The effect of phospholipases and proteases on the binding of gamma-aminobutyric acid to junctional complexes of rat cerebellum.

A preparation enriched in junctional complexes, as judged by marker enzymes and electron microscopy, was prepared from rat cerebellum. The junctional complexes were incubated with gamma-amino [14C]butyric acid at 25degreesC for 10 min, using [3H]sucrose as a marker for entrapped space, Total binding was determined in the absence of, and non-specific binding in the presence of, and excess of unlabelled gamma-aminobutyric acid. The difference bewteen the two binding values, i.e. the specific binding, was saturable and reversible, and showed positive cooperativity with a Hill number of about 2. The specific binding was inhibited by N-methylbicuculline, picrotoxinine and imidazole-4-acetic acid, but not by curare, strychnine or L-2,4-diaminobutyric acid. The above compounds had little effect on the non-specipic binding, but addition of ethylenediaminetetraacetic acid decreased non-specific binding by 80%. Trypsin, pronase, phospholipase A2 (EC 3.1.1.4), lysolecithin and sodium dodecyl sulfate decreased binding. Phospholipase C (EC 3.1.4.3) increased the specific binding by 260%. Phospholipids competed with gamma-aminobutyric acid for binding, with phosphatidylethanolamine being more potent than phosphatidylcholine. These results lend support for Watkins' hypothesis that phosphatidylethanolamine competes with gamma-aminobutyric acid for binding to the receptor protein.     

Differential modulation of two interferon-α binding proteins on a human lymphoblastoid cell line

The interaction between human interferon (IFN)-α or IFN-β with its receptor was originally described as the binding to a single class of high-affinity receptors. However, more recently, biphasic Scatchard plots as well as multiple IFN-α receptor cross-linked complexes have been reported. In this study using the Daudi B lymphoblastoid cell line, two primary IFN-α receptor cross-linked complexes with apparent M_r of 115 and 135 kilodaltons (kDa) were obtained. Both complexes were observed under a variety of cross-linking conditions, including the addition of a mixture of protease inhibitors throughout the binding reaction and solubilization of the cells. These two complexes appear to be caused by the binding and cross-linking of ¹²⁵I-rIFN-αA to two separate proteins because we also observed two IFN-α binding proteins using a ligand-blotting technique. At low concentrations of ¹²⁵I-rIFN-αA, it was found that the intensity of the signal in the 135-kDa cross-linked complex was greater than that of the 115-kDa complex. Addition of increasing concentrations of unlabeled rIFN-αA to a 4°C binding reaction reversed the ratio in intensities of the two complexes. Moreover, after pretreatment of the cells at 37°C with low concentrations of unlabeled rIFN-αA, there was preferential down-regulation of both the 135-kDa complex and the higher affinity binding component of the biphasic Scatchard plot. These results suggest that the 135-kDa complex represents the binding of ¹²⁵I-rIFN-αA to a protein having higher affinity for IFN than the protein that gives rise to the 115-kDa complex. These two proteins also appear to have different half lives in the plasma membrane in the absence of IFN because treatment with cycloheximide also caused a preferential decrease in the subsequent formation of the 135-kDa complex.     

DNA binding domains in Tn3 transposase

Summary Various segments of Tn3 transposase were fused individually to -galactosidase, and the resulting fusion proteins were examined for their DNA binding ability by a nitrocellulose filter binding assay. Analyses of a series of the fusion proteins revealed that the N-terminal segment of the transposase (amino acid positions 1–242; the transposase gene encodes 1004 residues in all) had specific DNA binding ability for the 38 bp terminal inverted repeat (IR) sequence, and the central segment (amino acid positions 243–632) had non-specific DNA binding ability. Further analyses of each of the two regions revealed that the N-terminal segment could be divided into at least two subsegments (amino acid positions 1–86 and 87–242), neither of which had specific DNA binding ability, but which both possessed nonspecific DNA binding ability. The central segment included two subsegments (amino acid positions 243–289 and 439–505) with non-specific DNA binding ability. These results and other observations suggest that Tn3 transposase has several domains including those responsible for non-specific DNA binding, and a combination of two or more domains gives rise to specific DNA binding activity. The C-terminal segment of the transposase (amino acid positions 633-1004), which is very well conserved among transposases encoded by Tn3 family transposons, had no DNA binding ability. This segment may represent the main part of the catalytic domain responsible for the initiation step of transposition.     

Binding of estrogens by α-fetoprotein in rat amniotic fluid

Amniotic fluid from 15–17-day rat fetuses bound estrone and 17β-estradiol specifically. Related steroids such as estriol, 6-ketoestradiol, 17α-estradiol and testosterone were not bound to any significant extent. The apparent K_a for 17β-estradiol was 2.6·10⁸ M⁻ at 4°C; 6 nmoles of 17β-estradiol were bound per ml of amniotic fluid. The binding component appears to be α-fetoprotein in that it migrates as an α₁-globulin upon polyacrylamide gel electrophoresis and has an isoelectric pH of 4.7 as determined by isoelectric focusing. Furthermore, binding activity was precipitated by antiserum which was shown by immuno-electrophoresis to be specific for α-fetoprotein. Binding activity, partially purified by isoelectric focusing of amniotic fluid, was associated with one of two bands seen by polyacrylamide gel electrophoresis. This band migrated as an α₁-globulin.     

Affinity labeling of high-affinity α-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase α-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). ¹²⁵I-labeled α-thrombin binds rapidly and specifically to CCL39 cells with high affinity (K_d ≈ 4 nM). Binding at 37°C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of α-thrombin receptors on CCL39 cells was identified by covalently coupling ¹²⁵I-α-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major α-thrombin-binding site of M_r ≈ 150 000 revealed as a ¹²⁵I-α-thrombin cross-linked complex of M_r ≈ 180 000. Independent of chemical cross-linking, ¹²⁵I-α-thrombin also formed a covalent complex with a minor, 35 000 M_r, membrane component identified as protease nexin. Two derivatives of α-thrombin modified at the active site are 1000-fold less than α-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native α-thrombin, and affinity-labeled the receptor subunit of M_r 150 000. When present in large excess, during incubation of cells with α-thrombin, these binding antagonists were ineffective in blocking α-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 M_r binding sites that display high affinity for α-thrombin do not mediate induction of the cellular mitogenic response.     

η-N-succinimidato complexes of iron, molybdenum and tungsten as reversible inhibitors of papain

Recently we have found that the metallocarbonyl complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-maleimidato) (M = Fe, Mo, W; x = 2 or 3) bearing a maleimide function were irreversible inhibitors of the enzyme papain. To get further insight into the binding mechanism of these compounds we synthesized the related complexes (η⁵-C₅H₅)M(CO)_x(η¹-N-succinimidato) (M = Fe, Mo, W; x = 2 or 3) that lacked the ethylenic bond responsible for alkylation of the cysteine 25 thiol group in the papain‘s catalytic pocket. We performed kinetic studies of the interaction of the synthesized complexes towards papain. We found that they act as reversible inhibitors of the enzyme with IC₅₀ values in the range 480–1700 μM. Docking experiments confirmed binding of these complexes to the enzyme’s catalytic pocket.     

Simultaneous saccharification and fermentation of cellulose: effect of β- -glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β- -glucosidase (β- -glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β- -glucosidase to filter paper activity was 1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ⁻¹, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β- -glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

Ability of growth hormone fragments to compete with I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with ¹²⁵I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with ¹²⁵I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.     

Characterization of the stimulatory effects of PGF2α on the release of arachidonic acid

Fibroblasts derived from a rat carrageenin granuloma were cultured in the presence of radioactive arachidonic acid, palmitic acid and linoleic acid. More than 90% of each labeled fatty acid was incorporated into a phospholipid fraction by the cells in 18 hrs. Arachidonic acid was evenly incorporated into phosphatidylcholine and phosphatidylethanolamine, while both palmitic acid and linoleic acid were almost entirely incorporated into phosphatidylcholine. The position of phosphatidylcholine where the fatty acids were incorporated was different for each fatty acid. The ratio of the amount of fatty acid incorporated into the 2-position to the amount incorporated into the 1-position of phosphatidylcholine for each fatty acid was >90% for arachidonic acid, 2:1 for palmitic acid and 5:1 for linoleic acid. In the case of phosphatidylethanolamine, most arachidonic acid (>90%) was incorporated into the 2-position. PGF_2^α caused the stimulation of arachidonic acid release but not of palmitic acid and linoleic acid from pre-labeled fibroblasts.The serum in the medium was completely replaceable by bovine serum albumin. The effect of PGF_2^α increased with an increasing concentration of bovine serum albumin, suggesting that serum only acts as a ‘trap’ for released arachidonic acid. The effect of PGF_2^α was greater than bradykinin, and no synergistic effect was seen, although an additive effect was observed.The effect of PGF_2^α depended on the concentration of calcium ions under magnesium-supplemented conditions.     

Characterization, size estimation and solubilization of α-macroglobulin complex receptors in liver membranes

Receptors for α₂-macroglobulin-proteinase complexes have been characterized in rat and human liver membranes. The affinity for binding of ¹²⁵I-labelled α₂-macroglobulin · trypsin to rat liver membranes was markedly pH-dependent in the physiological range with maximum binding at pH 7.8–9.0. The half-time for association was about 5 min at 37°C in contrast to about 5 h at 4°C. The half-saturation constant was about 100 pM at 4°C and 1 nM at 37°C (pH 7.8). The binding capacity was approx. 300 pmol per g protein for rat liver membranes and about 100 pmol per g for human membranes. Radiation inactivation studies showed a target size of 466 ± 71 kDa (S.D., n = 7) for α₂-macroglobulin · trypsin binding activity. Affinity cross-linking to rat and human membranes of ¹²⁵I-labelled rat α₁-inhibitor-3 · chymotrypsin, a 210 kDa analogue which binds to the α₂-macroglobulin receptors in hepatocytes (Gliemann, J. and Sottrup-Jensen, L. (1987) FEBS Lett. 221, 55–60), followed by SDS-polyacrylamide gel electrophoresis, revealed radioactivity in a band not distinguishable from that of cross-linked α₂-macroglobulin (720 kDa). This radioactivity was absent when membranes with bound ¹²⁵I-α₁-inhibitor-3 complex were treated with EDTA before cross-linking and when incubation and cross-linking were carried out in the presence of a saturating concentration of unlabelled complex. The saturable binding activity was maintained when membranes were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]profane sulfonate (CHAPS) and the size of the receptor as estimated by cross-linking experiments was shown to be similar to that determined in the membranes. It is concluded that liver membranes contain high concentrations of an approx. 400–500 kDa α₂-macroglobulin receptor soluble in CHAPS. The soluble preparation should provide a suitable material for purification and further characterization of the receptor.     

Structures, dynamic behaviour in solution and cross-coupling reactions of the α-palladiated (η-alkylarene)tricarbonylchromium complexes containing a palladium-chromium bond

Structures of the complexes (η³-C₃H₅)Pd(μ-η^6:1-CH₂PhCr(CO)₃ and (η³-C₃H₅)Pd[μ-η^6:1-CH(Ph)Ph]Cr(CO)₃ in solution were evaluated by NMR (¹H and ¹³C) and IR spectroscopy. The dynamic behaviour of the complexes was investigated. Quick rotation (on the NMR time scale) of the tricarbonylchromium groups around the axis passing through the centre of the η⁶-coordinated phenyl ring and the chromium atom takes place at room temperature and becomes slow on cooling. The η³-allylic ligand was proved to undergo no dynamic changes in solution. Unlike the solid state, the semi-bridging carbonyl groups between chromium and palladium atoms are absent or very weak in solution. Cross-coupling reactions of the complexes with organohalides are described.     

Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP cycling at high efficiency

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP⁺ was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD⁺ oxidoreductase, EC 1.1.1.176), and the regeneration of NADP⁺ was obtained through the glutamate dehydrogenase (l-glutamate:NADP⁺ oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained 70% of the initial activity after 2 months of continuous use.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [d-Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Interactions between proteins bound to the duck β-globin gene promoter and enhancer detected by the DNaseI footprinting

The duck β^A-globin (β^AGLB) enhancer was closely linked to the duck β^A-GLB promoter, and the construct was used to study binding of nuclear proteins to specific sites of these regulatory elements. DNaseI-footprint analysis showed that the presence of the enhancer induced binding of proteins to additional sites on the promoter. The results are consistent with the looping-out model, based on specific interactions of enhancer-bound and promoter bound-proteins.     

Extracellular calcium-inhibited alpha-amylase of Bacillus coagulans B 49

Extracellular α-amylase (EC 3.2.1.1) from Bacillus coagulans B 49 was purified to homogeneity by ion-exchange chromatography and gel filtration. The optimum pH and temperature for dextrinizing activity were 6–7 and 70°C and for saccharolytic activity were 7 and 60°C, respectively. Calcium inhibited α-amylase activity even at low concentrations (10 m ), and most of its activity could be restored by dialysis against EDTA. Other cations such as Mg²⁺, Fe²⁺, and Hg²⁺ also inhibited amylase activity, while Mn²⁺ exhibited a slight stimulatory effect. The activity of the enzyme was not affected by ethylenediaminetetraacetic acid (EDTA).     

Normal acid behavior for C(α)-proton transfer from a thiazolium lon

Rate constants for C(α)-proton transfer from racemic 2-(1-hydroxyethyl)-3,4-dimethylthi-oazolium ion catalyzed by lyoxide ion and various oxygen-containing and amine buffers were determined by iodination at 25°C and ionic strength 1.0 in H₂O. Thermodynamically unfavorable C(α)-proton transfer to oxygen-containing and amine bases shows general base catalysis with a Brønsted β value of ≥0.92 for bases of pK_a^′ ≤ 15; this indicates that the thermodynamically favorable protonation reaction in the reverse direction has a Brønsted α value ≤0.08, which is consistent with diffusion-controlled reprotonation of the C(α)-enamine by most acids. General base catalysis is detectable because there is an 85-fold negative deviation from the Brønsted correlation by hydroxide ion. Primary kinetic isotope effects of (k_H/k_D)_obsd = 1.0 for thermodynamically unfavorable proton transfer to buffer bases and hydroxide ion (ΔpK_a ≤ −6) and a secondary solvent isotope effect of k_DO⁻/k_HO⁻ = 2.3 for C(α)-proton transfer are consistent with a very late, enamine-like transition state and rate-limiting diffusional separation of buffer acids from the C(α)-enamine in the rate-limiting step, as expected for a “normal” acid. The second-order rate constants for catalysis by buffer bases were used to calculate a pK_a^′ of 21.8 for the C(α)-proton assuming a rate constant of 3 × 10^{9 −1} s⁻¹ for the diffusion-controlled reprotonation of the C(α)-enamine by buffer acids in the reverse direction. It is concluded (i) that C(α)-proton removal occurs at the maximum possible rate for a given equilibrium constant, and (ii) that C(α)-enamines can have a significant lifetime in aqueous solution and on thiamin diphosphate-dependent enzymes.     

Determination of β-glucosidase aggregating factor (BGAF) binding and polymerization regions on the maize β-glucosidase isozyme Glu1

β-Glucosidases (Glu1 and Glu2) in maize specifically interact with a lectin called β-glucosidase aggregating factor (BGAF). We have shown that the N-terminal (Glu⁵⁰–Val¹⁴⁵) and the C-terminal (Phe⁴⁶⁶–Ala⁵¹²) regions of maize Glu1 are involved in binding to BGAF. Sequence comparison between sorghum β-glucosidases (dhurrinases, which do not bind to BGAF) and maize β-glucosidases, and the 3D-structure of Glu1 suggested that the BGAF-binding site on Glu1 is much smaller than predicted previously. To define more precisely the BGAF-binding site, we constructed additional chimeric β-glucosidases. The results showed that a region spanning 11 amino acids (Ile⁷²–Thr⁸²) on Glu1 is essential and sufficient for BGAF binding, whereas the extreme N-terminal region Ser¹–Thr²⁹, together with C-terminal region Phe⁴⁶⁶–Ala⁵¹², affects the size of Glu1–BGAF complexes. The dissociation constants (K_d) of chimeric β-glucosidase–BGAF interactions also demonstrated that the extreme N-terminal and C-terminal regions are important but not essential for binding. To confirm the importance of Ile⁷²–Thr⁸² on Glu1 for BGAF binding, we constructed a chimeric sorghum β-glucosidase, Dhr2 (C-11, Dhr2 whose Val⁷²–Glu⁸² region was replaced with the Ile⁷²–Thr⁸² region of Glu1). C-11 binds to BGAF, indicating that the Ile⁷²–Thr⁸² region is indeed a major interaction site on Glu1 involved in BGAF binding.     

5-Phosphorylribose 1-α-methylenebisphosphonate: Properties of a substrate analog of 5-phosphorylribose 1-α-diphosphate

The enzymatic synthesis of 5-phosphorylribose 1-α-methylenebisphosphonate (PRPCP), an analog of 5-phosphorylribose 1-α-diphosphate (PRPP), has been achieved by incubating Mg²⁺, β,γ-methylene ATP, and ribose 5-phosphate with pure Salmonella typhimurium PRPP synthetase (EC 2.7.6.1). The PRPCP was purified from the reaction mixture by ion-exchange chromatography, and was isolated as the ammonium salt. It was characterized by phosphate and ribose contents, and by ³¹P NMR spectroscopy. A study of the rates of hydrolysis of PRPP and PRPCP at 37°C shows that the methylene analog is more stable to chemical hydrolysis at pH's 4, 7, and 10. The products of base hydrolysis of PRPCP are methylenebisphophonate and ribose 5-phosphate. PRPCP serves as a good alternate substrate for mammalian orotate phosphoribosyltransferase (EC 2.4.2.10), but is a very poor substrate for this enzyme derived from yeast. PRPCP should be a useful analog in kinetic studies of phosphoribosyl transferases because its chemical decomposition product, methylene bisphosphonate, is identical to the nonnucleotide product produced by these enzymes.     

Molecular determinants of desensitization and assembly of the chimeric GABAA receptor subunits (α1/γ2) and (γ2/α1) in combinations with β2 and γ2

Two γ-aminobutyric acid_A (GABA_A) receptor chimeras were designed in order to elucidate the structural requirements for GABA_A receptor desensitization and assembly. The (α1/γ2) and (γ2/α1) chimeric subunits representing the extracellular N-terminal domain of α1 or γ2 and the remainder of the γ2 or α1 subunits, respectively, were expressed with β2 and β2γ2 in Spodoptera frugiperda (Sf-9) cells using the baculovirus expression system. The (α1/γ2)β2 and (α1/γ2)β2γ2 but not the (γ2/α1)β2 and (γ2/α1)β2γ2 subunit combinations formed functional receptor complexes as shown by whole-cell patch–clamp recordings and [³H]muscimol and [³H]flunitrazepam binding. Moreover, the surface immunofluorescence staining of Sf-9 cells expressing the (α1/γ2)-containing receptors was pronounced, as opposed to the staining of the (γ2/α1)-containing receptors, which was only slightly higher than background. To explain this, the (α1/γ2) and (γ2/α1) chimeras may act like α1 and γ2 subunits, respectively, indicating that the extracellular N-terminal segment is important for assembly. However, the (α1/γ2) chimeric subunit had characteristics different from the α1 subunit, since the (α1/γ2) chimera gave rise to no desensitization after GABA stimulation in whole-cell patch–clamp recordings, which was independent of whether the chimera was expressed in combination with β2 or β2γ2. Surprisingly, the (α1/γ2)(γ2/α1)β2 subunit combination did desensitize, indicating that the C-terminal segment of the α1 subunit may be important for desensitization. Moreover, desensitization was observed for the (α1/γ2)β2γ2 receptor with respect to the direct activation by pentobarbital. This suggests differences in the mechanism of channel activation for pentobarbital and GABA.