Formulation of survival acceptor medium able to maintain the viability of skin explants over in vitro dermal experiments.

OBJECTIVE: In vitro assessments of skin absorption of xenobiotics are essential for toxicological evaluations and bioavailability studies of cosmetic and pharmaceutical ingredients. Since skin metabolism can greatly contribute to xenobiotic absorption, experiments need to be performed with skin explants kept viable in suitable survival media. Existing protocols for non-viable skin are modified to consider those conditions. The objective was to design a survival medium used as an acceptor fluid in Franz cells for testing cutaneous penetration of hydrophilic or lipophilic molecules. Their metabolism inside skin may be investigated under the same conditions. The determining factors involved in survival mechanisms in vitro are discussed. The consequences of short-term skin preservation at 4°C were also evaluated. METHODS: The metabolic activity of fresh skin samples mounted in Franz cells was studied by measurement of lactate release over 24 h in order to assess the impacts of pH, buffering, osmolality, ionic strength, initial glucose supply and the addition of ethanol or non-ionic surfactant in the acceptor part of Franz cells. CONCLUSION: Survival media must maintain physiological pH (>5.5) be isotonic with skin cells (300 mOsm kg-1 ) and contain at least 0.5 g L-1 glucose. Several compositions able to preserve skin metabolism are reported. Storage of skin explants overnight at 4°C impairs skin metabolic activity. The present work provides guidelines for designing survival media according to constraints related to the scientific requirements of the experiments.

OBJECTIFS: Les études d'absorption cutanée sont indispensables pour les évaluations toxicologiques et les études de biodisponibilité des ingrédients cosmétiques et pharmaceutiques. Etant donné que le métabolisme cutané peut contribuer à l'absorption cutanée des xénobiotiques, les études doivent être parfois menées sur les explants cutanés maintenus en survie à l'aide d'un milieu adapté. Les protocoles classiques utilisés avec des explants congelés non viables sont souvent modifiés pour prendre en compte ces conditions particulières. L'objectif de cette étude est d'étudier les conditions nécessaires à appliquer au milieu receveur des cellules de Franz pour maintenir la viabilité des explants, dans les études de pénétration cutanée de molécules hydrophiles et lipophiles. Leur métabolisme dans la peau peut être étudié dans ces mêmes conditions. Les facteurs déterminants à prendre en compte pour assurer la viabilité des explants in vitro sont discutés. Les conséquences de la conservation des explants cutanés durant une courte durée à 4°C, avant utilisation, ont été également évaluées. METHODES: L'activité métabolique des échantillons de peau, montés en cellules de Franz, a été évaluée grâce aux mesures du lactate produit durant 24h, durée de l'expérience. L'impact du pH, de solutions « tampon ¼, de l'osmolalité, de la force ionique, de la concentration initiale en glucose et de l'addition d'éthanol ou de tensioactifs non-ioniques, dans le milieu receveur de la cellule de Franz, a été étudié. CONCLUSION: Le milieu de survie doit maintenir un pH physiologique (>5.5), être isotonique par rapport aux cellules de la peau (300 mOsm kg-1 ) et contenir au moins 0.5 g L-1 de glucose. Plusieurs compositions capables de maintenir le métabolisme cutané sont décrites. La conservation des explants cutanés à 4°C, durant une nuit, perturbe l'activité métabolique de la peau. Ces travaux permettent de mettre en évidence des prérequis pour la formulation de milieux de survie adaptés aux expériences.

From the molecular characterization of iodide transporters to the prevention of radioactive iodide exposure.

In the event of a nuclear reactor accident, the major public health risk will likely result from the release and dispersion of volatile radio-iodines. Upon body exposure and food ingestion, these radio-iodines are concentrated in the thyroid, resulting in substantial thyroidal irradiation and accordingly causing thyroid cancers. Stable potassium iodide (KI) effectively blocks thyroid iodine uptake and is thus used in iodide prophylaxis for reactor accidents. The efficiency of KI is directly related to the physiological inhibition of the thyroid function in the presence of high plasma iodide concentrations. This regulation is called the Wolff-Chaikoff effect. However, to be fully effective, KI should be administered shortly before or immediately after radioiodine exposure. If KI is provided only several hours after exposure, it will elicit the opposite effect e.g. lead to an increase in the thyroid irradiation dose. To date, clear evaluation of the benefit and the potential toxicity of KI administration remain difficult, and additional data are needed. We outline in this review the molecular characterization of KI-induced regulation of the thyroid function. Significant advances in the knowledge of the iodide transport mechanisms and thyroid physiology have been made. Recently developed molecular tools should help clarify iodide metabolism and the Wolff-Chaikoff effect. The major goals are clarifying the factors which increase thyroid cancer risk after a reactor accident and improving the KI administration protocol. These will ultimately lead to the development of novel strategies to decrease thyroid irradiation after radio-iodine exposure.

Molecular biology and bacterial secondary transporters.

Bacterial permeases form a family of membrane proteins that actively transport nutrients according to a cation-solute cotransport mechanism. The purpose of this review is to consider the many perspectives offered by in vitro recombinant DNA and molecular biology techniques for analysis of structure-function relationships. In the first part of this review, the kinetic parameters that permit characterization of either the partial steps or the overall transport reaction are summarized. We then list the molecular properties (protein sequence and composition, secondary structure, biochemical identification) of the carriers readily available on cloning a structural gene into plasmids. Finally, the usefulness of site-specific, oligonucleotide-directed mutagenesis for investigation of the functional importance of given residues (cysteins and histidines) or domains of the transport proteins is illustrated.

Cloning of the mouse sodium iodide symporter and its expression in the mammary gland and other tissues.

Iodide concentration in milk by mammals is a necessary step for thyroid hormone synthesis by the newborn. With the purpose of using the mouse as an animal model to analyse the role of the sodium iodide symporter (NIS) in iodide transport and its regulation in the mammary gland, mouse NIS (mNIS) cDNA was isolated from lactating mice. The cloned sequence shows an open reading frame of 1854 nucleotides encoding a protein of 618 amino acids highly homologous to the rat and human NIS (95% and 81% identity respectively). Expression of mNIS in cultured mammalian cells induced cellular iodide accumulation. This iodide uptake process is sodium dependent and inhibited by thiocyanate and perchlorate. Tissue distribution analysis revealed that mNIS mRNAs are predominantly expressed in thyroid, stomach and in the lactating mammary gland and are present to a lower extent in several other tissues. Our data show for the first time that the level of mNIS mRNA is upregulated in the mammary gland during lactation.

Amplitude-based data selection for optimal retrospective reconstruction in micro-SPECT.

Respiratory motion can blur the tomographic reconstruction of positron emission tomography or single-photon emission computed tomography (SPECT) images, which subsequently impair quantitative measurements, e.g. in the upper abdomen area. Respiratory signal phase-based gated reconstruction addresses this problem, but deteriorates the signal-to-noise ratio (SNR) and other intensity-based quality measures. This paper proposes a 3D reconstruction method dedicated to micro-SPECT imaging of mice. From a 4D acquisition, the phase images exhibiting motion are identified and the associated list-mode data are discarded, which enables the reconstruction of a 3D image without respiratory artefacts. The proposed method allows a motion-free reconstruction exhibiting both satisfactory count statistics and accuracy of measures. With respect to standard 3D reconstruction (non-gated 3D reconstruction) without breathing motion correction, an increase of 14.6% of the mean standardized uptake value has been observed, while, with respect to a gated 4D reconstruction, up to 60% less noise and an increase of up to 124% of the SNR have been demonstrated.

Melibiose permease of Escherichia coli. Characteristics of co-substrates release during facilitated diffusion reactions.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was investigated by further analyzing the Na+ (H+ or Li+)-coupled facilitated diffusion reactions catalyzed by the carrier in de-energized membrane vesicles, with particular emphasis on the reaction of sugar exchange at equilibrium. It is first shown that melibiose exchange at equilibrium proceeds without concomitant movement of Na+, i.e. the coupled cation is kinetically occluded during the melibiose exchange reaction. These results provide further experimental support for the model of Na+ sugar co-transport of the physiological substrate melibiose previously suggested (Bassilana, M., Pourcher, T., and Leblanc, G. (1987) J. Biol. Chem. 262, 16865-16870) in which: 1) the mechanisms of co-substrate binding to (or release from) the carrier are ordered processes on both the outer (Na+ first, sugar last) and inner membrane surfaces (sugar first, Na+ last) and give rise to a mirror-type model; 2) release of Na+ from the carrier on the inner membrane surface is very slow and rate-limiting for carrier cycling but is fast on the opposite side, contributing to the asymmetrical functioning of the permease. On the other hand, analysis of the exchange of identical sugars (homologous exchange) and different sugar analogs (heterologous exchange) indicates that the overall rate of sugar exchange reaction coupled to Na+ or Li+ is limited by the rate of one (or more) partial step(s) associated with the inflow of co-substrates and most probably by the rate of sugar release into the intravesicular medium. It is proposed that the variability of the facilitated diffusion reactions catalyzed by the carrier in the presence of different coupled cations and/or sugar analogs reflects variations in the rate of co-substrate release from the carrier on the inner membrane surface.

Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles. Release of co-substrates is rate limiting for permease cycling.

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.

Evidence for intraprotein charge transfer during the transport activity of the melibiose permease from Escherichia coli.

Electrogenic activity associated with the activity of the melibiose permease (MelB) of Escherichia coli was investigated by using proteoliposomes containing purified MelB adsorbed onto a solid-supported membrane. Transient currents were selectively recorded by applying concentration jumps of Na+ ions (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-methyl galactoside, raffinose) using a fast-flow solution exchange system. Characteristically, the transient current response was fast, including a single decay exponential component (tau approximately 15 ms) on applying a Na+ (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the cation and sugar substrates, the electrical transients were biphasic and comprised both the fast and an additional slow (tau approximately 350 ms) decay components. Finally, selective inactivation of the cosubstrate translocation step by acylation of MelB cysteins with N-ethyl maleimide suppressed the slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within MelB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose binding in the absence of Na+ of k approximately 10 s(-1).

Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions.

An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.

Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215) is further analyzed using site-directed mutagenesis. To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine. The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied. The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue. Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport. Moreover, two groups of mutants could be distinguished. One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties. A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile. The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition. Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.

Melibiose permease of Escherichia coli: large scale purification and evidence that H+, Na+, and Li+ sugar symport is catalyzed by a single polypeptide.

As much as 20-30 mg of functional recombinant melibiose permease (Mel-6His permease) of Escherichia coli, carrying a carboxy-terminal affinity tag for metallic ions (six successive histidines), can be routinely purified from 10 g of cells (dry weight) by combining nickel chelate affinity chromatography and ion exchange chromatography. Mel-6His permease was constructed by modifying the permease gene (melB) in vitro and then overproduced in cells transformed with multicopy plasmids. The tagged permease was efficiently solubilized in the presence of 3-(laurylamido)-N,N'-dimethylaminopropylamine oxide (LAPAO) and high sodium salt concentration and then selectively adsorbed on a nickel nitrilotriacetic acid (Ni-NTA) affinity resin. After the replacement of LAPAO by n-dodecyl beta-D-maltoside to maintain the activity of the soluble permease in low ionic strength media, the permease-enriched fraction (> 90%) was eluted with 0.1 M imidazole and finally purified to homogeneity (> 99%) using ion exchange chromatography. Determination of the permease N-terminal sequence shows that an initiating methionine is missing and that a Ser-Ile-Ser stretch precedes the postulated primary amino acid sequence. Purified permeases, reconstituted in liposomes, display H(+)-, Na(+)-, or Li(+)-dependent sugar binding and active transport activities similar to those of the native permease in its natural environment, proving that all three modes of symport activity are mediated by one and the same polypeptide.

Melibiose permease of Escherichia coli: substrate-induced conformational changes monitored by tryptophan fluorescence spectroscopy.

Tryptophan fluorescence spectroscopy has been used to investigate the effects of sugars and coupling cations (H+, Na+, or Li+) on the conformational properties of purified melibiose permease after reconstitution in liposomes. Melibiose permease emission fluorescence is selectively enhanced by sugars, which serve as substrates for the symport reaction, alpha-galactosides producing larger variations (13-17%) than beta-galactosides (7%). Moreover, the sugar-dependent fluorescence increase is specifically potentiated by NaCl and LiCl (5-7 times), which are well-established activators of sugar binding and transport by the permease. The potentiation effect is greater in the presence of LiCl than NaCl. On their own, sodium and lithium ions produce quenching of the fluorescence signal (2%). Evidence suggesting that sugars and cations compete for their respective binding sites is also given. Both the sugar-induced fluorescence variation and the NaCl(or LiCl)-dependent potentiation effect exhibit saturation kinetics. In each ionic condition, the half-maximal fluorescence change is found at a sugar concentration corresponding to the sugar-binding constant. Also, half-maximal potentiation of the fluorescence change by sodium or lithium occurs at a concentration comparable to the activation constant of sugar binding by each ion. The sugar- and ion-dependent fluorescence variations still take place after selective inactivation of the permease substrate translocation capacity by N-ethylmaleimide. Taken together, the data suggest that the changes in permease fluorescence reflect conformational changes occurring upon the formation of ternary sugar/cation/permease complexes.

Membrane topology of the melibiose permease of Escherichia coli studied by melB-phoA fusion analysis.

In order to study the secondary structure of the melibiose permease of Escherichia coli, 57 melB-phoA gene fusions were constructed and assayed for alkaline phosphatase activity. In general agreement with a previously suggested secondary structure model of melibiose permease [Botfield, M. C., Naguchi, K., Tsuchiya, T., & Wilson, T.H. (1992) J. Biol. Chem. 267, 1818], clusters of fusions exhibiting low and high phosphatase activity fusions alternate along the primary sequence. Fusions with high activity generally cluster at residues predicted to be in the periplasmic half of transmembrane domains or in periplasmic loops, while fusions with low activity cluster at residues predicted to be in the cytoplasmic half of transmembrane domains or in cytoplasmic loops. Taken together, the findings strongly support the contention that melibiose permease contains 12 transmembrane domains that traverse the membrane in zigzag fashion connected by hydrophilic loops that are exposed alternatively on the periplasmic or cytoplasmic surfaces of the membrane with the N and C termini on the cytoplasmic face of the membrane. Moreover, on the basis of the finding that the cytoplasmic half of an out-going segment is sufficient for alkaline phosphatase export to the periplasm while the periplasmic half of an in-going segment prevents it [Calamia, T., & Manoil, C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4937], the activity profile of the melibiose permease-alkaline phosphatase fusions is consistent with the predicted topology of seven of 12 transmembrane segments. However, five transmembrane domains require adjustment, and as a consequence, the size of the central cytoplasmic loop is reduced and a significant number of charged residues are shifted from a hydrophilic to a hydrophobic domain in this region of the transporter.

Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. I. Effect on Na(+)-dependent transport and binding properties.

Four aspartic acids, distributed in different putative membrane-spanning segments of the NH2-terminal domain of melibiose (mel) permease (D31 in helix I, D51 and D55 in helix II, and D120 in helix IV) were individually replaced by either Asn or Cys using site-directed mutagenesis. mel permease with either neutral residues at position 51, 55, or 120 or permease with a Cys in place of D31 does not catalyze significant Na(+)-linked methyl-1-thio-beta-D-galactopyranoside (TMG) accumulation. Binding studies of a high affinity ligand (p-nitrophenyl-alpha-D-galactopyranoside (NPG)) on de-energized membrane vesicles indicate that these modified transporters (i) retain the ability to bind the alpha-galactosides NPG or melibiose and the beta-galactoside TMG and (ii) exhibit a Na(+)-independent sugar-binding phenotype. In contrast, mel permease with an Asn residue at position 31 mediates Na(+)-coupled TMG transport and displays a Na(+)-dependent sugar binding phenotype, but requires a higher concentration of sodium than wild-type permease to produce maximal stimulation of sugar binding. The observation that individual mutation of the Asp residue at position 31, 51, 55, or 120 systematically and selectively modifies the contribution of the coupling ion to the early step of the transport reaction, i.e. cosubstrate binding, raises the possibility that (i) these 4 aspartic residues are at or near the cationic binding site of mel permease, (ii) the NH2-terminal domain of mel permease in which they are distributed accommodates or is part of the cationic binding site, and (iii) the oxygen atoms of these Asp side chains contribute to coordination of the coupling ion.

Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. II. Effect on cationic selectivity and coupling properties.

Individual substitution of Cys or Asn for Asp-31, Asp-51, Asp-55, or Asp-120, distributed in different membrane spanning segments of the NH2-terminal domain of melibiose (mel) permease partially or completely inactivates Na(+)-linked sugar transport and stimulation of sugar binding on mel permease by Na+ ions (Pourcher, T., Zani, M.-L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215). To investigate further the effect of these substitutions on the cationic selectivity and coupling properties of mel permease, H(+)-melibiose coupled transport, coupling between H+ and melibiose movements, sugar counterflow, and zero-trans sugar efflux by the mutant permeases were analyzed. The results provide additional evidence indicating that manipulation of some of these Asp in the membrane-spanning segments of mel permease alters its cationic selectivity properties. The results also indicate that the individual mutations diversely affect mel permease-coupling properties. For example, only permease with Asn in place of Asp-31 or Cys in place of Asp-51 retains the capacity to actively transport melibiose. On the other hand, replacing Asp-55 by Cys produces uncoupling of cosubstrate flows by the carrier but does not hamper sugar translocation. These and other features of the mutant permeases are used to discuss the relative participation of Asp-31, Asp-51, Asp-55, or Asp-120 to the mel symport mechanism and to its ionic selectivity and also the existence of a possible gating mechanism that may contribute the obligatory coupling of cosubstrate flows by the symporter.

The melibiose/Na+ symporter of Escherichia coli: kinetic and molecular properties.

The role of the co-transported cation in the coupling mechanism of the melibiose permease of Escherichia coli has been investigated by analysing its sugar-binding activity, facilitated diffusion reactions and energy-dependent transport reactions catalysed by the carrier functioning either as an H+, Na+ or Li(+)-sugar symporter. The results suggest that the coupling cation not only acts as an activator for sugar-binding on the carrier but also regulates the rate of dissociation of the co-substrates in the cytoplasm by controlling the stability of the ternary complex cation-sugar-carrier facing the cell interior. Furthermore, there is some evidence that the membrane potential enhances the rate of symport activity by increasing the rate of dissociation of the co-substrates from the carrier in the cellular compartment. Identification of the melibiose permease as a membrane protein of 39 kDa by using a T7 RNA polymerase/promoter expression system is described. Site-directed mutagenesis has been used to replace individual carrier histidine residues by arginine to probe the functional contribution of each of the seven histidine residues to the symport mechanism. Only substitution of arginine for His94 greatly interferes with the carrier function. It is finally shown that mutations affecting the glutamate residue in position 361 inactivate translocation of the co-substrates but not their recognition by the permease.

Melibiose permease and alpha-galactosidase of Escherichia coli: identification by selective labeling using a T7 RNA polymerase/promoter expression system.

Identification and selective labeling of the melibiose permease and alpha-galactosidase in Escherichia coli, which are encoded by the melB and melA genes, respectively, have been accomplished by selectively labeling the two gene products with a T7 RNA polymerase expression system [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074]. Following generation of a novel EcoRI restriction site in the intergenic sequence between the two genes of the mel operon by oligonucleotide-directed, site-specific mutagenesis, melA and melB were separately inserted into plasmid pT7-6 of the T7 expression system. Expression of melB was markedly enhanced by placing a strong, synthetic ribosome binding site at an optimal distance upstream from the initiation codon of melB. Expression of cloned gene products was characterized functionally and by performing autoradiographic analysis on total cell, inner membrane, and cytoplasmic proteins from cells pulse labeled with (35S)methionine in the presence of rifampicin and resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results first confirm that alpha-galactosidase is a cytoplasmic protein with an Mr of 50K; in contrast, the membrane-bound melibiose permease is identified as a protein with an apparent Mr of 39K, a value significantly higher than that of 30K previously suggested [Hanatani et al. (1984) J. Biol. Chem. 259, 1807].

Histidine-94 is the only important histidine residue in the melibiose permease of Escherichia coli.

Oligonucleotide-directed, site-specific mutagenesis has been utilized to modify the melB gene of Escherichia coli such that each of the seven His residues in the melibiose permease has been replaced with Arg. Replacement of His-213, His-442, or His-456 has no significant effect on permease activity, while permease with Arg in place of His-198, His-318, or His-357 retains more than 70% of wild-type activity. In striking contrast, replacement of His-94 with Arg causes a complete loss of sugar binding and transport, although the cells contain a normal complement of permease molecules. Thus, as shown previously with lac permease, only a single His residue is important for activity, but, in the case of mel permease, the critical His residue is present in the 3rd putative transmembrane helix rather than the 10th.

Melibiose permease of Escherichia coli: mutation of histidine-94 alters expression and stability rather than catalytic activity.

Previous studies utilizing site-directed mutagenesis [Pourcher et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 468-472] indicate that out of seven histidinyl residues in the melibiose (mel) permease of Escherichia coli, only His94 is important. The role of His94 has now been investigated by replacing the residue with Asn, Gln, or Arg. Cells expressing mel permease with Asn94 or Gln94 retain 30% or 20% of wild-type activity, respectively, and surprisingly, immunological assays demonstrate that diminished transport activity is due to a proportional reduction in the amount of permease in the membrane. Moreover, kinetic analyses of transport and ligand binding studies with right-side-out membrane vesicles indicate that both substrate recognition and turnover (kcat) are comparable in the mutant permeases and the wild-type. Mel permease with Arg in place of His94 also binds ligand and catalyzes sugar accumulation, but only when the cells are grown at 30 degrees C, and evidence is presented that Arg94 permease is inactivated at 37 degrees C. Finally, labeling studies demonstrate that expression and/or insertion of the permease, but not degradation, is strongly dependent on the amino acid present at position 94 and temperature. The findings indicate that an imidazole group at position 94 is required for proper insertion and stability of mel permease, but not for transport activity per se. Since replacement of the other six histidinyl residues in mel permease with Arg has little or no effect on transport activity, it is concluded that histidinyl residues do not play a direct role in the mechanism of this secondary transport protein.

Phosphorylation of P-glycoprotein by PKA and PKC modulates swelling-activated Cl- currents.

Several proteins belonging to the ATP-binding cassette superfamily can affect ion channel function. These include the cystic fibrosis transmembrane conductance regulator, the sulfonylurea receptor, and the multidrug resistance protein P-glycoprotein (MDR1). We measured whole cell swelling-activated Cl- currents (ICl,swell) in parental cells and cells expressing wild-type MDR1 or a phosphorylation-defective mutant (Ser-661, Ser-667, and Ser-671 replaced by Ala). Stimulation of protein kinase C (PKC) with a phorbol ester reduced the rate of increase in ICl,swell only in cells that express MDR1. PKC stimulation had no effect on steady-state ICl,swell. Stimulation of protein kinase A (PKA) with 8-bromoadenosine 3',5'-cyclic monophosphate reduced steady-state ICl, swell only in MDR1-expressing cells. PKA stimulation had no effect on the rate of ICl,swell activation. The effects of stimulation of PKA and PKC on ICl,swell were additive (i.e., decrease in the rate of activation and reduction in steady-state ICl,swell). The effects of PKA and PKC stimulation were absent in cells expressing the phosphorylation-defective mutant. In summary, it is likely that phosphorylation of MDR1 by PKA and by PKC alters swelling-activated Cl- channels by independent mechanisms and that Ser-661, Ser-667, and Ser-671 are involved in the responses of ICl,swell to stimulation of PKA and PKC. These results support the notion that MDR1 phosphorylation affects ICl,swell.