Axonal transport of AAV9 in nonhuman primate brain.

A pilot study in nonhuman primates was conducted, in which two Rhesus macaques received bilateral parenchymal infusions of adeno-associated virus serotype 9 encoding green fluorescent protein (AAV9-GFP) into each putamen. The post-surgical in-life was restricted to 3 weeks in order to minimize immunotoxicity expected to arise from expression of GFP in antigen-presenting cells. Three main findings emerged from this work. First, the volume over which AAV9 expression was distributed (Ve) was substantially greater than the volume of distribution of MRI signal (Vd). This stands in contrast with Ve/Vd ratio of rAAV2, which is lower under similar conditions. Second, post-mortem analysis revealed expression of GFP in thalamic and cortical neurons as well as dopaminergic neurons projecting from substantia nigra pars compacta, indicating retrograde transport of AAV9. However, fibers in the substantia nigra pars reticulata, a region that receives projections from putamen, also stained for GFP, indicating anterograde transport of AAV9 as well. Finally, one hemisphere received a 10-fold lower dose of vector compared with the contralateral hemisphere (1.5 × 10(13) vg ml(-1)) and we observed a much stronger dose effect on anterograde-linked than on retrograde-linked structures. These data suggest that AAV9 can be axonally transported bi-directionally in the primate brain. This has obvious implications to the clinical developing of therapies for neurological disorders like Huntington's or Alzheimer's diseases.

Slow AAV2 clearance from the brain of nonhuman primates and anti-capsid immune response.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.

Adeno-associated virus type 6 is retrogradely transported in the non-human primate brain.

We recently demonstrated that axonal transport of adeno-associated virus (AAV) is serotype-dependent. Thus, AAV serotype 2 (AAV2) is anterogradely transported (e.g., from cell bodies to nerve terminals) in both rat and non-human primate (NHP) brain. In contrast, AAV serotype 6 (AAV6) is retrogradely transported from terminals to neuronal cell bodies in the rat brain. However, the directionality of axonal transport of AAV6 in the NHP brain has not been determined. In this study, two Cynomolgus macaques received an infusion of AAV6 harboring green fluorescent protein (GFP) into the striatum (caudate and putamen) by magnetic resonance (MR)-guided convection-enhanced delivery. One month after infusion, immunohistochemical staining of brain sections revealed a striatal GFP expression that corresponded well with MR signal observed during gene delivery. As shown previously in rats, GFP expression was detected throughout the prefrontal, frontal and parietal cortex, as well as the substantia nigra pars compacta and thalamus, indicating retrograde transport of the vector in NHP. AAV6-GFP preferentially transduced neurons, although a few astrocytes were also transduced. Transduction of non-neuronal cells in the brain was associated with the upregulation of the major histocompatibility complex-II and lymphocytic infiltration as previously observed with AAV1 and AAV9. This contrasts with highly specific neuronal transduction in the rat brain. Retrograde axonal transport of AAV6 from a single striatal infusion permits efficient transduction of cortical neurons in significant tissue volumes that otherwise would be difficult to achieve.

Convection-enhanced delivery improves distribution and efficacy of tumor-selective retroviral replicating vectors in a rodent brain tumor model.

In the present study, we compared the therapeutic effect of tumor-selective retroviral replicating vectors (RRV) expressing the yeast cytosine deaminase (CD) delivered by convection-enhanced delivery (CED) or simple injection, followed by systemic administration of the pro-drug, 5-fluorocytosine (5-FC). Treatment with RRV-CD and systemic 5-FC significantly increased survival in rodent U87MG glioma model in comparison with controls (P<0.01). Interestingly, CED of RRV-CD followed by 5-FC further enhanced survival in this animal model in comparison with intra-tumoral injection of RRV-CD, followed by systemic 5-FC (P<0.05). High expression levels of Ki-67 were found in untreated tumors compared with treated. Untreated tumors were also much larger than treated. CED resulted in excellent distribution of RRV while only partial distribution of RRV was obtained after injection. Furthermore, RRV-CD and CD were also found in tumors from treated rats at study end points. These results demonstrated that RRV vectors may efficiently transduce and stably propagate in malignant human glioma, thereby achieving a significant in situ amplification effect after initial administration. We conclude that delivery of RRV into the glioma by CED provides much wider vector distribution than simple injection, and this correlated with better therapeutic outcomes.

Axonal transport of adeno-associated viral vectors is serotype-dependent.

We have previously shown that adeno-associated virus type 2 (AAV2) undergoes anterograde axonal transport in rat and non-human primate brain. We screened other AAV serotypes for axonal transport and found that AAV6 is transported almost exclusively in a retrograde direction and, in the same way as AAV2, it is also neuron-specific in rat brain. Our findings show that axonal transport of AAV is serotype dependent and this has implications for gene therapy of neurological diseases such as Huntington's disease.

Magnetic resonance imaging-guided delivery of adeno-associated virus type 2 to the primate brain for the treatment of lysosomal storage disorders.

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.

Future applications: gene therapy.

Gene therapy for brain disorders is one of the most promising frontiers in the practice of restorative neurosurgery. There are significant experimental gene therapy initiatives underway that have led to currently active clinical trials using direct intracerebral delivery of viral vectors, and these treatments have been reported as safe and well tolerated. In the future, other clinical trials will likely use viral vectors to transfer genes that bestow on recipient tissue a desired enzymatic or neurotrophic activity relevant to the treatment of other neurodegenerative diseases, stroke, and traumatic brain injury.

Pharmacological differences between immunoisolated native brain and heterologously expressed rat alpha4beta2 nicotinic receptors.

Native brain and heterologously expressed rat alpha4beta2 nicotinic receptors (in Xenopus oocytes and CV-1 cells) were immunoisolated with the anti-alpha4 antibody mAb 299 and their pharmacological properties were compared using [3H](+/-)epibatidine, the novel N-alkylnicotinium analog N-n-octylnicotinium iodide (NONI), and the ganglionic antagonist trimethaphan (TRM). The equilibrium dissociation constant (K(d)) for [3H](+/-)epibatidine binding to the native and heterologously expressed receptors ranged from 13 to 21 pM. The Hill coefficients for [3H](+/-)epibatidine binding to the native and expressed receptors ranged from 0.8 to 1.1 and were consistent with a single high-affinity site. NONI inhibited 30 pM [3H](+/-)epibatidine binding to the native and expressed receptors with similar potency (IC(50) values of 6-7 microM). However, [3H](+/-)epibatidine dissociated 2-3 times more slowly from the native, than from the expressed receptors and TRM inhibited 30 pM [3H](+/-)epibatidine binding to the native receptors (IC(50) value of 330 microM) less potently than it did to the receptors expressed in oocytes (IC(50) value of 16 microM) or CV-1 cells (IC(50) value of 55 microM). The differences between the native and expressed [3H](+/-)epibatidine dissociation rate constants and IC(50) values for TRM were significant for both host cell types, although the values for the CV-1-expressed receptors were closer to the native ones than were those for the oocyte-expressed receptors. Thus, the epibatidine and trimethaphan binding sites in native and expressed alpha4beta2 receptors appear to have significantly different structural or chemical properties.

Acetylcholine receptors are required for agrin-induced clustering of postsynaptic proteins.

We have investigated the role of acetylcholine receptors (AChRs) in an early step of postsynaptic assembly at the neuromuscular synapse, the clustering of postsynaptic proteins induced by nerve-released agrin. To achieve this, we used two variants of C2 myotubes virtually lacking AChRs and C2 cells in which surface AChRs were down-regulated by AChR antibodies. In all cases, agrin caused normal clustering of the agrin receptor component MuSK, alpha-dystrobrevin and utrophin, but failed to aggregate AChRs, alpha- and beta-dystroglycan, syntrophin isoforms and rapsyn, an AChR-anchoring protein necessary for postsynaptic assembly and AChR clustering. In C2 variants, the stability of rapsyn was decreased, whereas in antibody-treated cells, rapsyn efficiently co-localized with remaining AChRs in microaggregates. Upon ectopic injection into myofibers in vivo, rapsyn did not form clusters in the absence of AChRs. These results show that AChRs and rapsyn are interdependent components of a pre-assembled protein complex that is required for agrin-induced clustering of a full set of postsynaptic proteins, thus providing evidence for an active role of AChRs in postsynaptic assembly.

UNCL, the mammalian homologue of UNC-50, is an inner nuclear membrane RNA-binding protein.

We isolated a mammalian homologue of the C. elegans gene unc-50 that we have named UNCL. The 777 kb rat UNCL cDNA encodes a 259 amino acid protein that is expressed in a wide variety of tissues with highest mRNA levels in brain, kidney and testis. Hydropathy plot analysis and in vitro translation experiments with microsomal membranes indicate that UNCL is a transmembrane protein. Hemagglutinin tagged UNCL was stably transfected into SaOS-2 osteosarcoma cells and exhibited a nuclear rim staining pattern which was retained following extraction with 1% Triton X-100, suggesting that UNCL localizes to the inner nuclear membrane. UNCL-HA was extractable in 350 mM NaCl, suggesting that UNCL is not associated with the nuclear matrix. Homopolymer RNA-binding assays performed on in vitro translated UNCL protein and 'structural modeling by homology' suggest that UNCL binds RNA via an amino-terminal RNA Recognition-like Motif. Since unc-50 is required for expression of assembled muscle-type nicotinic receptors in the nematode we investigated whether UNCL had a similar function for mammalian nicotinic receptors. When UNCL was co-expressed with neural nicotinic receptors in Xenopus oocytes or COS cells it increased expression of functional cell surface receptors up to 1. 6-fold. We conclude that UNCL is a novel inner nuclear membrane protein that associates with RNA and is involved in the cell-surface expression of neuronal nicotinic receptors. UNCL plays a broader role because UNCL homologues are present in two yeast and a plant species, none of which express nicotinic receptors and it is also found in tissues that lack nicotinic receptors.

Volatile anesthetics activate the human tandem pore domain baseline K+ channel KCNK5.

BACKGROUND: Previous studies have identified a volatile anesthetic-induced increase in baseline potassium permeability and concomitant neuronal inhibition. The emerging family of tandem pore domain potassium channels seems to function as baseline potassium channels in vivo. Therefore, we studied the effects of clinically used volatile anesthetics on a recently described member of this family. METHODS: A cDNA clone containing the coding sequence of KCNK5 was isolated from a human brain library. Expression of KCNK5 in the central nervous system was determined by Northern blot analysis and reverse-transcription polymerase chain reaction. Functional expression of the channel was achieved by injection of cRNA into Xenopus laevis oocytes. RESULTS: Expression of KCNK5 was detected in cerebral cortex, medulla, and spinal cord. When heterologously expressed in Xenopus oocytes, KCNK5 currents exhibited delayed activation, outward rectification, proton sensitivity, and modulation by protein kinase C. Clinical concentrations of volatile general anesthetics potentiated KCNK5 currents by 8-30%. CONCLUSION: Human KCNK5 is a tandem pore domain potassium channel exhibiting delayed activation and sensitivity to volatile anesthetics and may therefore have a role in suppressing cellular excitability during general anesthesia.

Multistep expression and assembly of neuronal nicotinic receptors is both host-cell- and receptor-subtype-dependent.

We tested the hypothesis that the folding, assembly and insertion of neuronal nicotinic receptors are critically dependent on the host cell line. We used recombinant adenoviruses encoding either the rat alpha7, alpha4 or beta2 subunits in which expression of the subunit is controlled by a tetracycline-dependent promoter to screen five cell lines (GH4C1, SH-EP1, CV1, SN-56, and CHO-CAR). All five lines do not express detectable nicotinic receptor but do express receptor for human adenovirus, and all expressed mRNA for alpha7, alpha4 and beta2 subunits when infected with viruses. Each cell line expressed varying levels of alpha4beta2 receptors that bound [3H]cytisine, but only the GH4C1 and SH-EP1 cell lines expressed either surface or internal alpha7 receptors that bound [125I]alpha-bungarotoxin ([125I]alpha-BGT). All five cell lines expressed a 60 kDa protein immunoblotted by anti-alpha7 antibodies when infected with the alpha7 virus, presumably representing unassembled alpha7 subunits. In addition, GH4C1 cells expressed over 10-fold more surface alpha7 receptor than SH-EP1 cells, even though the total alpha7 receptor in the two cell lines was similar. Sedimentation experiments indicate that SH-EP1 cells only partially assemble alpha7 receptors compared with GH4C1 cells and control alpha7 from rat brain. These data suggest that not only is surface alpha7 receptor expression a multistep process, but that each step may involve cell-specific assembly factors.

Pharmacological similarities between native brain and heterologously expressed alpha4beta2 nicotinic receptors.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat alpha4beta2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-alpha4 antibody mAb 299. 2 Immunodepletion with the anti-beta2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained beta2. 3 The association and dissociation rate constants for 30 pM +/-[3H]-epibatidine binding to alpha4beta2 receptors expressed in oocytes were 0.02+/-0.01 and 0.03+/-0.01 min-1 (+/-standard error, degrees of freedom=7 - 8) at 20 - 23 degrees C. 4 The Hill coefficients for +/-[3H]epibatidine binding to the native brain, alpha4beta2 receptors expressed in oocytes, and alpha4beta2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69 - 0.70 suggesting a heterogeneous receptor population. Fits of the +/-[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8 - 30 and 560 - 1,200 pM. The high-affinity sites comprised 73 - 74% of the native brain and oocyte alpha4beta2 receptor population, 85% of the CV-1 alpha4beta2 receptor population. 5 The expression of rat alpha4beta2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1. 0+/-0.2). Fits of the +/-[3H]-epibatidine binding data to a single-site model gave a KD of 40+/-3 pM. 6 DHbetaE (IC50=260-470 nM) and the novel nicotine analogue NDNI (IC50=7-10 microM) inhibited 30 pM+/-[3H]-epibatidine binding to the native brain and heterologously expressed alpha4beta2 receptors equally well. 7 The results show that alpha4beta2-containing nicotinic receptors in the rat brain and heterologously expressed rat alpha4beta2 receptors have similar affinities for +/-[3H]-epibatidine, DHbetaE, and NDNI.

Adenovirus-mediated overexpression of c-Jun and c-Fos induces intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 in human endothelial cells.

As distal targets and mediators of signal transduction pathways, activator protein-1 (AP-1), c-Jun, and c-Fos are among the primary regulators of genes involved in cell function, proliferation, and differentiation. By using adenovirus-mediated gene transfer, we show that overexpression of AP-1 proteins directly causes coinduction of gene expression of an adhesion molecule, intercellular adhesion molecule-1 (ICAM-1), and a chemokine, monocyte chemoattractant protein-1 (MCP-1), in human vascular endothelial cells (ECs). The AP-1-induced gene expression occurs through a mechanism independent of nuclear factor-kappaB. Because the induced expression of ICAM-1 and MCP-1 in ECs has been implicated in endothelial activation and a number of important vascular disorders, it is suggested that AP-1 activation may play an important role in the pathogeneses of inflammation, angiogenesis, and atherogenesis.

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family.

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.

Two mutations linked to nocturnal frontal lobe epilepsy cause use-dependent potentiation of the nicotinic ACh response.

1. We constructed rat homologues (S252F and +L264) of two human alpha4 nicotinic mutations - alpha4(S248F) and alpha4(777ins3) - that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat beta2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nM ACh pulses. Second, they delayed the rise times of the 5-15 nM (+L264) and 30 nM (S252F) ACh responses. Third, they reduced extracellular Ca2+-induced increases in the 30 microM ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured by antibody binding but the S252F mutation reduced the maximum ACh response. 4. The ACh concentration dependence of use-dependent potentiation and the delay in the rising phase of the mutant ACh response suggest that these effects are caused by a slow unblocking of the closed mutant receptors. Use-dependent potentiation of the mutant response during a series of high-frequency cholinergic inputs to the presynaptic terminal could trigger ADNFLE seizures by suddenly increasing nicotinic-mediated transmitter release.

TOK1 is a volatile anesthetic stimulated K+ channel.

BACKGROUND: Volatile anesthetic agents can activate the S channel, a baseline potassium (K+) channel, of the marine mollusk Aplysia. To investigate whether cloned ion channels with electrophysiologic properties similar to the S channel (potassium selectivity, outward rectification, and activation independent of voltage) also are modulated by volatile anesthetic agents, the authors expressed the cloned yeast ion channel TOK1 (tandem pore domain, outwardly rectifying K+ channel) in Xenopus oocytes and studied its sensitivity to volatile agents. METHODS: Standard two-electrode voltage and patch clamp recording methods were used to study TOK1 channels expressed in Xenopus oocytes. RESULTS: Studies with two-electrode voltage clamp at room temperature showed that halothane, isoflurane, and desflurane increased TOK1 outward currents by 48-65% in barium Frog Ringer's perfusate. The concentrations at which 50% potentiation occurred (EC50 values) were in the range of 768-814 microM (0.016-0.044 atm) and had a rank order of potency in atm in which halothane > isoflurane > desflurane. The potentiation of TOK1 by volatile anesthetic agents was rapid and reversible (onset and offset, 1-20 s). In contrast, the nonanesthetic 1,2-dichlorohexafluorocyclobutane did not potentiate TOK1 currents in concentrations up to five times the MAC value predicted by the Meyer-Overton hypothesis based on oil/gas partition coefficients. Single TOK1 channel currents were recorded from excised outside-out patches. The single channel open probability increased as much as twofold in the presence of isoflurane and rapidly returned to the baseline values on washout. Volatile anesthetic agents did not alter the TOK1 single channel current-voltage (I-V) relationship, however, suggesting that the site of action does not affect the permeation pathway of the channel. CONCLUSION: TOK1 is a potassium channel that is stimulated by volatile anesthetic agents. The concentrations over which potentiation occurred (EC50 values) were higher than those commonly used in clinical practice (approximately twice MAC).

An open rectifier potassium channel with two pore domains in tandem cloned from rat cerebellum.

Tandem pore domain K+ channels represent a new family of ion channels involved in the control of background membrane conductances. We report the structural and functional properties of a TWIK-related acid-sensitive K+ channel (rTASK), a new member of this family cloned from rat cerebellum. The salient features of the primary amino acid sequence include four putative transmembrane domains and, unlike other cloned tandem pore domain channels, a PDZ (postsynaptic density protein, disk-large, zo-1) binding sequence at the C terminal. rTASK has distant overall homology to a putative Caenorhabditis elegans K+ channel and to the mammalian clones TREK-1 and TWIK-1. rTASK expression is most abundant in rat heart, lung, and brain. When exogenously expressed in Xenopus oocytes, rTASK currents activate instantaneously, are noninactivating, and are not gated by voltage. Because rTASK currents satisfy the Goldman-Hodgkin-Katz current equation for an open channel, rTASK can be classified an open rectifier. Activation of protein kinase A produces inhibition of rTASK, whereas activation of protein kinase C has no effect. rTASK currents were inhibited by extracellular acidity. rTASK currents also were inhibited by Zn2+ (IC50 = 175 microM), the local anesthetic bupivacaine (IC50 = 68 microM), and the anti-convulsant phenytoin ( approximately 50% inhibition at 200 microM). By demonstrating open rectification and open probability independent of voltage, we have established that rTASK is a baseline potassium channel.

Detection of mycoplasma infection of mammalian cells.

Mycoplasma infection was detected in cultures of COS cells with a novel, simple assay that detects the conversion of arginine to citrulline by the enzyme, arginine deiminase, specific to all species of mycoplasma. Transfection of COS cells was inhibited in mycoplasma-infected cells, a phenomenon that was readily reversed by treatment with a mycoplasma removal agent. Cultures of cells used for transfection should be regularly monitored for evidence of mycoplasma by assay of arginine deiminase activity or by other methods.

Formation of oligomers containing the beta3 and beta4 subunits of the rat nicotinic receptor.

The role of the beta3 and beta4 subunits of the nicotinic acetylcholine receptor in brain is still unclear. We investigated nicotinic receptor structure with antibodies directed against unique regions of the beta3 and beta4 subunits of the rat nicotinic acetylcholine receptor. Anti-beta4 detected a single band of 66 kDa in most regions of the brain that was strongest in striatum and cerebellum. The 60 kDa beta3 subunit was detected primarily in striatum and cerebellum, and faintly in hippocampus. Immunoprecipitation experiments established that the two subunits were coassembled in the cerebellum along with the beta2 subunit. Antibodies against the alpha4, beta2, beta3, and beta4 subunits immunoprecipitated approximately 75% of the bungarotoxin-insensitive nicotinic receptor from cerebellar extracts as determined by nicotine-dependent acetylcholine binding. Transfection of COS cells with cDNAs for these four subunits induced expression of a high affinity nicotinic receptor. Omission of only a single subunit from the transfection affected either the Bmax or the apparent KD of the receptor. Our data suggest that the beta3 subunit functions as a structural entity that links a relatively unstable alpha4beta2 heterodimer to a more stable alpha4beta4 heterodimer. The agonist-binding site formed by alpha4beta2 has a much greater affinity than does that formed by alpha4beta4. In this respect, nicotinic receptors that contain the beta3 subunit are structurally homologous to the muscle nicotinic receptor.