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.     

Chronic Ethanol Treatment Decreases [3H]Epibatidine and [3H]Nicotine Binding and Differentially Regulates mRNA Levels of Nicotinic Acetylcholine Receptor Subunits Expressed in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing α4β2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing α3, α5, α7, β2, and β4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (−30–80%) in number of [³H]nicotine binding sites was observed in ethanol-treated (25–240 m M ) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 m M ) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [³H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 m M ethanol significantly decreased the mRNA level for the α3 nAChR subunit (−39%), while the mRNA levels for the α7 (+30%) and α4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the β2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

Identification of three cAMP-dependent protein kinase (PKA) phosphorylation sites within the major intracellular domain of neuronal nicotinic receptor alpha4 subunits

This study determined whether all protein kinase A (PKA) and protein kinase C (PKC) phosphorylation sites on the alpha4 subunit of rat alpha4beta2 neuronal nicotinic receptors could be localized to the M3/M4 cytoplasmic domain of the protein, and investigated specific amino acid substrates for the kinases through two-dimensional phosphopeptide mapping and site-directed mutagenesis. Experiments were conducted using alpha4beta2 receptors expressed in Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(333-594) ). When oocytes expressing alpha4beta2 receptors were incubated with [(32) P]orthophosphate in order to label endogenous ATP stores, phosphorylation of alpha4 subunits was evident. Incubation of either immunoprecipitated receptors or the fusion protein with [(32) P]ATP and either PKA or PKC followed by trypsinization of the samples demonstrated that the kinases phosphorylated alpha4 subunits on multiple phosphopeptides, and that the phosphorylated full-length alpha4 protein and fusion protein produced identical phosphopeptide maps. Site-directed mutagenesis of Ser365, Ser472 and Ser491 to alanines in the fusion protein eliminated phosphopeptides phosphorylated by PKA, but not by PKC. Other mutations investigated, Ser470, Ser493, Ser517 and Ser590, did not alter the phosphopeptide maps. Results indicate that Ser365, Ser472 and Ser491 on neuronal nicotinic receptor alpha4 subunits are phosphorylated by PKA and are likely to represent post-translational regulatory sites on the receptor.     

Selective Dephosphorylation of the Subunits of Skeletal Muscle Calcium Channels by Purified Phosphoprotein Phosphatases

Abstract: Multiple sites on the α1 and β subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both α1 and β subunits at similar rates, whereas calcineurin dephosphorylated β subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the α1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the α1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the β subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn²⁺ increased the rate and extent of dephosphorylation of all sites by calcineurin so that >80% dephosphorylation of both α1 and β sub-units was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the α1 and β subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.     

Cyclic AMP-dependent protein kinase (PKA) and protein kinase C phosphorylate sites in the amino acid sequence corresponding to the M3/M4 cytoplasmic domain of alpha4 neuronal nicotinic receptor subunits

To determine whether alpha4 subunits of alpha4beta2 neuronal nicotinic receptors are phosphorylated within the M3/M4 intracellular region by cyclic AMP-dependent protein kinase A (PKA) or protein kinase C (PKC), immunoprecipitated receptors from Xenopus oocytes and a fusion protein corresponding to the M3/M4 cytoplasmic domain of alpha4 (alpha4(336-597)) were incubated with ATP and either PKA or PKC. Both alpha4 and alpha4(336-597) were phosphorylated by PKA and PKC, providing the first direct biochemical evidence that the M3/M4 cytoplasmic domain of neuronal nicotinic receptor alpha4 subunits is phosphorylated by both kinases. When the immunoprecipitated receptors and the alpha4(336-597) fusion protein were phosphorylated and the labeled proteins subjected to phosphoamino acid analysis, results indicated that alpha4 and alpha4(336-597) were phosphorylated on the same amino acid residues by each kinase. Furthermore, PKA phosphorylated serines exclusively, whereas PKC phosphorylated both serines and threonines. To determine whether Ser(368) was a substrate for both kinases, a peptide corresponding to amino acids 356-371 was synthesized (alpha4(356-371)) and incubated with ATP and the kinases. The phosphorylation of alpha4(356-371) by both PKA and PKC was saturable with K(m)s of 15.3 +/- 3.3 microM and 160.8 +/- 26.8 microM, respectively, suggesting that Ser(368) was a better substrate for PKA than PKC.     

The alpha4beta2alpha5 nicotinic cholinergic receptor in rat brain is resistant to up-regulation by nicotine in vivo

We used immunoprecipitation with subunit-specific antibodies to examine the distribution of heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) that contain the α5 subunit in the adult rat brain. Among the regions of brain we surveyed, the α5 subunit is associated in ∼37% of the nAChRs in the hippocampus, ∼24% of the nAChRs in striatum, and 11–16% of the receptors in the cerebral cortex, thalamus, and superior colliculus. Sequential immunoprecipitation assays demonstrate that the α5 subunit is associated with α4β2* nAChRs exclusively. Importantly, in contrast to α4β2 nAChRs, which are increased by 37–85% after chronic administration of nicotine, the α4β2α5 receptors are not increased by nicotine treatment. These data thus indicate that the α4β2α5 nAChRs in rat brain are resistant to up-regulation by nicotine in vivo , which suggests an important regulatory role for the α5 subunit. To the extent that nicotine-induced up-regulation of α4β2 nAChRs is involved in nicotine addiction, the resistance of the α4β2α5 subtype to up-regulation may have important implications for nicotine addiction.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

Construction of SH-EP1-α4β2-hAPP695 Cell Line and Effects of Nicotinic Agonists on β-amyloid in the Cells

(1) Nicotinic acetylcholine receptors in central nervous system are thought to be new targets for Alzheimer’s disease. However, the most involved nicotinic receptor subtype in Alzheimer’s disease is unclear. α4β2 receptor is the most widely spread subtype in brain, involving in several important aspects of cognitive and other functions. We constructed cell line by transfecting human amyloid precursor protein (695) gene into SH-EP1 cells which have been transfected with human nicotinic receptor α4 subunit and β2 subunit gene, to observe effects of α4β2 receptors activation on β-amyloid, expecting to provide a new cell line for drug screening and research purpose. (2) Liposome transfection was used to express human amyloid precursor protein (695) gene in SH-EP1-α4β2 cells. Function of the transfected α4β2 receptors was tested by patch clamp. Effects of nicotine and epibatidine (selective α4β2 nicotinic receptor agonist) on β-amyloid were detected by Western blot and ELISA. Effects of nicotine and epibatidine on amyloid precursor protein (695) mRNA level were measured using real-time PCR. (3) Human amyloid precursor protein (695) gene was stably expressed in SH-EP1-α4β2 cells; Nicotine (1 μM) and epibatidine (0.1 μM) decreased intracellular and secreted β-amyloid in the cells; and activation of α4β2 receptors did not affect amyloid precursor protein (695) mRNA level. (4) These results suggest that the constructed cell line, expressing both amyloid precursor protein (695) gene and human nicotinic receptor α4 subunit and β2 subunit gene, might be useful for screening specific nicotinic receptor agonists against Alzheimer’s disease. Alteration of Aβ level induced by activation of α4β2 nAChR in our study might occur at a post-translational level.     

Regulation of Nicotinic Receptor Subtypes Following Chronic Nicotinic Agonist Exposure in M10 and SH-SY5Y Neuroblastoma Cells

Abstract: The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick α4β2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the K _i values for binding inhibition and EC₅₀ values for agonist-induced α4β2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing α3, α5, β2, and β4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.     

Dα3, a New Functional α Subunit of Nicotinic Acetylcholine Receptors from Drosophila

Abstract: Nicotinic acetylcholine (ACh) receptors (nAChRs) are important excitatory neurotransmitter receptors in the insect CNS. We have isolated and characterized the gene and the cDNA of a new nAChR subunit from Drosophila . The predicted mature nAChR protein consists of 773 amino acid residues and has the structural features of an ACh-binding α subunit. It was therefore named Dα3, for D rosophila α -subunit 3 . The dα3 gene maps to the X chromosome at position 7E. The properties of the Dα3 protein were assessed by expression in Xenopus oocytes. Dα3 did not form functional receptors on its own or in combination with any Drosophila β-type nAChR subunit. Nondesensitizing ACh-evoked inward currents were observed when Dα3 was coexpressed with the chick β2 subunit. Half-maximal responses were at ∼0.15 µ M ACh with a Hill coefficient of ∼1.5. The snake venom component α-bungarotoxin (100 n M ) efficiently but reversibly blocked Dα3/β2 receptors, suggesting that Dα3 may be a component of one of the previously described two classes of toxin binding sites in the Drosophila CNS.     

Multiple Determinants of Dihydro-β-Erythroidine Sensitivity on Rat Neuronal Nicotinic Receptor α Subunits

Abstract: Neuronal nicotinic acetylcholine receptors are differentially sensitive to blockade by the competitive antagonist dihydro-β-erythroidine. Both α and β subunits participate in determining sensitivity to this antagonist. The α subunit contribution to dihydro-β-erythroidine sensitivity is illustrated by comparing the α4β4 receptor and the α3β4 receptor, which differ in sensitivity to dihydro-β-erythroidine by ∼120-fold. IC₅₀ values for blocking α4β4 and α3β4, responding to EC₂₀ concentrations of acetylcholine, were 0.19 ± 0.06 and 23.1 ± 10.2 µ M , respectively. To map the sequence segments responsible for this difference, we constructed a series of chimeric α subunits containing portions of the α4 and α3 subunits. These chimeras were coexpressed with β4, allowing pharmacological characterization. We found determinants of dihydro-β-erythroidine sensitivity to be distributed throughout the N-terminal extracellular domain of the α subunit. These determinants were localized to sequence segments 1–94, 94–152, and 195–215. Loss of determinants within segment 1–94 had the largest effect, decreasing dihydro-β-erythroidine sensitivity by 4.3-fold.     

Heteromeric co-assembly of two insect nicotinic acetylcholine receptor α subunits: influence on sensitivity to neonicotinoid insecticides

Neonicotinoid insecticides, such as imidacloprid, are selective agonists of insect nicotinic acetylcholine receptors (nAChRs) and are used extensively in areas of crop protection and animal health to control a variety of insect pest species. Here, we describe studies performed with nAChR subunits Nlα1 and Nlα2 cloned from the brown planthopper Nilaparvata  lugens , a major insect pest of rice crops in many parts of Asia. The influence of Nlα1 and Nlα2 subunits upon the functional properties of recombinant nAChRs has been examined by expression in Xenopus oocytes. In addition, the influence of a Nlα1 mutation (Y151S), which has been linked to neonicotinoid lab generated resistance in N. lugens , has been examined. As in previous studies of insect α subunits, functional expression has been achieved by co-expression with the mammalian β2 subunit. This approach has revealed a significantly higher apparent affinity of imidacloprid for Nlα1/β2 than for Nlα2/β2 nAChRs. In addition, evidence has been obtained for the co-assembly of Nlα1 and Nlα2 subunits into 'triplet' nAChRs of subunit composition Nlα1/Nlα2/β2. Evidence has also been obtained which demonstrates that the resistance-associated Y151S mutation has a significantly reduced effect on neonicotinoid agonist activity when Nlα1 is co-assembled with Nlα2 than when expressed as the sole α subunit in a heteromeric nAChR. These findings may be of importance in assessing the likely impact of the target-site mutations such as Y151S upon neonicotinoid insecticide resistance in insect field populations.     

Contribution of Position α4S336 on Functional Expression and Up-regulation of α4β2 Neuronal Nicotinic Receptors

Phosphorylation of the nicotinic acetylcholine receptor (nAChR) is believed to play a critical role in its nicotine-induced desensitization and up-regulation. We examined the contribution of a consensus PKC site in the α4 M3/M4 intracellular loop (α4S336) on the desensitization and up-regulation of α4β2 nAChRs expressed in oocytes. Position α4S336 was replaced with either alanine to abolish potential phosphorylation at this site or with aspartic acid to mimic phosphorylation at this same site. Mutations α4S336A and α4S336D displayed a threefold increase in the ACh-induced response and an increase in ACh EC₅₀. Epibatidine binding revealed a three and sevenfold increase in surface expression for the α4S336A and α4S336D mutations, respectively, relative to wild-type, therefore, both mutations enhanced expression of the α4β2 nAChR. Interestingly, the EC₅₀’s and peak currents for nicotine activation remained unaffected in both mutants. Both mutations abolished the nicotine-induced up-regulation that is normally observed in the wild-type. The present data suggest that adding or removing a negative charge at this phosphorylation site cannot be explained by a simple straightforward on-and-off mechanism; rather a more complex mechanism(s) may govern the functional expression of the α4β2 nAChR. Along the same line, our data support the idea that phosphorylation at multiple consensus sites in the α4 subunit could play a remarkable role on the regulation of the functional expression of the α4β2 nAChR.     

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine-induced receptor up-regulation and anti-inflammatory effects

α4β2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of α4β2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 μM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in hα4β2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in ³[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1β and IL-6. Nicotine suppresses IL-1β and IL-6 expression at least in part by inhibiting NFκB activation. Antagonists dihydro-β-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing α4β2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal α4β2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of α4β2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate α4β2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic α4β2 receptor activation promotes anti-inflammatory effects similar to α7 receptor activation.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure

We have determined the subunit stoichiometry of chicken neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes by quantitation of the amount of radioactivity in individual subunits of [35S] methionine-labeled receptors. The chicken neuronal nicotinic acetylcholine receptor appears to be a pentamer of two alpha 4 acetylcholine-binding subunits and three beta 2 structural subunits. We also show that these expressed receptors bind L-[3H]nicotine with high affinity, are transported to the surface of the oocyte outer membrane, and cosediment on sucrose gradients with acetylcholine receptors isolated from chicken brain. Using this unique and generally applicable method of determining subunit stoichiometry of receptors expressed in oocytes, we obtained the expected (alpha 1) 2 beta 1 gamma delta stoichiometry for muscle-type acetylcholine receptors assembled from coexpression of either Torpedo alpha 1 or human alpha 1 subunits, with Torpedo beta 1, gamma, and delta subunits.     

Small N-Terminal Deletion by Splicing in Cerebellar α6 Subunit Abolishes GABAA Receptor Function

Abstract: Sequence variation was found in cDNA coding for the extracellular domain of the rat γ-aminobutyric acid type A (GABA_A) receptor α6 subunit. About 20% of polymerase chain reaction (PCR)-amplified α6 cDNA prepared from rat cerebellar mRNA lacked nucleotides 226–255 as estimated by counting single-stranded phage plaques hybridized specifically to the short (α6S) and long (wild-type) forms of the α6 mRNA. Genomic PCR revealed an intron located upstream of the 30-nucleotide sequence. Both splice forms were detected in the cerebellum by in situ hybridization. Recombinant receptors, resulting from coexpression of the α6S subunit with the GABA_A receptor β2 and γ2 subunits in human embryonic kidney 293 cells, were inactive at binding [³H]muscimol and [³H]Ro 15-4513. In agreement, injection of complementary RNAs encoding the same subunits into Xenopus oocytes produced only weak GABA-induced currents, indistinguishable from those produced by β2γ2 receptors. Therefore, the 10 amino acids encoded by the 30-nucleotide fragment may be essential for the correct assembly or folding of the α6 subunit-containing receptors.     

Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor-like subunit with homology to the Drosophila melanogaster Dβ1 subunit

The cloning, sequencing and functional expression of Sgβ1, a novel locust (Schistocerca gregaria) non-α nicotinic acetylcholine receptor (nAChR) subunit is described. This subunit shows 80% identity with the Drosophila melanogaster Dβ1 and 92% identity with the Locusta migratoria β1, non-α subunits but only 38% identity to Sgα1 (also referred to as αL1), a previously cloned S. gregaria nAChR α-subunit. When expressed in Xenopus laevis oocytes, Sgβ1 does not respond to nicotine. Responses to nicotine are observed, however, in oocytes co-expressing Sgα1 and Sgβ1, but the pharmacology is indistinguishable from that of currents produced by expressing Sgα1 alone. We conclude that either Sgβ1 does not co-assemble with Sgα1, or that it is unable to contribute to the functional properties of the receptor, in the Xenopus oocyte expression system.