Functional and idling rotatory motion within F1-ATPase

ATP synthase mediates proton flow through its membrane portion, F0, which drives the synthesis of ATP in its headpiece, F1. The F1-portion contains a hexagonal array of three subunits alpha and three beta encircling a central subunit gamma, that in turn interacts with a smaller epsilon and with F0. Recently we reported that the application of polarized absorption recovery after photobleaching showed the ATP-driven rotation of gamma over at least two, if not three, beta. Here we extend probes of such rotation aided by a new theory for assessing continuous versus stepped, Brownian versus unidirectional molecular motion. The observed relaxation of the absorption anisotropy is fully compatible with a unidirectional and stepping rotation of gamma over three equidistantly spaced angular positions in the hexagon formed by the alternating subunits alpha and beta. The results strongly support a rotational catalysis with equal participation of all three catalytic sites. In addition we report a limited rotation of gamma without added nucleotides, perhaps idling and of Brownian nature, that covers only a narrow angular domain.     

Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum

The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.     

G protein beta gamma subunits. Simplified purification and properties of novel isoforms

The beta and gamma subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) form tightly associated complexes. To examine functional differences among the large number of possible combinations of unique beta and gamma subunits, we have synthesized and characterized beta gamma complexes containing gamma 5 and gamma 7, two widely distributed gamma subunits. When either gamma 5 or gamma 7 is expressed concurrently with beta 1 or beta 2 subunits in a baculovirus/Sf9 cell system, all four subunit complexes support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 (where "r" indicates recombinant), indicating formation of functional complexes. Each of the complexes was purified by subunit exchange chromatography, using the G203A mutant of rGi alpha 1 as the immobilized ligand. The purified preparations were compared with other recombinant beta gamma subunits, including beta 1 gamma 1 and beta 1 gamma 2, for their ability to modulate type I and II adenylyl cyclase activities; stimulate phosphoinositide-specific phospholipase C beta; support pertussis toxin-catalyzed ADP-ribosylation of rGi alpha 1 and Go alpha; and inhibit steady-state GTP hydrolysis catalyzed by Gs alpha, Go alpha, and myristoylated rGi alpha 2. The results emphasize the unique properties of beta 1 gamma 1. The properties of the complexes containing gamma 5 or gamma 7 were similar to each other and to those of beta 1 gamma 2.     

Asymmetry of the alpha subunit of the chloroplast ATP synthase as probed by the binding of Lucifer Yellow vinyl sulfone

The catalytic portion of the chloroplast ATP synthase (CF1) is structurally asymmetric. Asymmetry of the otherwise symmetrical alpha3beta3 heterohexamer is induced by the presence of tightly bound nucleotides and interactions with the single-copy, smaller subunits. Lucifer Yellow vinyl sulfone (4-amino-N-[3-(vinylsulfonyl)phenyl]naphthalimide-3,6-disulfonic acid) rapidly and covalently binds to lysine 378 on one alpha subunit [Nalin, C. M., Snyder, B., and McCarty, R. E., (1985) Biochemistry 24, 2318-2324] [Shapiro, A. B. (1991) Ph.D. Thesis, Cornell University, Ithaca, NY). The asymmetrical binding of Lucifer Yellow to CF1 provides a method to investigate the cause of asymmetry in the alpha subunits. The reaction of CF1 with Lucifer Yellow was monitored by total fluorescence of bound Lucifer Yellow as well as by quantitative determination of Lucifer Yellow bound to the tryptic peptide that contains lysine 378 of the alpha subunit. The total binding of Lucifer Yellow to CF1 was not affected by the presence of tightly bound nucleotides or nucleotide in the medium. Neither the total binding of Lucifer Yellow to CF1 nor the reaction of alpha-lysine 378 with Lucifer Yellow was changed by the removal of the epsilon subunit, the delta subunit, or both subunits. The extent of incorporation of Lucifer Yellow into lysine 378 of the alpha subunit in (alphabeta)n was about three times that of Lucifer Yellow incorporation into CF1. Reconstitution of (alphabeta)n with gamma restored the binding of one Lucifer Yellow per alpha3beta3gamma. Therefore, the interactions between gamma and the alphabeta heterohexamer are important in conferring asymmetry to the alpha subunits of CF1.     

Changes of mitochondrial cytochrome c oxidase and FoF1 ATP synthase subunits in rat cerebral cortex during aging

The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.     

A subunit interaction in chloroplast ATP synthase determined by genetic complementation between chloroplast and bacterial ATP synthase genes

F1F0-ATP synthases utilize protein conformational changes induced by a transmembrane proton gradient to synthesize ATP. The allosteric cooperativity of these multisubunit enzymes presumably requires numerous protein-protein interactions within the enzyme complex. To correlate known in vitro changes in subunit structure with in vivo allosteric interactions, we introduced the beta subunit of spinach chloroplast coupling factor 1 ATP into a bacterial F1 ATP synthase. A cloned atpB gene, encoding the complete chloroplast beta subunit, complemented a chromosomal deletion of the cognate uncD gene in Escherichia coli and was incorporated into a functional hybrid F1 ATP synthase. The cysteine residue at position 63 in chloroplast beta is known to be located at the interface between alpha and beta subunits and to be conformationally coupled, in vitro, to the nucleotide binding site > 40 A away. Enlarging the side chain of chloroplast coupling factor 1 beta residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity or ADP binding in vitro. The in vivo coupling of nucleotide binding at catalytic sites to transmembrane proton movement may thus involve an interaction, via conformational changes, between the amino-terminal domains of the alpha and beta subunits.     

The stalk region of the Escherichia coli ATP synthase. Tyrosine 205 of the gamma subunit is in the interface between the F1 and F0 parts and can interact with both the epsilon and c oligomer

The soluble portion of the Escherichia coli F1F0 ATP synthase (ECF1) and E. coli F1F0 ATP synthase (ECF1F0) have been isolated from a novel mutant gammaY205C. ECF1 isolated from this mutant had an ATPase activity 3.5-fold higher than that of wild-type enzyme and could be activated further by maleimide modification of the introduced cysteine. This effect was not seen in ECF1F0. The mutation partly disrupts the F1 to F0 interaction, as indicated by a reduced efficiency of proton pumping. ECF1 containing the mutation gammaY205C was bound to the membrane-bound portion of the E. coli F1F0 ATP synthase (ECF0) isolated from mutants cA39C, cQ42C, cP43C, and cD44C to reconstitute hybrid enzymes. Cu2+ treatment or reaction with 5,5'-dithio-bis(2-nitro-benzoic acid) induced disulfide bond formation between the Cys at gamma position 205 and a Cys residue at positions 42, 43, or 44 in the c subunit but not at position 39. Using Cu2+ treatment, this covalent cross-linking was obtained in yields as high as 95% in the hybrid ECF1 gammaY205C/cQ42C and in ECF1F0 isolated from the double mutant of the same composition. The covalent linkage of the gamma to a c subunit had little effect on ATPase activity. However, ATP hydrolysis-linked proton translocation was lost, by modification of both gamma Cys-205 and c Cys-42 by bulky reagents such as 5,5'-dithio-bis (2-nitro-benzoic acid) or benzophenone-4-maleimide. In both ECF1 and ECF1F0 containing a Cys at gamma 205 and a Cys in the epsilon subunit (at position 38 or 43), cross-linking of the gamma to the epsilon subunit was induced in high yield by Cu2+. No cross-linking was observed in hybrid enzymes in which the Cys was at position 10, 65, or 108 of the epsilon subunit. Cross-linking of gamma to epsilon had only a minimal effect on ATP hydrolysis. The reactivity of the Cys at gamma 205 showed a nucleotide dependence of reactivity to maleimides in both ECF1 and ECF1F0, which was lost in ECF1 when the epsilon subunit was removed. Our results show that there is close interaction of the gamma and epsilon subunits for the full-length of the stalk region in ECF1F0. We argue that this interaction controls the coupling between nucleotide binding sites and the proton channel in ECF1F0.     

AutoCyte Interactive Screening System. Experience at a university hospital cytology laboratory

The F1-ATPase is a multimeric enzyme (alpha3 beta3 gamma delta epsilon) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding alpha-, beta-, gamma- or delta-subunits were unable to grow, while the strain with a null mutation in epsilon was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in gamma or delta became 100% rho0/rho- and the strain with the null mutation in gamma grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: Delta alpha Delta gamma, Delta beta Delta gamma, Deltaatp11 Delta gamma, Delta alpha Delta delta, Delta beta Delta delta or Deltaatp11 Delta delta. These results indicate that epsilon is not an essential component of the ATP synthase and that mutations in the genes encoding the alpha- and beta-subunits and in ATP11 are epistatic to null mutations in the genes encoding the gamma- and delta-subunits. These data suggest that the propensity to form rho0/rho- mutations in the gamma and delta null deletion mutant stains and the slow growing phenotypes of the null gamma mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells.     

Structural features of the human salivary mucin, MUC7

Specific mgi mutations in the alpha, beta or gamma subunits of the mitochondrial F1-ATPase have previously been found to suppress rho0 lethality in the petite-negative yeast Kluyveromyces lactis. To determine whether the suppressive activity of the altered F1 is dependent on the F0 sector of ATP synthase, we isolated and disrupted the genes KlATP4, 5 and 7, the three nuclear genes encoding subunits b, OSCP and d. Strains disrupted for any one, or all three of these genes are respiration deficient and have reduced viability. However a strain devoid of the three nuclear genes is still unable to lose mitochondrial DNA, whereas a mgi mutant with the three genes inactivated remains petite-positive. In the latter case, rho0 mutants can be isolated, upon treatment with ethidium bromide, that lack six major F0 subunits, namely the nucleus-encoded subunits b, OSCP and d, and the mitochondrially encoded Atp6, 8 and 9p. Production of rho0 mutants indicates that an F1-complex carrying a mgi mutation can assemble in the absence of F0 subunits and that suppression of rho0 lethality is an intrinsic property of the altered F1 particle.     

The role of the GABA(A) receptor alpha1 subunit N-terminal extracellular domain in propofol potentiation of chloride current

Propofol (2,6-diisopropylphenol), an intravenous general anesthetic in active clinical use today, potentiates the action of gamma-aminobutyric acid (GABA) at the type-A receptor and also directly induces current in the absence of GABA. We expressed different combinations of murine GABA(A) receptor alpha1, beta3 and gamma2 subunits in Xenopus oocytes to investigate the subunit dependence of propofol potentiation of pentobarbital-induced current. Pentobarbital induces current in all beta3-subunit-containing receptors, whereas current gating by GABA requires the presence of both alpha1 and beta3 subunits. Therefore, pentobarbital rather than GABA was used to induce current in order to separate the subunit dependence of current gating from the subunit dependence of potentiating action of propofol. alpha1beta3gamma2, alpha1beta3, beta3gamma2, or beta3 subunit combinations all responded to pentobarbital in a dose-dependent manner. True potentiation was defined as the current magnitude to simultaneous application of pentobarbital and propofol exceeding the additive responses to individual drug applications. A dose-dependent propofol potentiation of pentobarbital-induced current was observed in oocytes injected with alpha1beta3 or alpha1beta3gamma2 but not in beta3gamma2 or beta3 subunits, suggesting that the alpha1 subunit was necessary for this modulatory action of propofol. Further examination of the propofol potentiation in chimeras between the alpha1 and beta3 subunits showed that the extracellular amino-terminal half of the alpha1 subunit was sufficient to support propofol potentiation. The different requirements of the receptor structure for the agonistic (gating) and the potentiating actions suggest that these two actions of propofol are distinct processes mediated through its action at distinct sites.     

Direct observation of the rotation of epsilon subunit in F1-ATPase

Rotation of the epsilon subunit in F1-ATPase from thermophilic Bacillus strain PS3 (TF1) was observed under a fluorescence microscope by the method used for observation of the gamma subunit rotation (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302). The alpha3 beta3 gamma epsilon complex of TF1 was fixed to a solid surface, and fluorescently labeled actin filament was attached to the epsilon subunit through biotin-streptavidin. In the presence of ATP, the filament attached to epsilon subunit rotated in a unidirection. The direction of the rotation was the same as that observed for the gamma subunit. The rotational velocity was slightly slower than the filament attached to the gamma subunit, probably due to the experimental setup used. Thus, as suggested from biochemical studies (Aggeler, R., Ogilvie, I. , and Capaldi, R. A. (1997) J. Biol. Chem. 272, 19621-19624), the epsilon subunit rotates with the gamma subunit in F1-ATPase during catalysis.     

Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit

Run-down of L-type Ca2+ channels in CHO cells stably expressing alpha 1c, alpha 1c beta 1a, or alpha 1c beta 1a alpha 2 delta gamma subunits was studied using the patch-clamp technique (single channel recording). The channel activity (NPo) of alpha 1c channels was increased 4- and 8-fold by coexpression with beta 1a and beta 1a alpha 2 delta gamma, respectively. When membranes containing channels composed of different subunits were excised into basic internal solution, the channel activity exhibited run-down, the time-course of which was independent of the subunit composition. The run-down was restored by the application of calpastatin (or calpastatin contained in cytoplasmic P-fraction) + H-fraction (a high molecular mass fraction of bovine cardiac cytoplasm) + 3 mM ATP, which has been shown to reverse the run-down in native Ca2+ channels in the guinea-pig heart. The restoration level was 64.7, 63.5, and 66.4% for channels composed of alpha 1c, alpha 1c beta 1a, and alpha 1c beta 1a alpha 2 delta gamma, respectively, and was thus also independent of the subunit composition. We conclude that run-down of L-type Ca2+ channels occurs via the alpha 1 subunit and that the cytoplasmic factors maintaining Ca2+ channel activity act on the alpha 1 subunit.     

Direct interaction of the alpha and gamma subunits of the G proteins. Purification and analysis by limited proteolysis

The heterotrimeric G proteins are often regarded functionally as a heterodimer, consisting of a guanine nucleotide-binding alpha subunit and a beta gamma subunit complex. Since the tightly associated beta gamma subunit complex can be separated only under denaturing conditions, studies aimed at determining the individual contributions of the beta and gamma subunits in terms of binding to the various alpha subunits, interacting with receptors, and regulating effectors, have not been possible. To circumvent this problem, we have used baculovirus-infected cells to direct the individual expression of the beta 1 and gamma 2 subunits. Application of extracts from baculovirus-infected cells to an alpha subunit of G protein (G(o) alpha)-affinity matrix resulted in the selective retention and AMF-specific elution of the expressed gamma 2 subunit, but not the expressed beta 1 subunit. Overall, these and other data provide the first evidence of a direct association between the gamma and alpha subunits, which is dependent on prenylation of gamma. The apparent direct association between the gamma and alpha subunits was further probed by limited trypsin proteolysis. Upon addition of trypsin, the G(o) alpha subunit was rapidly cleaved to a 24-kDa fragment. However, in the presence of the purified gamma 2 subunit, trypsin cleavage of the G(o) alpha subunit was completely prevented. This demonstration of a direct association between the gamma and alpha subunits is particularly intriguing in light of the increasingly large number of known alpha, beta, and gamma subunits, which raises important questions regarding the assembly of these subunits into functionally distinct G proteins. Thus, a direct association between the gamma and alpha subunits, which exhibit the greatest structural diversity, may provide the basis for the selective assembly of these subunits into G proteins with functional diversity.     

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.     

Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

The GABA(A) receptor is a ligand gated chloride channel consisting of five membrane spanning proteins for which 13 different genes have been identified in the mammalian brain. The present review summarizes recent work from our laboratory on the characterization of the immunocytochemical distribution of these GABA(A) receptor subunits in the rat brain and changes in immunoreactivity and mRNA expression after kainic acid-induced status epilepticus. A heterogeneous distribution of immunoreactive GABA(A) receptor subunits was observed. The most abundant ones were: alpha1, alpha2, alpha4, alpha5, beta2, beta3, gamma2, and delta. Alpha1, beta2, and gamma2 were about equally distributed in all subfields of the hippocampus; alpha4- and delta-subunits were preferentially found in the dentate molecular layer and in CA1; alpha2 was localized to the dentate molecular layer and CA3; alpha5 was found in the dendritic areas of CA1 to CA3; and beta1 was preferentially seen in CA2. Alpha1, beta2, gamma2 and delta were highly concentrated in interneurons. Kainic acid-induced seizures caused acute and chronic changes in the expression of mRNAs and immunoreactive proteins. Acute changes included decreases in alpha2, alpha5, beta1, beta3, gamma2 and delta mRNA levels (by about 25-50%), accompanied by increases (by about 50%) in alpha1, alpha4, and beta2 messages in granule cells (after 6-12 h). Chronic changes, characterized by losses in mRNA and immunoreactive proteins in CA1 and CA3, are undoubtedly due to seizure-related cell damage. However, compensatory expression of alpha2 and beta3 subunits, especially in CA3b/c, was observed. Furthermore, increases in mRNAs and immunoreactive proteins were seen for alpha1, alpha2 alpha4, beta1, beta2, beta3 and gamma2 in granule cells and in the molecular layer of the dentate gyrus at 7-30 days after kainic acid injection. The changes in the expression of GABA(A) receptor subunits, observed in practically all hippocampal subfields, may reflect altered GABA-ergic transmission during development of the epileptic syndrome. Increased expression of GABA(A) receptor subunits in the dendritic field of granule cells and CA3 suggest that GABA-ergic inhibition may be augmented at these levels. However, the lasting preservation of alpha1-, beta2-, and gamma2-subunits in interneurons could provide a basis for augmented inhibition of GABA-ergic interneurons, leading to net disinhibition.     

Conformational transmission in ATP synthase during catalysis: search for large structural changes

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at beta Gly-149 was suppressed by the second mutations at the outer surface of the beta subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that beta/alpha and beta/gamma subunits interactions are important for the energy coupling between catalysis and H+ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the gamma subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.     

Purification of four forms of the beta gamma subunit complex of G proteins containing different gamma subunits

To investigate the physiological significance of the diversity of gamma subunits of G proteins, we purified four forms of beta gamma of G proteins from bovine brain (beta gamma-B1, beta gamma-B2, beta gamma-B3), and spleen (beta gamma-S1) by the sequential chromatography on columns of DEAE-Sephacel, Ultrogel AcA 34, heptylamine-Sepharose, phenyl-5PW, and DEAE-5PW. Electrophoretic analyses showed that each beta gamma mainly contained the 36-kDa beta and a distinct but homogeneous gamma. These beta gamma complexes were subjected directly to proteolytic digestion and subsequent amino acid sequence analyses of their fragments. It was revealed that beta gamma-B1, -B2, and -B3 were identical to beta 1 gamma 7 (with a low level of beta 2 gamma 7), beta 1 gamma 2 and beta 1 gamma 3, respectively, while beta gamma-S1 was composed of beta 1 and an unidentified form of gamma. Then we examined the functional differences among these beta gamma complexes and the beta gamma of transducin (beta gamma-T, beta 1 gamma 1). Few differences were observed among all beta gamma complexes to enhance pertussis toxin-catalyzed ADP-ribosylation of the alpha subunits of G(o) and Gt. The four forms of beta gamma complexes purified from brain and spleen showed indistinguishable inhibitory effects on the release of GDP from G(o) alpha, but beta gamma-T was much less effective. Brain and spleen beta gamma complexes were equally effective in inhibiting calmodulin-stimulated adenylyl-cyclase activity, but beta gamma-T had a very weak inhibitory effect. Five forms of beta gamma facilitated metarhodopsin II-catalyzed binding of GTP gamma S to Gt alpha in a concentration-dependent manner with the following rank order of effectiveness: beta gamma-S1 > beta gamma-T > beta gamma-B1 > beta gamma-B2 > beta gamma-B3. Because the beta gamma complexes used in this study mostly contained the same beta subunit, the functional differences must be dependent on the gamma subunits. Thus, it seems likely that the receptor, the alpha subunits, and the effector are able to distinguish between the various gamma subunits.     

Creating an education model for cardiac patients

gamma-Aminobutyric acidA (GABA(A)) gated chloride ion channels were expressed from human recombinant cDNA using the baculovirus/Sf-9 insect cell expression system. The electrophysiological effects in whole-cell currents of 5-(4-piperidyl) isoxazol-3-ol (4-PIOL), a GABA(A) receptor partial agonist, were investigated on GABA(A) receptor complexes of alpha1beta2gamma2S subunits as well as a slightly modified construct of alpha1(valine 121)beta2gamma2S subunits. Here we report that (1)4-PIOL induces an inward whole-cell current in a concentration-dependent manner in both alpha1(val 121)beta2gamma2S and alpha1(ile 121)beta2gamma2S receptor subunit combinations. (2) The 4-PIOL induced whole-cell currents were more pronounced in alpha1(val 121)beta2gamma2S than in alpha1(ile 121)beta2gamma2S receptor subunit combinations. (3) 4-PIOL inhibited GABA-induced responses on alpha1(ile 121)beta2gamma2S and alpha1(val 121)beta2gamma2S receptor combinations with similar potency.     

Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.