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1.
GABA A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA A receptors, α4/δ-containing GABA A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity. 相似文献
2.
GABA A receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ β? interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αβ, αβγ and αβδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and β subunits present within the receptors. Whereas the presence of a γ2 subunit within αβγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αβ receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1β2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ β? site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ β? interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1β3δ receptors was not observed at α4,6β3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1β3δ receptors was not observed with another α+ β? modulator, and that the efficacy of modulation by α+ β? ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand. 相似文献
3.
AbstractThe experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABA A receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABA A receptor α and β subunits. G 418 resistant colonies isolated at this step were screened for [ 3H] muscimol binding to select for those that coexpressed α- and β-subunits. The best α and β subunit expressing colony was then supertransfected with a plasmid coding for the γ rat GABA A receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary αβγ GABA A receptor combinations were distinguished using [ 3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABA A receptor clones, α 1β 2γ 2S, α 1β 2γ 2S and α 1β 2γ 2S, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABA A receptor subtypes. 相似文献
4.
We evaluated the effects of 6-methoxyflavanone and 6-methoxyflavone on wild-type α1/α2β2γ2L GABA A and ρ1 GABA C receptors and on mutant ρ1I307S, ρ1W328 M, ρ1I307S/W328 M GABA C receptors expressed in Xenopus oocytes using two-electrode voltage clamp and radioligand binding. 6-Methoxyflavanone and 6-methoxyflavone act as a flumazenil-insensitive positive allosteric modulator of GABA responses at human recombinant α1β2γ2L and α2β2γ2L GABA A receptors. However, unlike 6-methoxyflavone, 6-methoxyflavanone was relatively inactive at α1β2 GABA A receptors. 6-Methoxyflavanone inhibited [ 3H]-flunitrazepam binding to rat brain membranes. Both flavonoids were found to be inactive as modulators at ρ1, ρ1I307S and ρ1W328 M GABA receptors but acted as positive allosteric modulators of GABA at the benzodiazepine sensitive ρ1I307S/W328 M GABA receptors. This double mutant retains ρ1 properties of being insensitive to bicuculline and antagonised by TPMPA and THIP. Additionally, 6-methoxyflavanone was also a partial agonist at ρ1W328 M GABA receptors. The relative inactivity of 6-methoxyflavanone at α1β2 GABA A receptors and it’s partial agonist action at ρ1W328 M GABA receptors suggest that it exhibits a unique profile not matched by other flavonoids. 相似文献
5.
Exposure of female rats to estradiol during the perinatal period has profound effects on GABAergic neurotransmission that are crucial to establish sexually dimorphic brain characteristics. We previously showed that neonatal β-estradiol 3-benzoate (EB) treatment decreases brain concentrations of the neurosteroid allopregnanolone, a potent positive modulator of extrasynaptic GABA A receptors (GABA AR). We thus evaluated whether neonatal EB treatment affects GABA AR expression and function in the hippocampus of adult female rats. Neonatal EB administration increased the expression of extrasynaptic α4/δ subunit-containing GABA ARs and the modulatory action of THIP on tonic currents mediated by these receptors. The same treatment decreased the expression of synaptic α1/α4/γ2 subunit-containing receptors, as well as phasic currents. These effects of neonatal EB treatment are not related to ambient allopregnanolone concentrations per se, given that vehicle-treated rats in diestrus, which have opposite neurosteroid levels than EB-treated rats, show similar changes in GABA ARs. Rather, these changes may represent a compensatory mechanism to counteract the long-term reduction in allopregnanolone concentrations, induced by neonatal EB. Given that both α4/δ receptors and allopregnanolone are involved in memory consolidation, we evaluated whether neonatal EB treatment alters performance in the Morris water maze test during adulthood. Neonatal EB treatment decreased the latency and the cumulative search error to reach the platform, as well as thigmotaxis, suggesting improved learning, and also enhanced memory performance during the probe trial. These enduring changes in GABA AR plasticity may be relevant for the regulation of neuronal excitability in the hippocampus and for the etiology of psychiatric disorders that originate in development and show sex differences. 相似文献
6.
A dichloromethane extract of stems and roots of Pholidota chinensis (Orchidaceae) enhanced GABA-induced chloride currents ( IGABA) by 132.75 ± 36.69% when tested at 100 μg/mL in a two-microelectrode voltage clamp assay, on Xenopus laevis oocytes expressing recombinant α 1β 2γ 2S GABA A receptors. By means of an HPLC-based activity profiling approach, the three structurally related stilbenoids coelonin ( 1), batatasin III ( 2), and pholidotol D ( 3) were identified in the active fractions of the extract. Dihydrostilbene 2 enhanced IGABA by 1512.19 ± 176.47% at 300 μM, with an EC 50 of 52.51 ± 16.96 μM, while compounds 1 and 3 showed much lower activity. The relevance of conformational flexibility for receptor modulation by stilbenoids was confirmed with a series of 13 commercially available stilbenes and their corresponding semisynthetic dihydro derivatives. Dihydrostilbenes showed higher activity in the oocyte assay than their corresponding stilbenes. The dihydro derivatives of tetramethoxy-piceatannol ( 12) and pterostilbene ( 20) were the most active among these derivatives, but they showed lower efficiencies than compound 2. Batatasin III ( 2) showed high efficiency but no significant subunit specificity when tested on the receptor subtypes α 1β 2γ 2s, α 2β 2γ 2s, α 3β 2γ 2s, α 4β 2γ 2s, α 5β 2γ 2s, α 1β 1γ 2s, and α 1β 3γ 2s. Dihydrostilbenes represent a new scaffold for GABA A receptor modulators. 相似文献
7.
γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA A receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA A receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ~50 and ~52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABA A receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities. 相似文献
8.
Gamma‐aminobutyric acid type A receptors (GABA ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA ARs determine their function and pharmacological profile. GABA ARs are heteropentamers of subunits, and (α1) 2(β3) 2(γ2L) 1 is a common subtype. Biochemical and biophysical studies of GABA ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS) 3GK–1D4 GABA AR. These cells achieved expression levels of 70–90 pmol [ 3H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [ 3H]flunitrazepam to [ 3H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ~30%. Typical purifications yielded 1.0–1.5 nmoles of [ 3H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [ 3H]muscimol binding were maintained in the purified state. 相似文献
9.
BackgroundMagnolia bark preparations from Magnolia officinalis of Asian medicinal systems are known for their muscle relaxant effect and anticonvulsant activity. These CNS related effects are ascribed to the presence of the biphenyl-type neolignans honokiol and magnolol that exert a potentiating effect on GABA A receptors. 4- O-methylhonokiol isolated from seeds of the North-American M. grandiflora was compared to honokiol for its activity to potentiate GABA A receptors and its GABA A receptor subtype-specificity was established. MethodsDifferent recombinant GABA A receptors were functionally expressed in Xenopus oocytes and electrophysiological techniques were used determine to their modulation by 4- O-methylhonokiol. Results3 μM 4- O-methylhonokiol is shown here to potentiate responses of the α 1β 2γ 2 GABA A receptor about 20-fold stronger than the same concentration of honokiol. In the present study potentiation by 4- O-methylhonokiol is also detailed for 12 GABA A receptor subtypes to assess GABA A receptor subunits that are responsible for the potentiating effect. ConclusionThe much higher potentiation of GABA A receptors at identical concentrations of 4- O-methylhonokiol as compared to honokiol parallels previous observations made in other systems of potentiated pharmacological activity of 4- O-methylhonokiol over honokiol. General significanceThe results point to the use of 4- O-methylhonokiol as a lead for GABA A receptor potentiation and corroborate the use of M. grandiflora seeds against convulsions in Mexican folk medicine. 相似文献
11.
BackgroundWithin the GABA A-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA A-receptor subtypes. MethodsWe investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA A-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α 1 selective) and TPA023 (α 2/3 selective). In-vivo binding studies with [ 3H]flumazenil confirmed compounds occupied CNS GABA A receptors. ResultsChronic diazepam treatment resulted in tolerance to diazepam''s acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam''s anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam''s hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam''s hypothermic, anxiolytic or sedative effects. ConclusionsOur data indicate that: (i) GABA A-α 2/α 3 subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam''s sedative actions needs concomitant activation of GABA A-α 1/GABA A-α 5 receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA A α 1, α 2 or α 5 subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA A α 2, or α 3 receptors does not engender tolerance development, subtype-selective GABA A drugs might constitute a promising class of novel drugs. 相似文献
12.
Pyrazoloquinolinones (PQs) have been extensively studied as modulators of GABA A receptors with different subunit composition, exerting modulatory effects by binding at α+/β- interfaces of GABA A receptors. PQs with a substituent in position R7 have been reported to preferentially modulate α6- subunit containing GABA A receptors which are mostly expressed in the cerebellum but were also found in the olfactory bulb, in the cochlear nucleus, in the hippocampus and in the trigeminal sensory pathway. They are considered potentially interesting in the context of sensori-motor gating deficits, depressive-like behavior, migraine and orofacial pain. Here we explored the option to modify the lead ligands’ R7 position. In the compound series we observed two different patterns of allosteric modulation in recombinantly expressed α6β3γ2 receptors, namely monophasic and biphasic positive modulation. In the latter case the additional phase occurred in the nanomolar range, while all compounds displayed robust modulation in the micromolar range. Nanomolar, near silent binding has been reported to occur at benzodiazepine binding sites, but was not investigated at the diazepam insensitive α6+/γ2- interface. To clarify the mechanism underlying the biphasic effect we tested one of the compounds in concatenated receptors. In these constructs the subunits are covalently linked, allowing to form either the α6+/γ2- interface, or the α6+/β3- interface, to study the resulting modulation. With this approach we were able to ascribe the nanomolar modulation to the α6+/γ2- interface. While not all compounds display the nanomolar phase, the strong modulation at the α6+/β3 interface proved to be tolerant for all tested R7 groups. This provides the future option to introduce e.g. isotope labelled or fluorescent moieties or substituents that enhance solubility and bioavailability. 相似文献
13.
Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA A receptor (GABA ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3 arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His 102 binds at the γ2 subunit interface in proximity to γ2 residues Thr 142, Phe 77, and Met 130; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites. 相似文献
14.
Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA A) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β (2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100 mg/kg and was devoid of sedative properties. 相似文献
15.
Abstract: It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABA A receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABA A/benzodiazepine receptor complexes formed by different subunit compositions (α xβ yγ 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10 ?4M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (>200% of control values) the binding of [ 3H]flunitrazepam ([ 3H]FNM) to the α 3β 2γ 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [ 3H]FNM binding to α 1β xγ 2 and α 2β xγ 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the α 5β 2γ 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [ 3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [ 3H]FNM only if an α 1β 2γ 2 receptor combination was used. Given the heterogeneity of the GABA A/benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels. 相似文献
16.
Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na + and L-type Ca 2+ channels and GABA A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA A α 1β 3γ 2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA A receptor currents and suppressed allosteric modulation by flunitrazepam at α 1β 3γ 2S receptors. All effects were independent of the presence of a γ 2S subunit in the GABA A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA A receptor through changes in lipid bilayer elasticity. 相似文献
17.
Ethanol causes pathological changes in GABA A receptor trafficking and function. These changes are mediated in part by ethanol activation of protein kinase A (PKA). The current study investigated the expression of the GABA A α1 and α4 subunits and the kinase anchoring protein AKAP150, as well as bicuculline-induced seizure threshold, at baseline and following acute injection of ethanol (3.5 g/kg IP) in a mouse line lacking the regulatory RIIβ subunit of PKA. Whole cerebral cortices were harvested at baseline, 1 h, or 46 h following injection of ethanol or saline and subjected to fractionation and western blot analysis. Knockout (RIIβ?/?) mice had similar baseline levels of PKA RIIα and GABA A α1 and α4 subunits compared to wild type (RIIβ+/+) littermates, but had deficits in AKAP150. GABA A α1 subunit levels were decreased in the P2 fraction of RIIβ?/?, but not RIIβ+/+, mice following 1 h ethanol, an effect that was driven by decreased α1 expression in the synaptic fraction. GABA A α4 subunits in the P2 fraction were not affected by 1 h ethanol; however, synaptic α4 subunit expression was increased in RIIβ+/+, but not RIIβ?/? mice, while extrasynaptic α4 and δ subunit expression were decreased in RIIβ?/?, but not RIIβ+/+ mice. Finally, RIIβ knockout was protective against bicuculline-induced seizure susceptibility. Overall, the results suggest that PKA has differential roles in regulating GABA A receptor subunits. PKA may protect against ethanol-induced deficits in synaptic α1 and extrasynaptic α4 receptors, but may facilitate the increase of synaptic α4 receptors. 相似文献
18.
Abstract: The effect of calcium-phospholipid-dependent protein kinase (PKC) on GABA A receptor function was examined in Xenopus oocytes expressing recombinant human GABA A receptor using two-electrode voltage-clamp measurements. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, inhibited GABA-gated chloride currents by ~72% in oocytes expressing α lβ 1γ 2L subunit cDNAs. Phorbol 12-monomyristate (PMM), a negative control analogue of PMA, did not alter GABA A receptor responses. To investigate whether activation of PKC could alter the modulatory responses of the receptor complex, the effect of PMA on benzodiazepine and barbiturate potentiation of GABA responses was assessed. In oocytes expressing α lβ 1γ 2s subunit cDNAs, diazepam (300 nM) potentiated GABA responses by ~160%. Following PMA (5-25 nM/) treatment, diazepam potentiation was significantly increased to 333%. No effect of the inactive phorbol ester PMM (25 nM) was observed on diazepam potentiation of GABA responses. PMA enhancement of diazepam potentiation of GABA responses was also observed in oocytes expressing α lβ 1γ 2Ssubunit cDNAs, indicating that the unique PKC site present in the Tγ 2LL subunit is not required for observing the PMA effect. PMA (5-25 nM) also enhanced pentobarbital potentiation of GABA responses. In oocytes expressing α lβ 1γ 2L subunit cDNAs, pentobarbital (25 μM) potentiated GABA receptor responses by ~97%. Following treatment with PMA (5-25 nM), pentobarbital potentiation of GABA responses increased to ~ 156%. The present results suggest that protein phosphorylation may alter the coupling between the allosteric modulatory sites within the GABA A receptor complex. 相似文献
19.
ObjectiveChromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression. MethodsThe technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes. ResultsResults for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes. ConclusionsChromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes. 相似文献
20.
The brain's major inhibitory neuroreceptor is the ligand-gated ion channel γ-aminobutyric acid (GABA) type A receptor (GABAR). GABARs exist in a variety of different subunit combinations that act to modulate the physiological behavior of GABAR by altering its pharmacological profile, as well as its affinity for GABA. While the α(1)β(2)γ(2) subtype is one of the most prevalent GABARs, the less populous α(6)β(3)δ subtype has much higher GABA sensitivity. Previous studies identified residues crucial for GABA binding; however, the specific molecular differences responsible for this diverse sensitivity are not known. Furthermore, the role of loop F is a divisive subject, with conflicting evidence for ligand binding function. Using homology modeling, ligand docking, and molecular dynamics simulations, we investigated the GABA binding sites of the two receptor subtypes. Simulations identified seven residues that consistently interacted with GABA in both subtypes: αF65, αR132, βL99, βE155, βR/K196, βY205, and βR207. Residue substitution at position β196 (arginine in α(6)β(3)δ, lysine in α(1)β(2)γ(2)) resulted in a shift in GABA binding. However, the major difference between the two binding sites was the magnitude of loop F involvement, with a greater contribution in the α(6)β(3)δ receptor. Free energy calculations confirm that the α(6)β(3)δ binding pocket has an increased affinity for GABA. Thus, the possible role for loop F across the GABAR family is to modulate GABA affinity. 相似文献
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