copper to the culture medium increased the secretion of an active, quinone-containing enzyme. Furthermore, the inactive enzyme produced in a copper-deficient medium was activated considerably by subsequent incubation with excess cupric ions. These results strongly suggest that the Tyr-derived redox cofactor, 2,4,5-trihydroxyphenylalanylquinone (topa quinone, TPQ), is produced in the plant enzyme by post-translational modification that proceeds through the copper-dependent, self-processing mechanism, as in the enzymes from bacteria and yeast.

Amine Oxidase (Copper-Containing)↗
PubMed1998

Subtypes of odorant-binding proteins--heterologous expression and ligand binding.

Odorant-binding proteins (OBP) in the mucus of the olfactory epithelium are thought to transfer the hydrophobic odorous compounds through the aqueous barrier towards the chemo-sensory cells. To evaluate their binding properties, two distinct OBP subtypes of the rat were expressed as N-terminal His-tagged fusion proteins in Escherichia coli, thus allowing an efficient purification. Based on gel chromatography and CD spectroscopy analysis the recombinant OBP subtypes seem to share several structural features with other members of the lipocalin family. Approaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2-[3H]-isobutyl-3-methoxypyrazine whereas OBP2 did not shown any specific binding to this compound. In contrast, the chromophore 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) specifically interacted with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity revealed that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p-tert-butyl-alpha-methyl dihydrocinnamic aldehyde) and citralva (3,7-dimethyl-2,6-octadienenitrile), also displaced efficiently the chromophore, whereas pyrazine derivatives including 2-isobutyl-3-methoxypyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities.

Anilino Naphthalenesulfonates↗
PubMed2006

Molecular cloning and heterologous expression of a 10-deacetylbaccatin III-10-O-acetyl transferase cDNA from Taxus x media.

A full-length cDNA encoding 10-deacetylbaccatin III-10-O-acetyl transferase (designated as TmDBAT), which catalyzes the acetylation of the C-10 hydroxyl group of the advanced metabolite 10-deacetylbaccatin III (10-DAB) to yield baccatin III, the immediate diterpenoid precursor of Taxol, was isolated from Taxus x media. Heterologous expression of TmDBAT in E. coli demonstrated that TmDBAT was a functional gene. Tissue expression pattern analysis revealed that TmDBAT expressed strongly in leaves, weak in stems and no expression could be detected in fruits, implying that TmDBAT was tissue-specific. Expression profiling analysis of TmDBAT under different elicitor treatments including silver nitrate, ammonium ceric sulphate and methyl jasmonate indicated that TmDBAT was an elicitor-responsive gene. Southern blot analysis suggested that TmDBAT belonged to a small multigene family.

Acetyltransferases↗
PubMed2006

Heterologous expression of two trichothecene P450 genes in Fusarium verticillioides.

Fusarium graminearum Z-3639 and F. sporotrichioides NRRL3299 produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. These toxins differ in oxygenation at C-4, C-7, and C-8. In F. sporotrichioides, Tri1 (FsTri1) controls C-8 hydroxylation. To determine the function of an apparent F. graminearum Tri1 (FgTri1) homolog, both FsTri1 and FgTri1 genes were heterologously expressed in the trichothecene-nonproducing species F. verticillioides by fusing the Tri1 coding regions to the promoter of the fumonisin biosynthetic gene FUM8. FsTri1 and FgTri1 have been partially characterized by disruption analysis, and the results from these analyses suggest that FsTri1 most likely has a single function but that FgTri1 may have two functions. Transgenic F. verticillioides carrying the FsTri1 (FvF8FsTri1) converted exogenous isotrichodermin and calonectrin to 8-hydroxyisotrichodermin and 8-hydroxycalonectrin, respectively. Transgenic F. verticillioides carrying FgTri1 (FvF8FgTri1) converted isotrichodermin to a mixture of 7-hydroxyisotrichodermin and 8-hydroxyisotrichodermin but converted calonectrin to a mixture of 7-hydroxycalonectrin, 8-hydroxycalonectrin, and 3,15-diacetyldeoxynivalenol. A fourth compound, 7,8-dihydroxycalonectrin, was identified in large-scale F. verticillioides FvF8FgTri1 cultures fed isotrichodermin. Our results indicate that FgTri1 controls both C-7 and C-8 hydroxylation but that FsTri1 controls only C-8 hydroxylation. Our studies also demonstrate that F. verticillioides can metabolize some trichothecenes by adding an acetyl group to C-3 or by removing acetyl groups from C-4 or C-15. In addition, wild-type F. verticillioides can convert 7,8-dihydroxycalonectrin to 3,15-diacetyldeoxynivalenol.

Cytochrome P-450 Enzyme System↗
PubMed2002

A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: characterisation of recombinant sulphite:cytochrome c oxidoreductase from Starkeya novella.

The phototrophic purple non-sulfur bacterium Rhodobacter capsulatus expresses a wide variety of complex redox proteins in response to changing environmental conditions. Here we report the construction and evaluation of an expression system for recombinant proteins in that organism which makes use of the dor promoter from the same organism. A generic expression vector, pDorEX, was constructed and used to express sulphite:cytochrome c oxidoreductase from Starkeya novella, a heterodimeric protein containing both molybdenum and haem c. The recombinant protein was secreted to the periplasm and its biochemical properties were very similar to those of the native enzyme. The pDorEX system therefore seems to be potentially useful for heterologous expression of multi-subunit proteins containing complex redox cofactors.

Base Sequence↗
PubMed2003

A ubiquitous splice variant and a common polymorphism affect heterologous expression of recombinant human SCN5A heart sodium channels.

Amino acid sequence variations in SCN5A are known to affect function of wild-type channels and also those with coexisting mutations; therefore, it is important to know the exact sequence and function of channels most commonly present in human myocardium. SCN5A was analyzed in control panels of human alleles, demonstrating that the existing clones (hH1, hH1a, hH1b) each contained a rare variant and thus none represented the common sequence. Confirming prior work, the H558R polymorphism was present in approximately 30% of subjects. Quantitative mRNA analysis from human hearts showed that a shorter 2015 amino acid splice variant lacking glutamine at position 1077 (Q1077del) made up 65% of the transcript in every heart examined. Age, sex, race, or structural heart disease did not affect this proportion of Q1077del. Estimated population frequencies for the four common variants were 25% SCN5A, 10% [H558R], 45% [Q1077del], and 20% [H558R;Q1077del], where the reference sequence SCN5A is GenBank AC137587. When expressed in HEK-293 cells, these common variants had a more positive mid-point of the voltage dependence of inactivation than the standard clone hH1. Also, channels containing Q1077 expressed smaller currents. When H558R was present with Q1077 ([H558R]), current expression was profoundly reduced despite normal trafficking to the cell surface. Thus, four variant sequences for SCN5A are commonly present in human myocardium and they exhibit functional differences among themselves and with the previous standard clone. These results have implications for the choice of background sequence for experiments with heterologous expression systems, and possibly implications for electrophysiological function in vivo.

Black or African American↗
PubMed2006

Modulation of Ca2+ channels by heterologously expressed wild-type and mutant human micro-opioid receptors (hMORs) containing the A118G single-nucleotide polymorphism.

The most common single-nucleotide polymorphism (SNP) of the human mu-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca(2+) channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca(2+) channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration-response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC(50) values (nM) in cells expressing wild-type hMOR was: DAMGO (31) >> morphine (76) congruent with M-6-G (77) congruent with endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) >> morphine (39) >> endomorphin I (74) congruent with M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca(2+) current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.

Analgesics, Opioid↗
PubMedDate not supplied

Heterologous expression of bacteriocins using the mesentericin Y105 dedicated transport system by Leuconostoc mesenteroides.

Mesentericin Y105 (MesY105) is a class IIa anti-Listeria bacteriocin, produced by Leuconostoc (Ln.) mesenteroides Y105 and with potential food grade application. This bacterium produces a second bacteriocin, mesentericin B105 (MesB105), that does not belong to the same class. To study secretion of bacteriocins by the use of the MesY105 dedicated transport system (DTS), plasmids were constructed for heterologous expression by Ln. mesenteroides. pFBYC04 (Microbiology 144 (1998) 2845) harbours two divergent operons required for MesY105 secretion, i.e. the mesYI operon, encoding pre-MesY105 and immunity, respectively, and the mesCDE operon for secretion. A pFBYC04 derivative, pDMJF01 was constructed by divergent PCR to remove the mesY gene. Ln. mesenteroides DSM20484(pDMJF01) was unable to produce MesY105. The mesYI operon and mesB, mesH and mesF genes, encoding pre-MesB105, MesB105 immunity and a putative protein with unknown function, respectively, were cloned independently into a compatible pDMJF01 plasmid to produce, respectively, pDMJF:YI and pDMJF:BHF. DSM20484 transformed independently with these plasmids was unable to secrete any bacteriocin. MesY105 and MesB105 secretion was observed for DSM20484(pDMJF01) harbouring both pDMJF:YI and pDMJF:BHF. This indicates that the MesY105 DTS permits the transport of MesB105. MesY105 secretion machinery was used to secrete pediocin PA-1 (PedPA-1) by DSM20484 by an in-frame gene fusion strategy where the gene portions corresponding to the MesY105 leader peptide and the mature PedPA-1 were ligated. Thus, MesY105 secretion machinery appears to be a useful tool for secretion of class II bacteriocins by Leuconostoc.

Bacteriocins↗
PubMed2006

Protein kinase C-dependent enhancement of activity of rat brain NCKX2 heterologously expressed in HEK293 cells.

Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.

Animals↗