Structural requirement of the calcium-channel subunit alpha2delta for gabapentin binding.

Gabapentin [Neurontin, 1-(aminomethyl)cyclohexaneacetic acid] is a novel anticonvulsant drug with a high binding affinity for the Ca(2+)-channel subunit alpha(2)delta. In this study, the gabapentin-binding properties of wild-type and mutated porcine brain alpha(2)delta proteins were investigated. Removal of the disulphide bonds between the alpha(2) and the delta subunits did not result in a significant loss of gabapentin binding, suggesting that the disulphide linkage between the two subunits is not required for binding. Singly expressed alpha(2) protein remained membrane associated. However, alpha(2) alone was unable to bind gabapentin, unless the cells were concurrently transfected with the expression vector for delta, suggesting that both alpha(2) and delta are required for gabapentin binding. Using internal deletion mutagenesis, we mapped two regions [amino acid residues 339-365 (DeltaF) and 875-905 (DeltaJ)] within the alpha(2) subunit that are not required for gabapentin binding. Further, deletion of three other individual regions [amino acid residues 206-222 (DeltaD), 516-537 (DeltaH) and 583-603 (DeltaI)] within the alpha(2) subunit disrupted gabapentin binding, suggesting the structural importance of these regions. Using alanine to replace four to six amino acid residues in each of these regions abolished gabapentin binding. These results demonstrate that region D, between the N-terminal end and the first putative transmembrane domain of alpha(2), and regions H and I, between the putative splicing acceptor sites (Gln(511) and Ser(601)), may play important roles in maintaining the structural integrity for gabapentin binding. Further single amino acid replacement mutagenesis within these regions identified Arg(217) as critical for gabapentin binding.

Bacterial expression and characterization of human recombinant apolipoprotein(a) kringle IV type 9.

Elevated plasma lipoprotein(a) [Lp(a)] is an independent risk factor for several vascular diseases. Lp(a) particles are generated through the formation of a disulfide bond between Cys4057 of kringle IV type 9, (KIVt9), of the multikringle apolipoprotein(a) [apo(a)] and a cysteine in apoB-100 low-density lipoprotein (LDL). To better understand this interaction, we have expressed and purified KIVt9 from Escherichia coli as a His-Tag fusionprotein. Dithiothreitol (DTT)-treated purified KIVt9 migrated as a single approximately 17. 3-kDa band on SDS-PAGE gels. Without DTT, an additional band twice the molecular weight of KIVt9 was observed. The double-size band presumably resulted from dimerization of individual kringles, through their unpaired cysteine residues, since a mutation Cys4057 --> Ser ([Ser4057]KIVt9) abolished dimer formation. Using a gel-shift assay, we showed that KIVt9 could couple to 14-amino-acid apoB-100 synthetic peptides (apoB3732-3745 and apoB4319-4332) containing Cys3734 or Cys4326. Both of these apoB-100 cysteines have been reported to associate with apo(a) to generate Lp(a). In the presence of either apoB-100 peptide, KIVt9 was shifted to a higher molecular weight that was consistent with the covalent addition of a 1.2-kDa apoB-100 peptide. Identical apoB-100 peptides in which the cysteine residues were replaced by alanine ([Ala3734]apoB3732-3745 and [Ala4326]apoB4319-4332) had no effect in the gel-shift assay. Furthermore, [Ser4057]KIVt9 did not covalently interact with apoB3732-3745 or apoB4319-4332. These results indicated that KIVt9 couples to the Cys-apoB-100 peptides through a disulfide linkage. This system may be suitable for further investigating the apo(a)/apoB-100 coupling reaction and the structure of KIVt9 through X-ray crystallographic studies.

Troglitazone increases system A amino acid transport in 3T3-L1 cells.

System A is one of the most highly regulated transport systems for transport of neutral amino acids into mammalian cells. Stimulation of uptake of alpha-[3H]methylaminoisobutyric acid (MeAIB), a nonmetabolizable system A substrate, by a novel insulin-sensitizing agent, troglitazone, in 3T3-L1 adipocytes was investigated. Treating adipocytes with troglitazone alone resulted in a time- and dose-dependent increase in the uptake of MeAIB. The peak stimulation appeared about 24 h after troglitazone addition. Both troglitazone- and insulin-stimulated transport activities increased markedly after the induction of differentiation of preadipocytes into adipocytes, and declined to a steady state level in adipocytes. The stimulated MeAIB uptake exhibited substrate specificity typical of system A and was mediated by a single component as determined by Eadie-Hofstee plots. The stimulation by troglitazone and that by insulin were similarly sensitive to actinomycin D and cycloheximide, suggesting that both agents may induce de novo synthesis of the same type of system A transport. Apart from the insulin-independent effect, troglitazone also showed an insulin-dependent action characterized by enhanced sensitivity to insulin. The synergistic stimulation of MeAIB uptake by coadministration of insulin and troglitazone was most prominent at the early stages of adipocyte differentiation. Pretreating cells with troglitazone during the differentiation attenuated the sensitivity of insulin to inhibition by actinomycin D, suggesting that troglitazone may enhance the insulin action by stabilizing messenger RNA involved in system A function.

Regulation of system A amino acid transport in 3T3-L1 adipocytes by insulin.

The insulin-stimulated uptake of 2-(methylamino)isobutyric acid (MeAIB), a nonmetabolizable substrate for system A, in 3T3-L1 adipocytes was investigated. As cells took on a more adipogenic phenotype, the insulin-stimulated versus the saturable basal MeAIB uptake increased by 5-fold. The induced transport activity showed properties characteristic of system A, with a Km value of 190 microM. The half-life of the induced system A activity was independent of de novo mRNA and protein synthesis and was not accelerated by ambient amino acids, therefore, it was mechanistically distinct from the previously described adaptive and hormonal regulation of system A. Inhibition of mitogen-activated protein kinase kinase by PD98059, Ras farnesylation by PD152440 and B581, p70(S6K) by rapamycin, and phosphatidylinositol 3-kinase (PI 3'-K) by wortmannin and LY294002 revealed that only wortmannin and LY294002 inhibited the insulin-induced MeAIB uptake with IC50 values close to that previously reported for inhibition of PI 3'-K. These results suggest that the Ras/mitogen-activated protein kinase and pp70(S6K) insulin signaling pathways are neither required nor sufficient for insulin stimulation of MeAIB uptake, and activation of PI 3'-K or a wortmannin/LY294002-sensitive pathway may play an important role in regulation of system A transport by insulin in 3T3-L1 cells.

Glutamine transport in cerebellar granule cells in culture.

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K(m) = 2.6 mM) is the major transport system in the absence of Na+. The systems A and ASC represent the Na(+)-dependent transport routes, both with almost identical high affinity for glutamine (K(m) = 0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V(max)/K(m) = 20 min(-1) for system A as compared to the V(max)/K(m) ratio of 5 min(-1) in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.

The unpredicted high affinities of a large number of naturally occurring tachykinins for chimeric NK1/NK3 receptors suggest a role for an inhibitory domain in determining receptor specificity.

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide gamma, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently "improved" affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.

Effects of anticonvulsant drug gabapentin on the enzymes in metabolic pathways of glutamate and GABA.

Gabapentin is a novel anticonvulsant drug. The anticonvulsant mechanism of gabapentin is not known. Based on the amino acid structure of gabapentin we explored its possible effects on glutamate and gamma-aminobutyric acid (GABA) metabolism in brain as they may relate to its anticonvulsant mechanisms of action. Gabapentin was tested for its effects on seven enzymes in the metabolic pathways of these two neurotransmitters: alanine aminotransferase (AL-T), aspartate aminotransferase (AS-T), GABA aminotransferase (GABA-T), branched-chain amino acid aminotransferase (BCAA-T), glutamine synthetase (Gln-S), glutaminase (GLNase), and glutamate dehydrogenase (GDH). In the presence of 10 mM gabapentin, only GABA-T, BCAA-T, and GDH activities were affected by this drug. Inhibition of GABA-T by gabapentin was weak (33%). The Ki values for inhibition of cytosolic and mitochondrial forms of GABA-T (17-20 mM) were much higher than the Km values for GABA (1.5-1.9 mM). It is, therefore, unlikely that inhibition of GABA-T by gabapentin is clinically relevant. As with leucine, gabapentin stimulated GDH activity. The GDH activity in rat brain synaptosomes was activated 6-fold and 3.4-fold, respectively, at saturating concentrations (10 mM) of leucine and gabapentin. The half-maximal stimulation by gabapentin was observed at approximately 1.5 mM. Gabapentin is not a substrate of BCAA-T, but it exhibited a potent competitive inhibition of both cytosolic and mitochondrial forms of brain BCAA-T. Inhibition of BCAA-T by this drug was reversible. The Ki values (0.8-1.4 mM) for inhibition of transamination by gabapentin were close to the apparent Km values for the branched-chain amino acids (BCAA) L-leucine, L-isoleucine, and L-valine (0.6-1.2 mM), suggesting that gabapentin may significantly reduce synthesis of glutamate from BCAA in brain by acting on BCAA-T.

Transport of gabapentin, a gamma-amino acid drug, by system l alpha-amino acid transporters: a comparative study in astrocytes, synaptosomes, and CHO cells.

The system L transporter is generally considered to be one of the major Na(+)-independent carriers for large neutral alpha-amino acids in mammalian cells. However, we found that cultured astrocytes from rat brain cortex accumulate gabapentin, a gamma-amino acid, predominately by this alpha-amino acid transport system. Uptake of gabapentin by system L transporter was also examined in synaptosomes and Chinese hamster ovary (CHO) cells. The inhibition pattern displayed by various amino acids on gabapentin uptake in astrocytes and synaptosomes corresponds closely to that observed for the system L transport activity in CHO cells. Gabapentin and leucine have Km values that equal their Ki values for inhibition of each other, suggesting that leucine and gabapentin compete for the same system L transporter. By contrast, gabapentin exhibited no effect on uptake of GABA, glutamate, and arginine, indicating that these latter three types of brain transporters do not serve for uptake of gabapentin. A comparison of computer modeling analysis of gabapentin and L-leucine structures shows that although the former is a gamma-amino acid, it can assume a conformation that can resemble the L-form of a large neutral alpha-amino acid such as L-leucine. The steady-state kinetic study in astrocytes and CHO cells indicates that the intracellular concentrations of gabapentin are about two to four times higher than that of leucine. The uptake levels of these two substrates are inversely related to their relative exodus rates. The concentrating ability by system L observed in astrocytes is consistent with the substantially high accumulation gradient of gabapentin in the brain tissue as determined by microdialysis.

Evidence for a regulatory element controlling amino acid transport system L in Chinese hamster ovary cells.

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO-ts025C1 has a temperature-sensitive mutation in leucyl-tRNA synthetase. At the nonpermissive temperature of 39 degrees C, CHO-ts025C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell line, CHO-C11B6, that has constitutively elevated system L transport activity. The CHO-C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads to increased intracellular leucine levels, which compensate for the defective synthetase. Hybrid cells formed by fusion of the temperature-sensitive CHO-ts025C1 cells and the temperature-resistant CHO-C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO-C11B6 cell line.

The non-peptide tachykinin NK2 receptor antagonist SR 48968 interacts with human, but not rat, cloned tachykinin NK3 receptors.

SR 48968, a non-peptide tachykinin NK2 receptor antagonist, has been shown to possess sub-micromolar affinity for NK3 receptors present in the guinea pig. In the present study, we have compared the binding affinities of SR 48968 to the cloned human and rat NK3 receptors expressed in CHO cells. Using [125I]-[MePhe7]-neurokinin B as the radioligand, SR48968 displayed an IC50 value of 350 nM for the human NK3 receptor as compared with a value of greater than 10 microM for the rat NK3 receptor. Exposure of cells transfected with human NK3 receptor cDNA to [Pro7]-neurokinin B increased inositol phospholipid turnover in a concentration-dependent manner and this response was blocked competitively by SR 48968. Our results demonstrate that SR 48968 is an antagonist at the human NK3 receptor and may be a useful tool for elucidating the species-dependent variations in the non-peptide antagonist binding site(s) on the NK3 receptor.

Identification of methionine134 and alanine146 in the second transmembrane segment of the human tachykinin NK3 receptor as reduces involved in species-selective binding to SR 48968.

We have shown that SR 48968 possess sub-micromolar affinity for the human tachyknin NK3 receptor; however, its affinity for the rat NK3 receptor is greater than 10 microM. To determine the functional domain(s) responsible for the species variation in binding affinities, we have constructed several human/rat chimeric NK3 receptors. Based on studies of these chimeric receptors, the species-specific binding sites for SR 48968 were localized to five residues in the 1st and 2nd transmembrane segments of the human NK3 receptor. We have individually mutated all five residues in the rat receptor to their corresponding residues in human. Only two single-substituted mutants (V121M and G133A) show a small increase in their binding affinities for SR 48968. However, a mutant containing both substitutions was shown to have the same affinity for SR 48968 as the wild type human NK3 receptor. It is concluded that collectively these two amino acid changes are responsible for the species difference in binding affinities for SR 48968.

Chinese hamster ovary mRNA-dependent, Na(+)-independent L-leucine transport in Xenopus laevis oocytes.

In freshly prepared uninjected folliculated oocytes, Na(+)-independent leucine uptake is mediated predominantly by a system L-like transport system. Removal of follicular cells, however, results in an irreversible loss of this transport activity. When total poly(A)+ mRNA derived from Chinese hamster ovary (CHO) cells was injected into prophase-arrested stage V or VI Xenopus laevis oocytes, enhanced expression of Na(+)-independent leucine transport was observed. The injected mRNAs associated with increased levels of leucine uptake were between 2 and 3 kb in length. The newly expressed leucine transport activity exhibited important differences from the known characteristics of system L, which is the dominant Na(+)-independent leucine transporter in CHO cells as well as in freshly isolated folliculated oocytes. The CHO mRNA-dependent leucine uptake in oocytes was highly sensitive to the cationic amino acids lysine, arginine, and and ornithine (> 95% inhibition). As with the leucine uptake, an enhanced lysine uptake was also observed in size-fractionated CHO mRNA-injected oocytes. The uptakes of leucine and lysine were mutually inhibitable, suggesting that the newly expressed transporter was responsible for uptakes of both leucine and lysine. The inhibition of uptake of lysine by leucine was Na+ independent, thus clearly distinguishing it from the previously reported endogenous system y+ activity. Furthermore, the high sensitivity to tryptophan of the CHO mRNA-dependent leucine transport was in sharp contrast to the properties of the recently cloned leucine transport-associated gene from rat kidney tissue, although leucine transport from both sources was sensitive to cationic amino acids. Our results suggest that there may be a family of leucine transporters operative in different tissues and possibly under different conditions.

Intragenic complementation between Escherichia coli trp repressors with different defects in the tryptophan-binding pocket.

Site-directed mutagenesis of the trpR gene (encoding the trp repressor, TrpR) was used to replace Gly85 with tryptophan (Trp or W), in order to place Trp near its normal location in the L-tryptophan(L-W)-binding pocket. The resulting mutant protein (G85W) exhibits weak, but significant repressor activity in vivo that is independent of the presence of L-W in the media. This mutant negatively complements the chromosomal wild type (wt), but does not negatively complement either the wt or the super-repressor, E49K, when any of these alleles is expressed on a multicopy plasmid. Activity of the mutant repressor, G85W, when produced in vivo together with T44M, approaches that of the wt repressor. This result presumably reflects complementation between the two mutant polypeptides. Similar results are obtained when G85R or G85K are combined with T44M in vivo, but not when G85W is replaced by G85E. The level of repression is dependent on the presence of L-W in the media. The TrpR with two mutations altering both Gly85 (G85W, G85R, G85E or G85K) and Thr44 (T44M) has no repressor activity. These results suggest a type of site-specific intragenic complementation where only certain alterations at Gly85 complement T44M. In this study, a positive charge or an indole ring appears to be required for the observed intragenic complementation.

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.

Premature termination of in vivo transcription of a gene encoding a branched-chain amino acid transport protein in Escherichia coli.

Previous studies have suggested that control of expression of genes of the LIV-I permease system for the high-affinity transport of branched-chain amino acids in Escherichia coli involves modulation in the frequency of mRNA elongation. Mutation of the Rho transcription termination factor and shortages of charged leucyl-tRNA have been shown to alter LIV-I transport activity. Rho-dependent transcription termination regulated by shortages of charged leucyl-tRNA at sites preceding structural genes has been proposed to account for their role in regulation of LIV-I transport. Transcription of the livJ-binding protein gene, encoding one of the periplasmic components of the LIV-I system, was analyzed in vivo with strains which lack repression of the LIV-I genes and harbor a temperature-sensitive allele for either leucyl-tRNA synthetase or Rho factor. Analysis of mRNA synthesis by DNA-RNA hybridization in the various mutant strains indicated that both shortages of leucyl-tRNA caused by inactivation of the temperature-sensitive leucyl-tRNA synthetase and inactivation of the Rho factor were associated with increased synthesis of livJ mRNA. Nuclease protection and gel electrophoresis studies detected prematurely terminated transcripts corresponding in size to the leader region of livJ mRNA. Accumulations of these short transcripts were suppressed in strains harboring temperature-sensitive alleles for either leucyl-tRNA synthetase or Rho factor. These results provide support for the hypothesis that expression of livJ involves Rho-dependent transcription termination in which antitermination is associated with the intracellular availability of aminoacyl leucyl-tRNA.

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.

A bacterial system for investigating transport effects of cystic fibrosis--associated mutations.

LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). CF-associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes. Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM. A mutation equivalent to a benign polymorphism had no effect on transport. The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins.