Effects of Redox Reagents and Arsenical Compounds on [3H]-Cytisine Binding to Immunoisolated Nicotinic Acetylcholine Receptors from Chick Brain Containing α4 β2 Subunits

All known nicotinic receptor α subunits include a conserved disulfide bond that is essential for function and is a site for labeling via biochemical modification. In an effort to develop a universal ligand for all subtypes of nicotinic receptors, we previously studied the effects of arsenylation with two compounds, ρ-aminophenyldichloroarsine (APA) and bromoacetyl-ρ-aminophenylarsenòxide (BAPA) on nicotinic receptors from Torpedo electroplax. Here we apply these reagents to immunoisolated receptors containing α4, β2, and possibly other subunits from chick brain that bind [³H]cytisine with high affinity (K_D∼5 nM). These are distinct from another receptor subtype that also binds [³H]cytisine and [³H]nicotine and can be arsenylated with APA, but instead contains α5,β2, and probably other subunits. Reduction of α4 β2 receptors with dithiothreitol blocked [³H]cytisine binding and this effect was reversed upon reoxidation by dithiobisnitrobenzoic acid. APA or BAPA prevented the dithiobisnitrobenzoic acid reactivation of dithiothreitol-treated receptors with IC₅₀ values of 15 and 70 n M , respectively. However, the antiarsenical dimercaptopropanesulfonic acid restored function to APA- or BAPA- "arsenylated" receptors (EC₅₀∼100 μ M ). APA-treated receptors remained blocked for up to 24 h, but treatment with dimercaptopropanesulfonic acid at any time restored [³H]cytisine binding. APA treatment of reduced receptors protected against irreversible alkylation by Bromoacetyl choline, indicating that arsenylation occurs at least in part in the agonist binding site. Thus, these reagents have similar effects on different nicotinic receptor subtypes from both muscle and nerves.     

Nicotine Enhances the Cyclic AMP-Dependent Protein Kinase-Mediated Phosphorylation of α4 Subunits of Neuronal Nicotinic Receptors

Abstract: Studies determined whether α4β2 or α3β2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 n M for α4β2 and 500 n M for α3β2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing α4β2 receptors were incubated with [γ-³²P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the α4 subunit was present. Phosphorylation of α4 subunits of α4β2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing α3β2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the α3 subunit. Results suggest that the PKA-mediated phosphorylation of α4 and not α3 subunits may explain the differential inactivation by nicotine of these receptors subtypes expressed in oocytes.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

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.     

Modulation of recombinant, α2*, α3* or α4*-nicotinic acetylcholine receptor (nAChR) function by nAChR β3 subunits

The nicotinic acetylcholine receptor (nAChR) β3 subunit is thought to serve an accessory role in nAChR subtypes expressed in dopaminergic regions implicated in drug dependence and reward. When β3 subunits are expressed in excess, they have a dominant-negative effect on function of selected nAChR subtypes. In this study, we show, in Xenopus oocytes expressing α2, α3 or α4 plus either β2 or β4 subunits, that in the presumed presence of similar amounts of each nAChR subunit, co-expression with wild-type β3 subunits generally (except for α3*-nAChR) lowers amplitudes of agonist-evoked, inward peak currents by 20-50% without having dramatic effects (≤ 2-fold) on agonist potencies. By contrast, co-expression with mutant β3(V9'S) subunits generally (except for α4β2*-nAChR) increases agonist potencies, consistent with an expected gain-of-function effect. This most dramatically demonstrates formation of complexes containing three kinds of subunit. Moreover, for oocytes expressing nAChR containing any α subunit plus β4 and β3(V9'S) subunits, there is spontaneous channel opening sensitive to blockade by the open channel blocker, atropine. Collectively, the results indicate that β3 subunits integrate into all of the studied receptor assemblies and suggest that natural co-expression with β3 subunits can influence levels of expression and agonist sensitivities of several nAChR subtypes.     

Expression and functional properties of α7 acetylcholine nicotinic receptors are modified in the presence of other receptor subunits

Although α7 nicotinic receptors are predominantly homopentamers, previous reports have indicated that α7 and β2 subunits are able to form heteromers. We have studied whether other nicotinic receptor subunits can also assemble with α7 subunits and the effect of this potential association. Coexpression of α7 with α2, α3, or β4 subunits reduced to about half, surface α‐bungarotoxin binding sites and acetylcholine‐gated currents. This is probably because of inhibition of membrane trafficking, as the total amount of α7 subunits was similar in all cases and a significant proportion of mature α7 receptors was present inside the cell. Only β4 subunits appeared to directly associate with α7 receptors at the membrane and these heteromeric receptors showed some kinetic and pharmacological differences when compared with homomeric α7 receptors. Finally, we emulated the situation of bovine chromaffin cells in Xenopus laevis oocytes by using the same proportion of α3, β4, α5, and α7 mRNAs, finding that α‐bungarotoxin binding was similarly reduced in spite of increased currents, apparently mediated by α3β4(α5) receptors.     

Similarity Between Rat Brain Nicotinic α-Bungarotoxin Receptors and Stably Expressed α-Bungarotoxin Binding Sites

Abstract: The present results demonstrate stable expression of α-bungarotoxin (α-BGT) binding sites by cells of the GH₄C₁ rat pituitary clonal line. Wild-type GH₄C₁ cells do not express α-BGT binding sites, nor do they contain detectable mRNA for nicotinic receptor α2, α3, α4, α5, α7, β2, or β3 subunits. However, GH₄C₁ cells stably transfected with rat nicotinic receptor α7 cDNA (α7/GH₄C₁ cells) express the transgene abundantly as mRNA, and northern analysis showed that the message is of the predicted size. The α7/GH₄C₁ cells also express saturable, high-affinity binding sites for ¹²⁵I-labeled α-BGT, with a K_D of 0.4 nM and B_max of 3.2 fmol/10⁶ intact cells. ¹²⁵I-α-BGT binding affinities and pharmacological profiles are not significantly different for sites in membranes prepared either from rat brain or α7/GH₄C₁ cells. Furthermore, K_D and K_i values for ¹²⁵I-α-BGT binding sites on intact α7/GH₄C₁ cells are essentially similar to those for hippocampal neurons in culture. Sucrose density gradient analysis showed that the size of the α-BGT binding sites expressed in α7/GH₄C₁ cells was similar to that of the native brain α-BGT receptor. Chronic exposure of α7/GH₄C₁ cells in culture to nicotine or an elevated extracellular potassium concentration induces changes in the number of α-BGT binding sites comparable to those observed in cultured neurons. Collectively, the present results show that the properties of α-BGT binding sites in transfected α7/GH₄C₁ cells resemble those for brain nicotinic α-BGT receptors. If the heterologously expressed α-BGT binding sites in the present study are composed solely of α7 subunits, the results could suggest that the rat brain α-BGT receptor has a similar homooligomeric structure. Alternatively, if α-BGT binding sites exist as heterooligomers of α7 plus some other previously identified or novel subunit(s), the data would indicate that the α7 subunits play a major role in determining properties of the α-BGT receptor.     

The loop between β-strands β2 and β3 and its interaction with the N-terminal α-helix is essential for biogenesis of α7 nicotinic receptors

Recently, we have shown that the α-helix present at the N-termini of α7 nicotinic acetylcholine receptors plays a crucial role in their biogenesis. Structural data suggest that this helix interacts with the loop linking β-strands β2 and β3 (loop 3). We studied the role of this loop as well as its interaction with the helix in membrane receptor expression. Residues from Asp62 to Val75 in loop 3 were mutated. Mutations of conserved amino acids, such as Asp62, Leu65 and Trp67 abolished membrane receptor expression in Xenopus oocytes. Others mutations, at residues Asn68, Ala69, Ser70, Tyr72, Gly74, and Val 75 were less harmful although still produced significant expression decreases. Steady state levels of wild-type and mutant α7 receptors (L65A, W67A, and Y72A) were similar but the formation of pentameric receptors was impaired in the latter (W67A). Mutation of critical residues in subunits of heteromeric nicotinic acetylcholine receptors (α3β4) also abolished their membrane expression. Complementarity between the helix and loop 3 was evidenced by studying the expression of chimeric α7 receptors in which these domains were substituted by homologous sequences from other subunits. We conclude that loop 3 and its docking to the α-helix is an important requirement for receptor assembly.     

Sustained Nicotine Exposure Differentially Affects α3β2 and α4β2 Neuronal Nicotinic Receptors Expressed in Xenopus Oocytes

Abstract: To determine whether prolonged exposure to nicotine differentially affects α3β2 versus α4β2 nicotinic receptors expressed in Xenopus oocytes, oocytes were coinjected with subunit cRNAs, and peak responses to agonist, evoked by 0.7 or 7 µ M nicotine for α4β2 and α3β2 receptors, respectively, were determined before and following incubation for up to 48 h with nanomolar concentrations of nicotine. Agonist responses of α4β2 receptors decreased in a concentration-dependent manner with IC₅₀ values in the 10 n M range following incubation for 24 h and in the 1 n M range following incubation for 48 h. In contrast, responses of α3β2 receptors following incubation for 24–48 h with 1,000 n M nicotine decreased by only 50–60%, and total ablation of responses could not be achieved. Attenuation of responses occurred within the first 5 min of nicotine exposure and was a first-order process for both subtypes; half-lives for inactivation were 4.09 and 2.36 min for α4β2 and α3β2 receptors, respectively. Recovery was also first-order for both subtypes; half-lives for recovery were 21 and 7.5 h for α4β2 and α3β2 receptors, respectively. Thus, the responsiveness of both receptors decreased following sustained exposure to nicotine, but α4β2 receptors recovered much slower. Results may explain the differential effect of sustained nicotine exposure on nicotinic receptor-mediated neurotransmitter release.     

β-Lumicolchicine Interacts with the Benzodiazepine Binding Site to Potentiate GABAA Receptor-Mediated Currents

Abstract: An analogue of colchicine,β-lumicolchicine, does not bind tubulin or disrupt microtubules. However, this compound is not pharmacologically completely inactive. β-Lumicolchicine was found to competitively inhibit [³H]flunitrazepam binding and to enhance muscimol-stimulated ³⁶Cr-uptake in mouse cerebral cortical microsacs. It also markedly potentiated GABA responses in Xenopusoocytes expressing human α₁β₂γ_2S, but not α₁β₂, GABA_A receptor subunits; this potentiation was reversed by the benzodiazepine receptor antagonist flumazenil. These results strongly suggest a direct effect of β-Lumicolchicine on the GABA_A receptor/chloride channel complex and caution that it possesses pharmacological effects, despite its inability to disrupt microtubules. Furthermore, β-Lumicolchicine is structurally unrelated to benzodiazepines or quinolines and may provide a novel approach to the synthesis of ligands for this receptor.     

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.     

Structure of α6β3δ GABAA receptors and their lack of ethanol sensitivity

Delta (δ) subunit containing GABA_A receptors are expressed extra‐synaptically and mediate tonic inhibition. In cerebellar granule cells, they often form a receptor together with α₆ subunits. We were interested to determine the architecture of these receptors. We predefined the subunit arrangement of 24 different GABA_A receptor pentamers by subunit concatenation. These receptors (composed of α₆, β₃ and δ subunits) were expressed in Xenopus oocytes and their electrophysiological properties analyzed. Currents elicited in response to GABA were determined in presence and absence of 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one and to 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. α₆‐β₃‐α₆/δ receptors showed a substantial response to GABA alone. Three receptors, β₃‐α₆‐δ/α₆‐β₃, α₆‐β₃‐α₆/β₃‐δ and β₃‐δ‐β₃/α₆‐β₃, were only uncovered in the combined presence of the neurosteroid 3α, 21‐dihydroxy‐5α‐pregnan‐20‐one with GABA. All four receptors were activated by 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]‐pyridin‐3‐ol. None of the functional receptors was modulated by physiological concentrations (up to 30 mM) of ethanol. GABA concentration response curves indicated that the δ subunit can contribute to the formation of an agonist site. We conclude from the investigated receptors that the δ subunit can assume multiple positions in a receptor pentamer composed of α₆, β₃ and δ subunits.     

Differential Inhibition by α-Conotoxin-MII of the Nicotinic Stimulation of [3H]Dopamine Release from Rat Striatal Synaptosomes and Slices

Abstract: The presynaptic nicotinic modulation of dopamine release from striatal nerve terminals is well established, but the subtype(s) of neuronal nicotinic acetylcholine receptor (nAChR) underlying this response has not been identified. Recently, α-conotoxin-MII has been reported to inhibit potently and selectively the rat α3/β2 combination of nAChR subunits. Here we have synthesised the peptide, confirmed its specificity, and examined its effect on the (±)-anatoxin-a-evoked release of [³H]dopamine from rat striatal synaptosomes and slices. α-Conotoxin-MII (112 nM) completely blocked acetylcholine-evoked currents of α3β2 nAChRs expressed in Xenopus oocytes (IC₅₀ = 8.0 ± 1.1 nM). Pairwise combinations of other nicotinic subunits were not blocked by 112 nMα-conotoxin-MII. On perfused striatal synaptosomes and slices, α-conotoxin-MII dose-dependently inhibited [³H]dopamine release evoked by 1 µM (±)-anatoxin-a with IC₅₀ values of 24.3 ± 2.9 and 17.3 ± 0.1 nM, respectively. The dose-response curve was shifted to the right with increasing agonist concentrations. However, the maximal inhibition of responses achieved by α-conotoxin-MII (112 nM) was 44.9 ± 5.4% for synaptosomes and 25.0 ± 4.1% for slices, compared with an inhibition by 10 µM mecamylamine of 77.9 ± 3.7 and 88.0 ± 2.1%, respectively. These results suggest the presence of presynaptic α3β2-like nAChRs on striatal dopaminergic terminals, but the incomplete block of (±)-anatoxin-a-evoked [³H]dopamine release by α-conotoxin-MII also supports the participation of nAChRs composed of other subunits. The lower inhibition found in slices is consistent with an additional indirect nicotinic stimulation of dopamine release via an α-conotoxin-MII-insensitive nAChR.     

Identification of N-terminal extracellular domain determinants in nicotinic acetylcholine receptor (nAChR) α6 subunits that influence effects of wild-type or mutant β3 subunits on function of α6β2*- or α6β4*-nAChR

Despite the apparent function of naturally expressed mammalian α6*-nicotinic acetylcholine receptors (α6*-nAChR; where * indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type β3 subunits abolishes the small amount of function typically seen for all-human or all-mouse α6β4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of α6 and β4 subunits with mutant β3 subunits harboring valine-to-serine mutations at 9'- or 13'-positions. There is no function when α6 and β2 subunits are expressed alone or in the presence of wild-type or mutant β3 subunits. Interestingly, hybrid nAChR containing mouse α6 and human (h) β4 subunits have function potentiated rather than suppressed by coexpression with wild-type hβ3 subunits and potentiated further upon coexpression with hβ3(V9'S) subunits. Studies using nAChR chimeric mouse/human α6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the α6 subunit N-terminal domain. More specifically, nAChR hα6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR hβ3 subunits on hα6hβ4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR hα6 subunits are involved in the gain-of-function effects of nAChR hβ3(V9'S) subunits on α6β2*-nAChR function. These studies illuminate the structural bases for effects of β3 subunits on α6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of α6 subunits are involved.     

\4 integrin expression on<Emphasis Type="Italic">in vitro</Emphasis> and<Emphasis Type="Italic">in vivo</Emphasis> metastatic variants of lewis lung carcinoma

The expression of β4, α6, and β1 integrin subunits has been investigated on somein vitro andin vivo murine metastatic variants derived from Lewis lung carcinoma (3LL). By the use of monoclonal antibodies which recognizes different epitopes of α6, β1, and β4 subunits we demonstrate that α6 and β1 subunits are expressed in all metastatic variants of 3LL irrespective of their metastatic potential, whereas β4 subunit is expressed only in highly metastasizing cells of 3LL. Northern blots of different metastatic variants probed with β1 and β4 subunits demonstrate thata) significant amounts of β1 mRNA were detected in all metastatic variants of 3LL;b) mRNA corresponding to the described entire coding sequence of β4 subunit is expressed only on highly metastasizing cells of 3LL. We conclude that β4 subunit is specifically expressed in highly metastasizig cells of 3LL while is undetectable in lower metastasizing ones.     

Apparent homomeric NR1 currents observed in Xenopus oocytes are caused by an endogenous NR2 subunit

Functional N-methyl-d-aspartate receptors NMDARs are thought to be heteromeric receptor complexes consisting of NR1 and NR2 subunits. However, recombinant NR1 subunits expressed in Xenopus oocytes assemble functional ion channels even without exogenous NR2 subunits and with a different pharmacology, suggesting a homomeric subunit stoichiometry. To explain this phenomenon, we screened oocytes for Xenopus NR2 subunits and found all four subunit-encoding mRNAs (XenNR2A-XenNR2D) to be present endogenously, with those encoding the XenNR2B subunit being particularly abundant. We cloned the full-length XenNR2B cDNA and co-expressed it with NR1 in oocytes. A detailed electrophysiological characterization revealed that the pharmacology of NR1/XenNR2B was identical with that of the presumed homomeric NMDARs expressed from NR1 subunits. By contrast, heteromeric receptors containing the rat NR2B subunit showed significant pharmacological differences compared with NR1/XenNR2B receptors. These results demonstrate that recombinant NR1 subunits expressed in Xenopus oocytes interact with an endogenously expressed NR2B subunit and form hybrid heteromeric NMDARs. These findings confirm the current views that NMDARs are obligatory heteromeric complexes and that functional homomeric NMDARs do not exist.     

Molecular and pharmacological properties of GABA-ρ subunits from white perch retina

Five γ-aminobutyric acid (GABA)-ρ subunits were cloned from a white perch retinal cDNA library and expressed in Xenopus oocytes. The deduced amino acid sequences indicated that all are highly homologous to the GABA-ρ subunits cloned from mammalian retinas; two clones (perch-ρ1A and perch-ρ1B) were in the ρ1 family, two (perch-ρ2A and perch-ρ2B) were in the ρ2 family, and one clone has been tentatively identified as a perch-ρ3 subunit. When expressed in Xenopus oocytes, all but one of the subunits (ρ3) formed functional homooligomeric receptors. However, the receptors expressed by each of the GABA-ρ subunits displayed unique response properties that distinguished one from the other. For example, receptors formed by perch-ρ1B subunits were more sensitive to GABA than the receptors formed by other GABA-ρ subunits, the dose–response curves for the various receptors revealed different Hill coefficients, and there were differences in the kinetics of the GABA-induced currents. In addition, the GABA-mediated current–voltage curve for ρ2 receptors was approximately linear, whereas the responses from ρ1 receptors showed outward rectification. A further division in the properties of the GABA-ρ subunits was revealed in their responses to imidazole-4-acetic acid (I4AA); the drug behaved as an antagonist on A-type ρ receptors and a partial agonist on the B-type ρ receptors. These results suggest that there is a large diversity of GABA_c receptors in the vertebrate retina, probably formed by homooligomeric and heterooligomeric combinations of GABA ρ subunits, that exhibit different functional properties. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 305–320, 1998