Hetero-oligomeric Proteins Research Articles

Display of a functional hetero-oligomeric catalytic antibody on the yeast cell surface.

A functional hetero-oligomeric protein was, for the first time, displayed on the yeast cell surface. A hetero-oligomeric Fab fragment of the catalytic antibody 6D9 can hydrolyze a non-bioactive chloramphenicol monoester derivative to produce chloramphenicol. The gene encoding the light chain of the Fab fragment of 6D9 was expressed with the tandemly-linked C-terminal half of alpha-agglutinin. At the same time, the gene encoding the Fd fragment of the heavy chain of the Fab fragment was expressed as a secretion protein. The combined Fab fragment displayed and associated on the yeast cell surface had an intermolecular disulfide linkage between the light and heavy chains. This protein fragment catalyzed the hydrolysis of a chloramphenicol monoester derivative and exhibited high stability in binding with a transition-state analog (TSA). The catalytic reaction was also inhibited by the TSA. The successful display of a functional hetero-oligomeric catalytic antibody provides a useful model for the display of hetero-oligomeric proteins and enzymes.

Activation of Human Meprin-α in a Cell Culture Model of Colorectal Cancer Is Triggered by the Plasminogen-activating System

The activation of latent proenzymes is an important mechanism for the regulation of localized proteolytic activity. Human meprin-alpha, an astacin-like zinc metalloprotease expressed in normal colon epithelial cells, is secreted as a zymogen into the intestinal lumen. Here, meprin is activated after propeptide cleavage by trypsin. In contrast, colorectal cancer cells secrete meprin-alpha in a non-polarized way, leading to accumulation and increased activity of meprin-alpha in the tumor stroma. We have analyzed the activation mechanism of promeprin-alpha in colorectal cancer using a co-culture model of the intestinal mucosa composed of colorectal adenocarcinoma cells (Caco-2) cultivated on filter supports and intestinal fibroblasts grown in the companion dish. We provide evidence that meprin-alpha is activated by plasmin and show that the presence of plasminogen in the basolateral compartment of the co-cultures is sufficient for promeprin-alpha activation. Analysis of the plasminogen-activating system in the co-cultures revealed that plasminogen activators produced and secreted by fibroblasts converted plasminogen to active plasmin, which in turn generated active meprin-alpha. This activation mechanism offers an explanation for the observed meprin-alpha activity in the tumor stroma, a prerequisite for a potential role of this protease in colorectal cancer.

Localisation of GABA A receptor ϵ-subunit in cholinergic and aminergic neurones and evidence for co-distribution with the θ-subunit in rat brain

In situ hybridisation and immunohistochemical methodologies suggest the existence of a large diversity of GABA A receptor subtypes in the brain. These are hetero-oligomeric proteins modulated by a number of clinically important drugs, depending on their subunit composition. We recently cloned and localised the rat GABA A receptor ϵ-subunit by in situ hybridisation and immunohistochemical procedures. Here, in a dual-labelling immunohistochemical study in the rat brain, we used our affinity-purified antiserum to ϵ with antisera to markers of cholinergic, catecholaminergic, and serotonergic neurones. As far as cholinergic systems were concerned, ϵ-immunoreactivity was expressed in all forebrain cell-groups, as well as in the caudal lateral pontine tegmentum and dorsal motor nucleus of the vagus nerve. As far as dopaminergic systems were concerned, ϵ-immunoreactivity was found to be expressed in a great number of hypothalamic cell-groups (A15, A14 and A12) and in the substantia nigra pars compacta. The noradrenergic, and to a lesser extent, adrenergic cell-groups were all ϵ-immunoreactive. Also, ϵ-immunoreactivity was detected in all serotonergic cell-groups. We also revealed by in situ hybridisation in a monkey brain that ϵ mRNA was expressed in the locus coeruleus, as previously observed in rats. Finally, by using in situ hybridisation in rat brains, we compared the distribution of the mRNA of ϵ with that of the recently cloned θ-subunit of the GABA A receptor. Both subunits showed strikingly overlapping expression patterns throughout the brain, especially in the septum, preoptic areas, various hypothalamic nuclei, amygdala, and thalamus, as well as the aforementioned monoaminergic cell-groups. No θ-mRNA signals were detected in cholinergic cell-groups. Taken together with previously published evidence of the presence of the α3-subunit in monoamine- or acetylcholine-containing systems, our data suggest the existence of novel GABA A receptors comprising α3/ϵ in cholinergic and α3/θ/ϵ in monoaminergic cell-groups.

Alternate Use of Distinct Intersubunit Contacts Controls GABAAReceptor Assembly and Stoichiometry

GABA(A) receptors are the major inhibitory transmitter receptors in the CNS. Recombinant GABA(A) receptors composed of alpha(1)beta(3)gamma(2) subunits have been demonstrated to assemble as pentamers consisting of two alpha(1), two beta(3), and one gamma(2) subunit. Using truncated and chimeric alpha(1) subunits, we identified the alpha(1)(80-100) sequence as a major binding site for gamma(2) subunits. In addition, we demonstrated its direct interaction with gamma(2)(91-104), a sequence that previously has been identified to form the contact to alpha(1) subunits. The observation that the amino acid residues alpha(1)P96 and alpha(1)H101, which can be photolabeled by [(3)H]flunitrazepam, are located within or adjacent to the alpha(1)(80-100) sequence, indicates that the benzodiazepine binding site of GABA(A) receptors is located close to this intersubunit contact. The observation that alpha(1)(80-100) interacts with gamma(2) but not with beta(3) subunits indicates the existence of an additional beta(3) binding site on alpha(1) subunits. The preferred alternate use of the gamma(2) and beta(3) binding sites in two different alpha(1) subunits of the same receptor ensures the incorporation of only a single gamma(2) subunit and thus, determines subunit stoichiometry of alpha(1)beta(3)gamma(2) receptors. Distinct binding sites and their alternate use can therefore explain how subunits of hetero-oligomeric transmembrane proteins assemble into a defined protein complex.

Nucleoside triphosphate-dependent restriction enzymes.

The known nucleoside triphosphate-dependent restriction enzymes are hetero-oligomeric proteins that behave as molecular machines in response to their target sequences. They translocate DNA in a process dependent on the hydrolysis of a nucleoside triphosphate. For the ATP-dependent type I and type III restriction and modification systems, the collision of translocating complexes triggers hydrolysis of phosphodiester bonds in unmodified DNA to generate double-strand breaks. Type I endonucleases break the DNA at unspecified sequences remote from the target sequence, type III endonucleases at a fixed position close to the target sequence. Type I and type III restriction and modification (R-M) systems are notable for effective post-translational control of their endonuclease activity. For some type I enzymes, this control is mediated by proteolytic degradation of that subunit of the complex which is essential for DNA translocation and breakage. This control, lacking in the well-studied type II R-M systems, provides extraordinarily effective protection of resident DNA should it acquire unmodified target sequences. The only well-documented GTP-dependent restriction enzyme, McrBC, requires methylated target sequences for the initiation of phosphodiester bond cleavage.

Separate Paths to the Surface

AMPA glutamate receptors (GluR) are hetero-oligomeric proteins and multiple combinations of subunits are expressed in the same neuron. The different combinations have been implicated in different types of neuronal activity, for example, receptors formed from GluR1/GluR2 subunits increase in response to neuronal activity and are important for mediating long-term potentiation, a process implicated in learning and memory. Receptors formed from GluR2/GluR3 subunits are involved in controlling basal neuronal excitability. Shi et al. used cultured neurons and organotypic slice preparations to express various AMPA GluR subunits and modified subunits or portions thereof. The delivery of the proteins was monitored by fusion with green fluorescent protein, the inclusion of a point mutation that altered their electrophysiological properties, or both. Based on the results from expression of the various combinations of proteins, two independent pathways for delivery of the receptors to the cell surface were identified. The GluR1-containing receptors, including GluR1/GluR2 receptors, were not inserted in the plasma membrane unless the synapses were stimulated or unless activated calcium-calmodulin-dependent protein kinase II was also expressed. GluR2 homo-oligomers or GluR2/GluR3 hetero-oligomers were constitutively inserted into the membrane, and the endogenous receptors were continuously endocytosed. Trafficking of GluR-containing receptors depended on the binding domain for PDZ group 1 proteins, and trafficking of the GluR2/GluR2 or GluR2/GluR3 receptors depended on the GluR2 PDZ group II binding domain and interaction with N-ethylmaleimide-sensitive factor (NSF). The independence of the trafficking pathways was confirmed by expression of the COOH-domains of GluR1 or GluR2, which showed only selective inhibition of the cognate receptor's delivery. These two independently regulated pathways contribute to the control of synaptic activity.S.-H. Shi, Y. Hayashi, J. A. Esteban, R. Malinow, Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105, 331-343 (2001). [Online Journal]

Separate Paths to the Surface

AMPA glutamate receptors (GluR) are hetero-oligomeric proteins and multiple combinations of subunits are expressed in the same neuron. The different combinations have been implicated in different types of neuronal activity, for example, receptors formed from GluR1/GluR2 subunits increase in response to neuronal activity and are important for mediating long-term potentiation, a process implicated in learning and memory. Receptors formed from GluR2/GluR3 subunits are involved in controlling basal neuronal excitability. Shi et al. used cultured neurons and organotypic slice preparations to express various AMPA GluR subunits and modified subunits or portions thereof. The delivery of the proteins was monitored by fusion with green fluorescent protein, the inclusion of a point mutation that altered their electrophysiological properties, or both. Based on the results from expression of the various combinations of proteins, two independent pathways for delivery of the receptors to the cell surface were identified. The GluR1-containing receptors, including GluR1/GluR2 receptors, were not inserted in the plasma membrane unless the synapses were stimulated or unless activated calcium-calmodulin-dependent protein kinase II was also expressed. GluR2 homo-oligomers or GluR2/GluR3 hetero-oligomers were constitutively inserted into the membrane, and the endogenous receptors were continuously endocytosed. Trafficking of GluR-containing receptors depended on the binding domain for PDZ group 1 proteins, and trafficking of the GluR2/GluR2 or GluR2/GluR3 receptors depended on the GluR2 PDZ group II binding domain and interaction with N -ethylmaleimide-sensitive factor (NSF). The independence of the trafficking pathways was confirmed by expression of the COOH-domains of GluR1 or GluR2, which showed only selective inhibition of the cognate receptor's delivery. These two independently regulated pathways contribute to the control of synaptic activity. S.-H. Shi, Y. Hayashi, J. A. Esteban, R. Malinow, Subunit-specific rules governing AMPA receptor trafficking to synapses in hippocampal pyramidal neurons. Cell 105 , 331-343 (2001). [Online Journal]

Molecular determinants of glycine receptor subunit assembly.

The inhibitory glycine receptor (GlyR) is a pentameric transmembrane protein composed of homologous alpha and beta subunits. Single expression of alpha subunits generates functional homo-oligomeric GlyRs, whereas the beta subunit requires a co-expressed alpha subunit to assemble into hetero-oligomeric channels of invariant stoichiometry (alpha(3)beta(2)). Here, we identified eight amino acid residues within the N-terminal region of the alpha1 subunit that are required for the formation of homo-oligomeric GlyR channels. We show that oligomerization and N-glycosylation of the alpha1 subunit are required for transit from the endoplasmic reticulum to the Golgi apparatus and later compartments, and that addition of simple carbohydrate side chains occurs prior to GlyR subunit assembly. Our data are consistent with both intersubunit surface and conformational differences determining the different assembly behaviour of GlyR alpha and beta subunits.

CD40-tumor necrosis factor receptor-associated factor (TRAF) interactions: regulation of CD40 signaling through multiple TRAF binding sites and TRAF hetero-oligomerization.

CD40 is a TNF receptor superfamily member that provides activation signals in antigen-presenting cells such as B cells, macrophages, and dendritic cells. Multimerization of CD40 by its ligand initiates signaling by recruiting TNF receptor-associated factors (TRAFs) to the CD40 cytoplasmic domain. Recombinant human TRAF proteins overexpressed in insect cells were biochemically characterized and used to finely map TRAF binding regions in the human CD40 cytoplasmic domain. TRAF1, TRAF2, TRAF3, and TRAF6, but not TRAF4 or TRAF5, bound directly to the CD40 cytoplasmic domain. CD40 interactions with TRAF2 and TRAF3 were stronger than the interactions with TRAF1 and TRAF6. Full-length TRAF3 and TRAF5 formed hetero-oligomers, presumably through their predicted isoleucine zippers. TRAF3-TRAF5 hetero-oligomers interacted with CD40, indicating that TRAF5 can be indirectly recruited to the CD40 cytoplasmic domain. Overlapping peptides synthesized on cellulose membranes were used to map each TRAF interaction region. TRAF1, TRAF2, and TRAF3 interacted with the same region. The recognition site for TRAF6 was a nonoverlapping membrane proximal region. Using peptides with progressive deletions, a minimal TRAF1, TRAF2, and TRAF3 binding region was mapped to the PVQET sequence in the CD40 cytoplasmic domain. The minimal region for TRAF6 binding was the sequence QEPQEINF. These studies demonstrate that the CD40 cytoplasmic domain contains two nonoverlapping TRAF binding regions and suggest that TRAF1, TRAF2, and TRAF3 could bind competitively to one site. Relative affinities and competition of individual and hetero-oligomeric TRAF proteins for CD40 binding sites may contribute to receptor specificity and cell-type selectivity in CD40-dependent signaling.

Relative Functions of the α and β Subunits of the Proteasome Activator, PA28

PA28 is a 180,000-dalton protein that activates hydrolysis of small nonubiquitinated peptides by the 20 S proteasome. PA28 is composed of two homologous subunits, alpha and beta, arranged in alternating positions in a ring-shaped oligomer with a likely stoichiometry of (alphabeta)3. Our previous work demonstrated that the carboxyl terminus of the alpha subunit was necessary for PA28 to bind to and activate the proteasome. The goals of this work were to define the exact structural basis for this effect and to determine the relative roles of the alpha and beta subunits in proteasome activation. Each subunit and various mutants of the alpha subunit were expressed in Escherichia coli and purified. PA28alpha stimulated the proteasome, but had a much greater Kact than native heteromeric PA28. In contrast, PA28beta was unable to stimulate the proteasome. Mutants of the alpha subunit in which the carboxyl-terminal tyrosine residue was deleted or substituted with charged amino acids could neither bind to nor activate the proteasome. However, substitution of the carboxyl-terminal tyrosine with other amino acids resulted in proteins which could stimulate the proteasome to various extents. Tryptophan mutants stimulated the proteasome as well as did native PA28, whereas serine or phenylalanine mutants stimulated the proteasome much poorer than did wild type PA28alpha. Deletion of the "KEKE" motif, a 28-amino acid domain near the amino terminus of PA28alpha, had no effect on proteasome stimulatory activity. Hetero-oligomeric PA28 proteins were reconstituted from isolated wild type and mutant subunits. PA28 reconstituted from wild type subunits had structural and functional properties that were indistinguishable from those of the native hetero-oligomeric protein. PA28 molecules reconstituted from inactive alpha subunits and wild type beta subunits remained inactive. However, PA28 molecules reconstituted from suboptimally active alpha mutants and wild type beta subunits had the same activity as native heteromeric PA28. These results indicate that the beta subunit modulates PA28 activity, perhaps by influencing the affinity of PA28 for the proteasome.

MalFGK complex assembly and transport and regulatory characteristics of MalK insertion mutants.

MalK is a peripheral cytoplasmic membrane protein that has multiple activities in Escherichia coli. It associates with integral cytoplasmic membrane proteins MalF and MalG to form the maltose transport complex (MalFGK), a member of the ATP-binding cassette (ABC) superfamily of proteins. In addition, MalK participates in two different regulatory pathways which modulate mal gene expression and MalFGK transport activity. We have created a set of malK mutations for analysis of the protein's structure and folding. These mutations, distributed throughout malK, are all similar insertions of 31 codons. The ability of each mutant to function in maltose transport and MalK-dependent regulation was characterized. Furthermore, we have exploited a sensitive biochemical assay to classify our MalK insertion mutants into two additional categories: MalFGK complex assembly proficient and complex assembly defective. The regions containing the insertions in the assembly-proficient class should correspond to areas within MalK that are surface exposed within the MalFGK complex. Affected regions in assembly-deficient mutants may be involved in critical structural contacts within the complex. One mutant apparently blocks assembly at an intermediate stage prior to oligomerization of the final MalFGK complex. This work contributes to the analysis of ABC transport proteins and to the study of the assembly process for hetero-oligomeric membrane proteins.

Subunit Stoichiometry of Oligomeric Membrane Proteins: GABAA Receptors Isolated by Selective Immunoprecipitation from the Cell Surface

GABAA receptors are hetero-oligomeric proteins of unknown subunit stoichiometry. In this study α1β3 GABAA receptor channels were functionally expressed in Xenopus oocytes. Direct immunoprecipitation from the oocyte surface was used to exclusively isolate mature GABAA receptors. The subunit ratio was determined by quantitation of the amount of [35S]methionine incorporated into individual receptor subunits. Antibody released from the antigen or antibody not reacted was prevented from reassociation with labeled antigen by addition of excess unlabeled antigen. Variation of the α1β3 ratio of injected cRNAs only slightly affected the subunit ratio in mature receptors. This indicates that the subunit stoichiometry generated is independent of the pools of newly synthesized subunit monomers and supports the view that the receptor assembly is a regulated process. The ratio of α1β3 subunits was found to be 1.1 ± 0.1 (SEM, n = 6). Our data are in best agreement with a tetrameric receptor with the composition 2α2β. For a pentameric receptor the ratio found slightly favors a receptor with the composition 3α2β. The method developed here is applicable to the determination of the subunit stoichiometry of other recombinant oligomeric membrane proteins. Copyright © 1996 Elsevier Science Ltd

Co-regulation of two gene activities by tetracycline via a bidirectional promoter.

Recently, we have described a regulatory system that allows the stringent control of individual gene activities in higher eukaryotic cell lines (1), in plants (2) and in animals (3,4). The essential components of this system are (i) an RNA polymerase II minimal promoter placed downstream of multiple operator sequences (tetO) of the Escherichia coli TnlO tetracycline resistance operon and (ii) a fusion between the Tet repressor (TetR) and the herpes simplex virus protein 16 (VP16), named tTA (1). In the absence of tetracycline (Tc), tTA binds to the tet operators to activate transcription from the minimal promoter, whereas in the presence of Tc its association and consequently its transcription activation is prevented. After the binding of tTA, minimal promoters derived from the cytomegalovirus IE promoter (PhCMv; 5) and fused to seven tetO sequences reach the remarkable strength of the parent promoter in HeLa cells when compared in transient expression assays (6). This high activation potential of tTA and the arrangement of its binding sites within PhCMV*-i [(!)'»see Fig. 1A] suggested the design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences (Fig. 1A). Such a promoter should be useful for a number of experimental approaches. First, it may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts, frequently a prerequisite for the production of heterodimeric (or hetero-oligomeric) proteins. Second, by fusing minimal promoters of differing efficiencies to the centrally located tetO sequences, two gene products may be co-regulated at different but defined levels. Third, by integrating an appropriate reporter gene at one side of the bidirectional promoter, the regulation of a not-readily-assayable gene of interest may be monitored via the reporter function. This latter possibility may also facilitate—at the cellular as well as at the organismal level—the screening for properly integrated expression units controlling the gene of interest.

Use of a purified heterodimer to test negative cooperativity as the basis of substrate inactivation of Escherichia coli thymidylate synthase (Asn177-->Asp).

Thymidylate synthase (TS) converts deoxyuridylate to thymidylate, an essential DNA precursor. Replacement of Asn177 with aspartate (Asn177-->Asp) in Escherichia coli TS creates a novel ability to methylate 2'-deoxycytidylate (dCMP). The dCMP-methylase activity of TS(Asn177-->Asp) is transiently inactivated by reaction with deoxyuridylate and methylene-tetrahydrofolate, the methyl donor. We have tested the possibility that the inactivation is due to negative cooperativity, created in the TS dimer by the Asn177-->Asp mutation. A heterodimeric form of TS, containing one wild type and one Asn177-->Asp active site, was created to test for negative cooperativity. Substrate inactivation still occurred, even with the mutation present at only one active site. Inactivation of TS(Asn177-->Asp) by deoxyuridylate is not due to negative cooperativity created by the mutation. The 'artificial isozyme' method we have developed for purifying heterodimers away from the progenitor homodimers is generally applicable to other hetero-oligomeric proteins.

Protein Kinase C-Mediated Regulation of L-Type Ca Channels from Skeletal Muscle Requires Phosphorylation of the α1 Subunit

Dihydropyridine-sensitive, L-type Ca channels are hetero-oligomeric proteins that are modulated in certain cell types by protein kinase C (PKC). In native skeletal muscle membranes, PKC phosphorylates the α1 and β subunits of these Ca channels and modulates channel activity. However, it is unknown if phosphorylation of both subunits is necessary for PKC-mediated channel regulation. Here we report that stoichiometric phosphorylation of the α1 subunit was required for activation of these Ca channels by PKC, while PKC-mediated phosphorylation of the β subunit alone did not modify channel activity. Furthermore, reversal of the functional effects of PKC by protein phosphatase-1c was quantitatively correlated with dephosphorylation of the α1 subunit.

Dissection of a class II tRNA synthetase: determinants for minihelix recognition are tightly associated with domain for amino acid activation.

The ten class II aminoacyl-tRNA synthetases are large homo- and hetero-oligomeric proteins that share three conserved sequence motifs. Within this class, Escherichia coli alanyl-tRNA synthetase is the only homotetramer and is comprised of subunits of 875 amino acids. The enzyme aminoacylates sequence-specific RNA oligonucleotides that recreate as few as four base pairs of the acceptor stem of tRNA(Ala). A monomeric 461 amino acid N-terminal fragment (461N) was previously shown to have full adenylate synthesis activity. However, fragment 461N has significant, but reduced, efficiency of charging of tRNA(Ala), when compared to native enzyme [Ho, C., Jasin, M., & Schimmel, P. (1985) Science 229, 389-393]. We show here that, in contrast, the fragment and the native enzyme aminoacylate a 12 base pair acceptor-T psi C stem minihelix and a four base pair RNA tetraloop with the same efficiency. We also show that fragment 461N makes footprint contacts both on and outside the acceptor helix of bound tRNA(Ala). With one possible exception, the contacts observed with fragment 461N are identical to those seen with the native enzyme. In spite of contacts outside the acceptor helix, fragment 461N charges the native tRNA, minihelix, and tetraloop with similar efficiency. Thus, all minihelix contacts required for activation for charging are tightly associated with the adenylate synthesis domain and, at least in the fragment, are not influenced by additional RNA-protein contacts outside the minihelix domain.(ABSTRACT TRUNCATED AT 250 WORDS)

The Xenopus Oocyte as an Ectopic Expression System for the Selection of Protein Isoform-Specific Antibodies

A panel of Xenopus oocytes, each injected with cRNA coding for one specific isoform of the rat brain RCK family of voltage gated potassium channel proteins, was employed to screen for isoform-specific monoclonal antibodies. Several days after injection, cryosections of embedded oocytes were produced and were employed in immunohistochemical analysis of antibody binding. Of the advantageous properties of the assay, it employs the native antigen, it can be applied to homooligomeric and heterooligomeric proteins, and cryosections of the same batch can be stored frozen for later tests. The method may be advantageous also for the selection of isoform-specific antibodies of other protein families.

Molecular mechanisms of the partial allosteric modulatory effects of bretazenil at gamma-aminobutyric acid type A receptor.

In central nervous system gamma-aminobutyric acid (GABA) inhibits neuronal activity by acting on GABA type A (GABAA) receptors. These heterooligomeric integral membrane proteins include a GABA-gated Cl- channel and various allosteric modulatory sites where endogenous modulators and anxiolytic drugs act to regulate GABA action. In vivo, various anxiolytic drugs exhibit a wide range of variability in their modulatory efficacy and potency of GABA action. For instance, bretazenil modulatory efficacy is much lower than that of diazepam. Such low efficacy could be due either to a preferential modulation of specific GABAA receptor subtypes or to a low modulatory efficacy at every GABAA receptor subtype. To address these questions we studied drug-induced modifications of GABA-activated Cl- currents in native GABAA receptors of cortical neurons in primary cultures and in recombinant GABAA receptors transiently expressed in transformed human embryonic kidney cells (293) after transfection with cDNAs encoding different molecular forms of alpha, beta, and gamma subunits of GABAA receptors. In cortical neurons the efficacy of bretazenil was lower than that of diazepam, whereas the potency of the two drugs was similar. In cells transfected with gamma 2 subunits and various molecular forms of alpha and beta subunits bretazenil efficacy was always lower than that of diazepam. However, in cells transfected with gamma 1 or gamma 3 subunits and various forms of alpha and beta subunits the efficacy of both diazepam and bretazenil was lower and always of similar magnitude. When bretazenil and diazepam were applied together to GABAA receptors including a gamma 2 subunit, the action of diazepam was curtailed in a manner related to the dose of bretazenil.

Dihydropyridine-sensitive calcium channels from skeletal muscle. I. Roles of subunits in channel activity.

Dihydropyridine-sensitive Ca2+ channels from skeletal muscle are hetero-oligomeric proteins. Little is known about the functional roles of the various subunits, except that the alpha 1 subunit is the essential channel unit. We have reconstituted both partially purified holomeric channels and the separated subunits into liposomes and measured their properties using an assay based on the Ca2+ indicator dye fluo-3. The holomeric channels exhibited Ca2+ influx that was sensitive to membrane potential achieved by the addition of valinomycin in the presence of a K+ gradient. Dissipation of the K+ gradient resulted in the loss of the valinomycin-sensitive Ca2+ flux. In addition, the reconstituted channels were: 1) activated by the dihydropyridine Ca2+ channel activator Bay K 8644 in a dose-dependent manner with a Kd of 20 nM; 2) inhibited by various types of Ca2+ channel inhibitors including the dihydropyridine (+)-PN 200-110, the phenylalkylamine verapamil, and the benzothiazepine d-cis-diltiazem; and 3) modulated in a stereoselective manner by the enantiomers of the dihydropyridine S-202-791. The purified channels used in this work possessed an alpha 1 subunit of 165 kDa and did not appear to contain a larger alpha 1 subunit of approximately 210 kDa, suggesting that channel activity with properties similar to those observed in intact cells can be supported with an alpha 1 subunit of 165 kDa. Reconstituted channels that were 85% depleted in the alpha 2/delta subunits showed a significant decrease in the initial rate of Ca2+ influx induced by valinomycin, but retained responsiveness to Bay K 8644 and (+)-PN 200-110. When the separated alpha 2 and delta subunits were added back to the alpha 1 subunit-containing preparation, the channels exhibited their normal rate of Ca2+ influx. These results demonstrated that the dihydropyridine-sensitive Ca2+ channels from skeletal muscle require the presence of the alpha 2.gamma complex in stoichiometric amounts to exhibit full activity.