Biomedical subjects

M Quick

Publications and source records attributed to M Quick.

17 recordsLinked to original sources

PubMed2001

Amino acid transporter CAATCH1 is also an amino acid-gated cation channel.

CAATCH1 (cation-amino acid transporter/channel) is a recently cloned insect epithelial membrane protein related to mammalian Na(+)-, Cl(-)-coupled neurotransmitter transporters (Feldman, D. H., Harvey, W. R., and Stevens, B. R. (2000) J. Biol. Chem. 275, 24518-24526). In the present study we analyze the relationship between CAATCH1-mediated amino acid transport and ion fluxes by utilizing the Xenopus oocyte expression system in conjunction with electrophysiology and radiotracer uptake. Simultaneous flux measurements reveal that electrical currents and amino acid transport are thermodynamically uncoupled. This observation is supported by measuring significant uptake even in the absence of external alkali cations. Remarkably, CAATCH1-associated Na(+) or K(+) currents are large and do not saturate with voltage nor with cation concentration. These currents reverse in Nernstian fashion, thereby conferring channel activity in CAATCH1. Upon step-changes in the membrane potential, CAATCH1-expressing oocytes exhibit transient currents. Detailed analyses of these transients in the absence and presence of amino acids reveal direct ligand-protein interaction, demonstrating that binding by different amino acids (e.g. proline, threonine, methionine) differentially affects the state probability of CAATCH1 but has no effect on the maximal charge movement (Q(max)). Together these data suggest that CAATCH1 is a multifunction membrane protein that mediates thermodynamically uncoupled amino acid uptake but functions predominantly as an amino acid-gated alkali cation channel.

Amino Acids↗

PubMed2000

Neutralization of a conserved amino acid residue in the human Na+/glucose transporter (hSGLT1) generates a glucose-gated H+ channel.

The role of conserved Asp204 in the human high affinity Na+/glucose cotransporter (hSGLT1) was investigated by site-directed mutagenesis combined with functional assays exploiting the Xenopus oocyte expression system. Substitution of H+ for Na+ reduces the apparent affinity of hSGLT1 for glucose from 0.3 to 6 mm. The apparent affinity for H+ (7 microm) is about three orders of magnitude higher than for Na+ (6 mm). Cation/glucose cotransport exhibits a coupling ratio of 2 Na+ (or 2 H+):1. Pre-steady-state kinetics indicate that similar Na+ - or H+ -induced conformational changes are the basis for coupled transport. Replacing Asp204 with Glu increases the apparent affinity for H+ by >20-fold with little impact on the apparent Na+ affinity. This implies that the length of the carboxylate side chain is critical for cation selectivity. Neutralization of Asp204 (Asp --> Asn or Cys) reveals glucose-evoked H(+) currents that were one order of magnitude greater than Na(+) currents. These phlorizin-sensitive H+ currents reverse and are enhanced by internal acidification of oocytes. Together with a H(+) to sugar stoichiometry as high as 145:1, these results favor a glucose-gated H+ channel activity of the mutant. Our observations support the idea that cotransporters and channels share common features.

Base Sequence↗

PubMed1999

Role of conserved Arg40 and Arg117 in the Na+/proline transporter of Escherichia coli.

The Na+/proline transporter of Escherichia coli (PutP) is a member of a large family of Na+/solute symporters. To investigate the role of Arg residues which are conserved within this family, Arg40 at the cytoplasmic end of transmembrane domain (TM) II and Arg117 in cytoplasmic loop 4 of PutP are subjected to amino acid substitution analysis. Removal of the positive charge at position 40 (PutP-R40C, Q, E) leads to a dramatic decrease of the V(max) of Na(+)-coupled proline uptake (1-10% of PutP-wild-type). The reduced transport rates are accompanied by decreased apparent affinities of the transporter for Na+ and Li+ while the apparent affinity for proline is only slightly altered. Furthermore, single Cys PutP-R40C reacts with N-ethylmaleimide (NEM), and this reaction is partially inhibited by proline and more efficiently by Na+ ions. Remarkably, NEM modification of Cys40 inhibits Na(+)-driven proline uptake almost completely while facilitated influx of proline into deenergized cells is stimulated by this reaction, suggesting an at least partially uncoupled phenotype under these conditions. These results suggest that Arg40 is located close to the site of ion binding and is important for the coupling of ion and proline transport. The observations confirm the functional importance of TM II described in earlier studies [M. Quick and H. Jung (1997) Biochemistry 36, 4631-4636]. In contrast to Arg40, Arg117 is apparently not important for function of the mature protein. The low transport rates observed upon substitution of Arg117 (PutP-R117C, K, Q) can at least partially be attributed to reduced amounts of PutP in the membrane. However, once inserted into the membrane, PutP containing Arg117 replacements shows a stability comparable to the wild-type as indicated by pulse-chase experiments. These observations suggest that Arg117 plays a crucial role at a stage prior to complete functional insertion of PutP into the membrane, i. e., by stabilizing a folding intermediate.

Amino Acid Sequence↗

PubMed1998

Topology of the Na+/proline transporter of Escherichia coli.

Hydropathy profile analysis of the amino acid sequence of the Na+/proline transporter of Escherichia coli (PutP) suggests that the protein consists of 12 transmembrane domains (TMs) which are connected by hydrophilic loops (Nakao, T., Yamato, I., and Anraku, Y. (1987) Mol. Gen. Genet. 208, 70-75). We have tested this prediction by applying a gene fusion approach in combination with a Cys accessibility analysis and site-specific proteolysis. Characterization of a series of PutP-alkaline phosphatase (PhoA) and PutP-beta-galactosidase (LacZ) hybrid proteins yields a reciprocal activity pattern of the reporter proteins that is in agreement with the topology of TMs III to XII of the 12-helix model. Placement of the PutP-PhoA and PutP-LacZ junction sites closer to the N terminus does not yield conclusive results. As a prerequisite for further topology studies, a functional PutP molecule devoid of all five native Cys residues (Cys-free PutP) is generated. Subsequently, amino acids in Cys-free PutP are replaced individually with Cys, and the accessibility of the sulfhydryl groups is analyzed. Surprisingly, Cys residues placed close to the N terminus of PutP (Ile-3 --> Cys, Thr-5 --> Cys) or into putative TM II (Ser-71 --> Cys, Glu-75 --> Cys) are highly accessible to membrane permeant and impermeant thiol reagents in intact cells. In contrast, Cys at the C terminus (Ser-502 --> Cys) reacts only with the membrane permeant but not with the impermeant reagent in intact cells. These results contradict the 12-helix motif and indicate a periplasmic location of the N terminus whereas the C terminus faces the cytoplasm. In addition, a transporter with Cys in place of Leu-37 (putative periplasmic loop (pL2) shows the same accessibility pattern as the Cys at the C terminus. Furthermore, PutP which has been purified and reconstituted into proteoliposomes in an inside-out orientation, is readily cleaved by the endoproteinase AspN before Asp-33 (pL2), Asp-112 (putative cytoplasmic loop (cL3), Asp-262 (cL7), and Asp-356 (cL9). These results suggest a cytosolic location of Asp-33 and Leu-37, thereby implying the formation of an additional TM formed by amino acids of pL2. Based on these observations, a new secondary structure model is proposed according to which the protein consists of 13 TMs with the N terminus on the outside and the C terminus facing the cytoplasm. The 13-helix structure is discussed as a common topological motif for all members of the Na+/solute cotransporter family.

Amino Acid Sequence↗

PubMed1998

A conserved aspartate residue, Asp187, is important for Na+-dependent proline binding and transport by the Na+/proline transporter of Escherichia coli.

Asp187 in the Na+/proline transporter of Escherichia coli (PutP) is conserved within the Na+/solute cotransporter family. Information on the role of this residue has been gained by amino acid substitution analysis. PutP with Glu, Asn, or Cys in place of Asp187 catalyzed Na+-coupled proline uptake at 75%, 25%, and 1.5%, respectively, of the Vmax of PutP-wild-type while the apparent Km for proline was only slightly altered. Importantly, acetylation or amidoacetylation of an engineered transporter containing a single Cys at position 187 stimulated proline uptake. Strikingly, PutP-D187C exhibited high-affinity proline binding even at very low Na+ concentrations (2 microM) while proline binding to PutP-wild-type, -D187E, and -D187N was strictly dependent on the Na+ concentration. The apparent independence of proline binding from the Na+ concentration can at least partially be attributed to an enhanced Na+ affinity of PutP-D187C. In addition, reaction of PutP containing a single Cys at position 187 with N-ethylmaleimide was inhibited by Na+ but not by Li+ or proline. The results indicate that electrostatic interactions of the amino acid side chain at position 187 in PutP with other parts of the transporter and/or the coupling ion are crucial for active proline transport. It is suggested that Asp187 is located close to the pathway of the coupling ion through the membrane and may be involved in the release of Na+ on the cytoplasmic side of the membrane.

Amino Acid Sequence↗

PubMed1997

Aspartate 55 in the Na+/proline permease of Escherichia coli is essential for Na+-coupled proline uptake.

Four acidic residues in the N-terminal domain of Na+/proline permease of Escherichia coli (Asp33, Asp34, and Asp55 in putative loop 2, Glu75 in putative transmembrane domain II) were individually replaced with neutral or charged amino acid residues. Replacement of Glu75, the only residue in the permease presumed to be in the middle of a transmembrane domain, Asp33, or Asp34 had little or no influence on the kinetics of Na+-coupled proline transport. In contrast, removal of the carboxylate at position 55 (Asp55 --> Asn or Asp55 --> Cys permease) impaired proline uptake completely while lengthening of the side chain at this position by one methylene group (Asp55 --> Glu permease) allowed transport at a reduced initial rate. Importantly, all permease molecules were present in the membrane at concentrations comparable to the wild-type protein. Kinetic analysis of Na+-coupled proline transport catalyzed by Asp55 --> Glu permease revealed a 5-fold increase of the K(m) for proline and a 30-fold decrease of the V(max) compared to wild-type. Remarkably, replacement of Asp55 by Glu led to a 50-fold decrease of the apparent affinity of the permease for Na+. Furthermore, replacement of Asp55 with Cys or Asn blocked proline-induced Na+ uptake whereas significant Na+ transport was observed with Asp55 --> Glu permease. In addition, transport of proline down its concentration gradient was not detectable with deenergized cells containing Asp55 --> Glu permease at low Na+ concentrations. However, downhill transport activity was observed in the presence of high Na+ concentrations. Replacement of Asp55 with Asn or Cys impaired downhill transport under all conditions tested. The observations demonstrate that a carboxylate at position 55 of proline permease is essential for Na+-coupled proline transport. It is suggested that Asp55 may be involved in binding of the coupling ion.

Amino Acid Sequence↗

PubMed1996

Ser57 in the Na+/proline permease of Escherichia coli is critical for high-affinity proline uptake.

Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azetidine-2-carboxylate and 3.4-dehydro-D.L-proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18 respectively, relative to wild-type. In contrast, all of the Ser57 replacements analyzed cause only small changes in Vmax values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein.

Amino Acid Sequence↗

PubMed1995

Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase.

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.

Amino Acid Sequence↗

PubMed1993

Steady states, charge movements, and rates for a cloned GABA transporter expressed in Xenopus oocytes.

Voltage-clamp analysis was applied to study the currents associated with the uptake of extracellular gamma-aminobutyric acid (GABA) by the cloned transporter GAT1 expressed at high efficiency in Xenopus oocytes. Steady-state GABA currents were increased at higher extracellular [GABA], [Na+], and [Cl-] and at more negative potentials. The Hill coefficient for Na+ exceeded unity, suggesting the involvement of two Na+ ions. In the absence of GABA, voltage jumps produced transient currents that behaved like capacitive charge movements; these were suppressed by the uptake inhibitor SKF-89976A, were shifted to more negative potentials at lower external [Na+] and [Cl-], and had an effective valence of 1.1 elementary charge. A turnover rate per transporter of 6-13/s at maximal [GABA] (-80 mV, 96 mM NaCl, 22 degrees C) is given both by the kinetics of voltage jump relaxations and by the ratio between the maximal GABA currents and the charge movements. These quantitative data are necessary for evaluating the roles of GAT1 in synaptic function.

Animals↗

PubMed1993

Receptors that couple to 2 classes of G proteins increase cAMP and activate CFTR expressed in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), a Cl- channel activated by phosphorylation, was expressed in Xenopus oocytes along with various combinations of several other components of the cAMP signalling pathway. Activation of the coexpressed beta 2 adrenergic receptor increased cAMP and led to CFTR activation. The activation of CFTR (1) requires only short (15 s) exposure to isoproterenol, (2) occurs for agonist concentrations 100-1000 fold lower than those that produce cAMP increases detectable by a radioimmunoassay, (3) requires injection of only 5 pg of receptor cRNA per oocyte, and (4) can be increased further by coexpression of cRNA for adenylyl cyclase type II or III or for Gs alpha. In addition, CFTR activation and cAMP increases by beta 2 activation were enhanced by activation of the coexpressed 5HT1A receptor, which is thought to couple to Gi. The additional activation by the 5HT1A receptor was enhanced by coexpression of adenylyl cyclase type II but not with type III and may proceed via the beta gamma subunits of a G protein. The sensitivity of the assay system is also demonstrated by responses to vasoactive intestinal peptide and to pituitary adenylate cyclase-activating polypeptide in oocytes injected with cerebral cortex mRNA.

Adenylyl Cyclases↗

PubMed1990