Individually monitoring ligand-induced changes in the structure of the GABAA receptor at benzodiazepine binding site and non-binding-site interfaces.

The mechanisms by which the GABA and benzodiazepine (BZD) binding sites of the GABA-A receptor are allosterically coupled remain elusive. In this study, we separately monitored ligand-induced structural changes in the BZD binding site (alpha/gamma interface) and at aligned positions in the alpha/beta interface. alpha(1)His101 and surrounding residues were individually mutated to cysteine and expressed with wild-type beta2 and gamma2 subunits in Xenopus laevis oocytes. The accessibilities of introduced cysteines to modification by methanethiosulfonate ethylammonium (MTSEA)-Biotin were measured in the presence and absence of GABA-site agonists, antagonists, BZDs, and pentobarbital. The presence of flurazepam or the BZD-site antagonist flumazenil (Ro15-1788) decreased the rate of modification of alpha(1)H101C at the BZD binding site. GABA and muscimol each increased MTSEA-Biotin modification of alpha(1)H101C located at the BZD-site, gabazine (SR-95531, a GABA binding site antagonist) decreased the rate, whereas pentobarbital had no effect. Modification of alpha(1)H101C at the alpha/beta interface was significantly slower than modification of alpha(1)H101C at the BZD site, and the presence of GABA or flurazepam had no effect on its accessibility, indicating the physicochemical environments of the alpha/gamma and alpha/beta interfaces are different. The data are consistent with the idea that GABA-binding site occupation by agonists causes a GABA binding cavity closure that is directly coupled to BZD binding cavity opening, and GABA-site antagonist binding causes a movement linked to BZD binding cavity closure. Pentobarbital binding/gating resulted in no observable movements in the BZD binding site near alpha(1)H101C, indicating that structural mechanisms underlying allosteric coupling between the GABA and BZD binding sites are distinct.

Effects of gamma2S subunit incorporation on GABAA receptor macroscopic kinetics.

GABA(A) receptors, the major inhibitory neurotransmitter receptors in the mammalian central nervous system, are heteropentameric proteins. We are interested in understanding the contribution of the gamma subunit to the kinetic properties of GABA(A) receptors. Studies in Xenopus oocytes have suggested that co-expression of alpha1, beta2, and gamma 2S subunits results in the formation of both alpha beta and alpha betagamma receptors (Boileau et al. 2002a; Boileau et al., 1998). Here, we have used an excess of the gamma 2S subunit in transfections of HEK293 cells to bias expression toward alpha beta gamma-containing receptors. Using rapid application and whole cell patch clamp techniques, we found that incorporation of the gamma subunit eliminated the rapid phases of desensitization and accelerated deactivation, consistent with a proposed role of desensitization in slowing deactivation. In addition, alpha betagamma receptors had an increased GABA EC(50), reduced sensitivity to block by Zn(2+), and did not display outward rectification as compared to alpha beta receptors.

The relative amount of cRNA coding for gamma2 subunits affects stimulation by benzodiazepines in GABA(A) receptors expressed in Xenopus oocytes.

Benzodiazepine (BZD) potentiation of GABA-activated Cl(-)-current (I(GABA)) in recombinant GABA(A) receptors requires the presence of the gamma subunit. When alpha1, beta2 and gamma2S cRNA are expressed in a 1:1:1 ratio in Xenopus oocytes, BZD potentiation of I(GABA) is submaximal, variable and diminishes over time. Potentiation by BZDs is increased, more reproducible and is stabilized over time by increasing the relative amount of cRNA coding for the gamma2S subunit. In addition, GABA EC(50) values for alpha1beta2gamma2 (1:1:1) receptors are intermediate to values measured for alpha1beta2 (1:1) and alpha1beta2gamma2 (1:1:10) receptors. We conclude that co-expression of equal ratios of alpha1, beta2 and gamma2 subunits in Xenopus oocytes produces a mixed population of alpha1beta2 and alpha1beta2gamma2 receptors. Therefore, for accurate measurements of BZD potentiation it is necessary to inject a higher ratio of gamma2 subunit cRNA relative to alpha1 and beta2 cRNA. This results in a purer population of alpha1beta2gamma2 receptors.

A (beta)-strand in the (gamma)2 subunit lines the benzodiazepine binding site of the GABA A receptor: structural rearrangements detected during channel gating.

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma(2)Y72-gamma(2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma(2)T73-gamma(2)T81 adopts a beta-strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma(2)A79 and gamma(2)T81, that line the BZD binding site. Sulfhydryl modification of gamma(2)A79C or gamma(2)T81C allosterically shifts the GABA EC(50) of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

Protein mobility and GABA-induced conformational changes in GABA(A) receptor pore-lining M2 segment.

Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABAA receptor alpha1 and beta1 M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at alpha1N275C/beta1E270C (20') and alpha1S272C/beta1H267C (17'), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at alpha1T261C/beta1T256C (6'), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.

Structure and dynamics of the GABA binding pocket: A narrowing cleft that constricts during activation.

Photo-affinity labeling and mutagenesis studies have identified several amino acids that may contribute to the ligand binding domains of ligand-gated ion channels. These types of studies, however, only generate a one-dimensional, static description of binding site structure. In this study, we used the substituted cysteine accessibility method not only to identify binding pocket residues but also to elicit information about binding site dynamics and structure. Residues surrounding the putative loop C ligand binding domain of the GABA(A) receptor (beta(2)V199 to beta(2)S209) were individually mutated to cysteine, and the mutant subunits were coexpressed with wild-type alpha(1) subunits in Xenopus oocytes. N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) reacts with cysteines introduced at positions G203, S204, Y205, P206, R207, and S209. This accessibility pattern is not consistent with either an alpha-helix or beta-strand. Instead, G203-S209 seems to form a water-accessible extended coil, whereas V199-T202 appears to buried in the protein or membrane. Coapplication of either GABA or the competitive antagonist SR-95531 significantly slows MTSEA-biotin modification of cysteines introduced at positions S204, Y205, R207, and S209, demonstrating that these residues line and face into the GABA binding pocket. MTSEA-biotin reaction rates reveal a steep accessibility gradient from G203-S209 and suggests that the binding pocket is a deep narrowing cleft. Pentobarbital activation of the receptor significantly slows MTSEA-biotin modification of cysteines at S204, R207, and S209, suggesting that the binding site may constrict during gating.

Identification of benzodiazepine binding site residues in the gamma2 subunit of the gamma-aminobutyric acid(A) receptor.

gamma-Aminobutyric acid(A) receptor gamma-subunits are important for benzodiazepine (BZD) binding and modulation of the gamma-aminobutyric acid-mediated Cl(-) current. Previously, by using gamma2/alpha1 chimeric subunits, we identified two domains of the gamma2-subunit, Lys-41-Trp-82 and Arg-114-Asp-161, that are, in conjunction, necessary and sufficient for high-affinity BZD binding. In this study, we generated additional gamma2/alpha1 chimeric subunits and gamma2 point mutants to identify specific residues within the gamma2 Lys-41-Trp-82 region that contribute to BZD binding. Mutant gamma2 and gamma2/alpha1 chimeric subunits were expressed with wild-type alpha1 and beta2 subunits in HEK 293 cells, and the binding of several BZDs was measured. We present evidence that the gamma2 region Met-57-Ile-62 is important for flunitrazepam binding and that, in particular, gamma2 Met-57 and gamma2 Tyr-58 are essential determinants for conferring high-affinity binding. Furthermore, we identify an additional residue, gamma2 Ala-79, that not only is important for high-affinity binding by flunitrazepam (a strong positive modulator) but also plays a crucial role in the binding of the imidazobenzodiazepines Ro15-1788 (a zero modulator) and Ro15-4513 (a weak negative modulator) in the BZD binding pocket. Results from site-directed mutagenesis of gamma2 Ala-79 suggest that this residue may be part of a microdomain within the BZD binding site that is important for binding imidazobenzodiazepines. This separation of drug-specific microdomains for competitive BZD ligands lends insight into the structural determinants governing the divergent effects of these compounds.

Rapid and direct modulation of GABAA receptors by halothane.

BACKGROUND: Hypotheses regarding the nature of channel modulation by volatile anesthetics have focused primarily on "membrane actions" of anesthetics and more recently on direct actions of volatile agents on receptor proteins themselves. With the recognition that many channels are subject to modulation by intracellular enzymes, such as protein kinases and phosphatases, and recent demonstrations that the activity of these modulators themselves may be altered by anesthetic agents, a third possibility has been suggested:-anesthetic actions on channels may be indirect, produced, for example, via direct effects on intracellular enzyme systems. METHODS: To determine the contribution of indirect versus direct modulation, the authors compared effects of the volatile anesthetic halothane on gamma-aminobutyric acid A receptors under two conditions: in the whole cell configuration with intact intracellular regulatory systems, and in the excised patch configuration, in which intracellular signaling systems have been disrupted. They also evaluated the effects of rapid application and withdrawal of anesthetic to determine the time course of onset and offset of the anesthetic actions on these channels. RESULTS: Characteristic changes in gamma-aminobutyric acid A receptor function occurred in excised patches as in whole cells, did not require alteration of receptor phosphorylation, and were rapid (onset and offset of anesthetic action occurred within milliseconds). CONCLUSIONS: These results are not consistent with indirect modulation but rather indicate that volatile agents modulate gamma-aminobutyric acid A receptors by direct action on the channel complex or surrounding lipid membrane.

Identification of transduction elements for benzodiazepine modulation of the GABA(A) receptor: three residues are required for allosteric coupling.

Modulation of GABA(A) receptors by benzodiazepines (BZDs) is believed to involve two distinct steps: a recognition step in which BZDs bind and a conformational transition step in which the affinity of the receptor for GABA changes. Previously, using gamma(2)/alpha(1) chimeric subunits (chi), we demonstrated that although the N-terminal 167 gamma(2) amino acid residues confer high-affinity BZD binding, other gamma(2) domains couple BZD binding to potentiation of the GABA-mediated Cl(-) current (I(GABA)). To determine which gamma(2) regions couple binding to potentiation, we generated chis with longer N-terminal gamma(2) segments for voltage-clamp experiments in Xenopus oocytes. Chimeras containing greater than the N-terminal 167 gamma(2) residues showed incremental gains in maximal potentiation for diazepam enhancement of I(GABA). Residues in gamma(2)199-236, gamma(2)224-236 (pre-M1), and particularly gamma(2)257-297 (M2 and surrounding loops) are important for BZD potentiation. For several positive BZD modulators tested, the same regions restored potentiation of I(GABA). In contrast, beta-carboline inverse-agonism was unaltered in chimeric receptors, suggesting that structural determinants for positive and negative BZD allosteric modulation are different. Dissection of the gamma(2)257-297 domain revealed that three residues in concert, gamma(2)T281, gamma(2)I282 (M2 channel vestibule), and gamma(2)S291 (M2-M3 loop) are necessary to impart full BZD potentiation to chimeric receptors. Thus, these residues participate in coupling distant BZD-binding events to conformational changes in the GABA(A) receptor. The location of these novel residues provides insight into the mechanisms underlying allosteric coupling for other members of the ligand-gated ion channel superfamily.

Mapping the agonist binding site of the GABAA receptor: evidence for a beta-strand.

GABAA receptors, along with the receptors for acetylcholine, glycine, and serotonin, are members of a ligand-gated ion channel superfamily (Ortells and Lunt, 1995). Because of the paucity of crystallographic information for these ligand-gated channels, little is known about the structure of their binding sites or how agonist binding is transduced into channel gating. We used the substituted cysteine accessibility method to obtain secondary structural information about the GABA binding site and to systematically identify residues that line its surface. Each residue from alpha1 Y59 to K70 was mutated to cysteine and expressed with wild-type beta2 subunits in Xenopus oocytes or HEK 293 cells. The sulfhydryl-specific reagent N-biotinylaminoethyl methanethiosulfonate (MTSEA-Biotin) was used to covalently modify the cysteine-substituted residues. Receptors with cysteines substituted at positions alpha1 T60, D62, F64, R66, and S68 reacted with MTSEA-Biotin, and alpha1 F64C, R66C, and S68C were protected from reaction by agonist. We conclude that alpha1 F64, R66, and S68 line part of the GABA binding site. The alternating pattern of accessibility of consecutive engineered cysteines to reaction with MTSEA-Biotin indicates that the region from alpha1 Y59 to S68 is a beta-strand.

A GABAA receptor alpha1 subunit tagged with green fluorescent protein requires a beta subunit for functional surface expression.

gamma-Aminobutyric acid, type A (GABAA) receptors, the major inhibitory neurotransmitter receptors in the central nervous system, are heteropentameric proteins assembled from distinct subunit classes with multiple subtypes, alpha(1-6), beta(1-4), gamma(1-3), delta(1), and epsilon(1). To examine the process of receptor assembly and targeting, we tagged the carboxyl terminus of the GABAA receptor alpha1 subunit with red-shifted enhanced green fluorescent protein (EGFP). Xenopus oocytes were injected with cRNA of this fusion protein, alpha1-EGFP, alone or in combination with cRNA of GABAA receptor beta2, gamma2, or beta2+gamma2 subunits. Within 72 h after injection, EGFP fluorescence was visible in all fusion protein-injected cells. The fluorescence was associated with the plasmalemma only when the beta2 subunit was co-injected with alpha1-EGFP. Texas Red-conjugated immunolabeling of EGFP on nonpermeabilized cells demonstrated that EGFP was localized extracellularly. Hence, the COOH terminus of the alpha1 subunit is extracellular. Two-electrode voltage clamp of alpha1-EGFPbeta2- and alpha1-EGFPbeta2 gamma2-injected oocytes demonstrates that these cells express functional receptors, with EC50 values for GABA and diazepam similar to wild-type receptors. Thus, a COOH-terminal tag of the alpha1 subunit appears to be functionally silent, providing a useful marker for studies of GABAA receptor expression, assembly, transport, targeting, and clustering. Moreover, the beta2 subunit is required for receptor assembly and surface expression.

Molecular dissection of benzodiazepine binding and allosteric coupling using chimeric gamma-aminobutyric acidA receptor subunits.

Although gamma-aminobutyric acid (GABA)A receptor alpha subunits are important for benzodiazepine (BZD) binding and GABA-current potentiation by BZDs, the presence of a gamma subunit is required for high affinity BZD effects. To determine which regions unique to the gamma2S subunit confer BZD binding and potentiation, we generated chimeric protein combinations of rat gamma2S and alpha1 subunits using a modified protocol to target crossover events to the amino-terminal extracellular region of the subunits. Several chimeras with full open reading frames were constructed and placed into vectors for either voltage-clamp experiments in Xenopus laevis oocytes or radioligand binding experiments in human embryonic kidney 293 cells. Chimeras (chi) containing at least the amino-terminal 161 amino acids of gamma2S bound BZDs with wild-type affinity when coexpressed with alpha1 and beta2 subunits. Further analysis of the gamma2S binding site region uncovered two areas, gamma2S K41-W82 and gamma2S R114-D161, that together are necessary and sufficient for high affinity BZD binding. Surprisingly, although the 161-amino acid residue amino terminus of the gamma2S subunit is sufficient for high affinity BZD binding, it is not sufficient for efficient allosteric coupling of the GABA and BZD binding sites, as demonstrated by reduced diazepam potentiation of the GABA-gated current and GABA potentiation of [3H]flunitrazepam binding. Thus, by using gamma/alpha chimeras, we identified two gamma2 subunit regions required for BZD binding that are distinct from domain or domains responsible for allosteric coupling of the BZD and GABA binding sites.

Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

The contributions of aspartyl residues in the acetylcholine receptor gamma and delta subunits to the binding of agonists and competitive antagonists.

The acetylcholine (ACh) receptors in muscle have the composition alpha2betagammadelta and contain two ACh binding sites. One is formed between an alpha subunit and the gamma subunit, and the other is formed between an alpha subunit and the delta subunit. Among the residues in the ACh binding sites are alphaCys-192 and alphaCys-193. The negatively charged deltaAsp-180 is at an appropriate distance from alphaCys-192/193 also to be in the ACh binding site and to interact electrostatically with the positively charged ammonium group common to agonists and competitive antagonists. Mutation to Asn of either deltaAsp-180 or the aligned residue in the gamma subunit, gammaAsp-174, decreased the affinities of three agonists, acetylcholine, tetramethylammonium, and succinyldicholine 170-560-fold. By contrast, these mutations decreased the affinities of three competitive antagonists, (+)-tubocurarine, hexamethonium, and dihydro-beta-erythroidine, only 2-15-fold. Agonists, but not antagonists, promote the transitions of the receptor from the resting state to the higher affinity active and desensitized states, and the greater effects of the mutations of gammaAsp-174 and deltaAsp-180 on the apparent affinities of agonists could reflect the involvement of these residues in the conformational changes of the receptor corresponding to its transitions to higher affinity states. In these transitions, one possibility is that gammaAsp-174 and deltaAsp-180 move closer to bound agonist.

Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding.

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between alpha-subunit residues Cys192 and Cys193. The cross-linker S-(2-[3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced alpha Cys192/Cys193 and an amide bond with an acidic residue in the delta subunit (Czajkoswski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH2CONHCH2CH2S- is is 0.9 nm long. After the disulfide bond linking -NHCH2CONHCH2CH2S- to the alpha subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the delta subunit by an amide bond. The delta subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to delta Asp165, delta Asp180, and delta Glu182. The mutation of delta Asp180 to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either delta Asp165, delta Glu182, or 8 other acidic residues in the same region of delta decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A. (1993) Proc. Natl. Acad. Sci. U.S.A 90, 6285-6289). Because delta Asp180 both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the alpha and the delta subunits.

The roles of bicarbonate and CO2 in transendothelial fluid movement and control of corneal thickness.

PURPOSE: To determine whether maintenance of corneal hydration is dependent on bicarbonate ions and whether these ions can be derived from metabolic or exogenous CO2, and to investigate the relationship of transendothelial fluid movement to control of hydration. METHODS: The thickness of intact or deepithelialized rabbit corneas was measured while superfused on the endothelial surface with either 33 mM HGO3-/5% CO2 buffered media or 10 mM HPO4- buffered media in the presence and absence of inhibitors of ion transport and respiration. The corneal surface was covered with either silicone oil ("normal" corneas) or with the same media used for superfusion ("swollen" corneas). ATP and Na+,K(+)-ATPase activity were measured in endothelia scraped from the tissues after superfusion. RESULTS: Intact and deepithelialized corneas covered with oil swelled at a negligible rate (4 to 8 microns/hour) in 33 mM HCO3- medium but at 45 to 60 microns/hour in HPO4- medium. Antimycin A altered neither of these swelling rates, but ethoxzolamide (0.1 mM) caused swelling in HCO3-/CO2 (approximately 12 microns/hour above controls) with no change of rate in HPO4-. Ouabain (0.1 mM) increased swelling to 45 to 50 microns/hour in HCO3-/CO2 but had no effect in HPO4-. Saturating the oil on deepithelialized corneas with 5% CO2, or putting HCO3-/CO2 medium on the epithelial surface of intact corneas, did not alter the swelling rates seen with HPO4- superfusion. The equilibrium thickness of deepithelialized corneas swollen with HCO3-/CO2 on both surfaces was 35 microns less than that of corneas swollen in HPO4-. The difference was abolished by ouabain, which caused corneas in HCO3-/CO2 to swell an additional 30 microns but did not alter the equilibrium thickness of corneas swollen in HPO4-. Ethoxzolamide and DIDS (0.2 mM) increased the thickness in HCO3-/CO2 but not in HPO4-. Na+,K(+)-ATPase activities of endothelia were similar after HCO3-/CO2 and HPO4- superfusions, but the concentration of ATP in the HPO4(-)-superfused tissues was increased 35%. CONCLUSIONS: Normal corneal thickness can be maintained in vitro only in media that contain HCO3- at concentrations of more than 20 mM. Neither metabolic CO2 nor CO2 present in air-equilibrated, nominally HCO3(-)-free media can supply this requirement for HCO3-, even though these sources support the presumably related processes of transendothelial fluid movement and intracellular pH regulation.

Relationship between fluid transport and in situ inhibition of Na(+)-K+ adenosine triphosphatase in corneal endothelium.

PURPOSE: To examine the relationship between the activity of the sodium pump of the corneal endothelium and corneal thickness. It was postulated that because inhibition pressure of the stroma decreases as thickness increases, a partially inhibited sodium pump would result in a new steady-state thickness of the cornea when reduced rates of fluid influx and efflux were equal. Measurements of physiologic behavior and biochemical activity were to be made in the same tissue and thus establish the relationship directly. METHODS: Rabbit corneas were superfused with a bicarbonate Ringer solution containing different concentrations of ouabain. Exposure to ouabain was either continuous for 4 hours or for an initial 10 minutes followed by ouabain-free superfusion. Thickness was measured, and, after superfusion, endothelium was removed from the corneas, sonicated, and assayed for Na(+)-K+ adenosine triphosphatase (ATPase) activity without further addition of ouabain to the assay medium. Thickness was also measured during superfusion with suboptimal concentrations of Na+ or HCO3- and with brefeldin A, an inhibitor of protein trafficking. RESULTS: Continuous exposure to ouabain caused corneas to swell, but no new steady-state thickness was reached. At low concentrations, swelling rates increased with time, as did the extent of inhibition of the Na(+)-K+ ATPase. With only a 10-minute exposure to ouabain, swelling rates with 10(-4) M to 10(-5) M decreased with the duration of ouabain-free superfusion. Similar swelling curves were obtained by reductions in Na+ or HCO3- concentrations in the superfusion medium, indicating that partial inhibition of the endothelial fluid transport processes, whether via the Na(+)-K+ ATPase or by suboptimal ionic conditions, led toward a new equilibrium thickness of the cornea. However, when superfusion was continued for more than 4 hours, the corneas exposed for 10 minutes to 3 x 10(-5) M or lower-concentration ouabain showed increasing Na(+)-K+ ATPase activity and began to thin, indicating a recovery of fluid transport capability. This recovery was blocked by addition of brefeldin A during the ouabain-free superfusion. CONCLUSIONS: Inhibition of Na(+)-K+ ATPase by low concentrations of ouabain increases with time. Temporary exposure to ouabain causes swelling at rates that decline with time as ouabain dissociates from enzyme sites. This dissociation, together with the turnover of Na(+)-K+ ATPase in the plasma membrane, can lead to recovery of normal thickness in ouabain-exposed corneas. Twenty percent of Na(+)-K+ ATPase in the endothelium is estimated to be intracellular, and about 20% of the activity can be inhibited without inducing swelling.

Negatively charged amino acid residues in the nicotinic receptor delta subunit that contribute to the binding of acetylcholine.

In nicotinic receptors, the binding sites for acetylcholine are likely to contain negatively charged amino acid side chains that interact with the positively charged quaternary ammonium group of acetylcholine and of other potent agonists. We previously found that a 61-residue segment of the delta subunit contains aspartate or glutamate residues within 1 nm of cysteines in the acetylcholine binding site on the alpha subunit. We have now mutated, one at a time, the 12 aspartates and glutamates in this segment of the mouse muscle delta subunit and have expressed the mutant receptors in Xenopus oocytes. Both the concentration of acetylcholine eliciting half-maximal current (Kapp) and the Ki for the inhibition by acetylcholine of alpha-bungarotoxin binding were increased 100-fold by the mutation of delta Asp180 to Asn and 10-fold by the mutation of delta Glu189 to Gln. These two residues, and their homologs in the gamma and epsilon subunits, are likely to contribute to the acetylcholine binding sites.

Characterization of transport-mediated methotrexate resistance in human tumor cells with antibodies to the membrane carrier for methotrexate and tetrahydrofolate cofactors.

An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.