Patricia W. Lamb

Rat nicotinic ACh receptor α7 and β2 subunits co‐assemble to form functional heteromeric nicotinic receptor channels

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7‐containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co‐assembly of the α7 and β2 subunits might occur, these subunits were co‐expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two‐electrode voltage clamp. Co‐expression of the β2 subunit, both wild‐type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near‐full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC50 values for all three agonists significantly increased when the β2 subunit was co‐expressed with the α7 subunit. Co‐expression with the β2 subunit did not result in any significant change in the current‐voltage curve. Biochemical evidence for the co‐assembly of the α7 and β2 subunits was obtained by co‐immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co‐assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co‐assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system.

Mechanisms of arsenic-induced cell transformation

Arsenic is a well-established carcinogen in humans, but there is little evidence for its carcinogenicity in animals and it is inactive as an initiator or tumor promoter in two-stage models of carcinogenicity in mice. Studies with cells in culture have provided some possible mechanisms by which arsenic and arsenical compounds may exert a carcinogenic activity. Sodium arsenite and sodium arsenate were observed to induce morphological transformation of Syrian hamster embryo cells in a dose-dependent manner. The trivalent sodium arsenite was greater than tenfold more potent than the pentavalent sodium arsenate. The compounds also exhibited toxicity; however, transformation was observed at nontoxic as well as toxic doses. At low doses, enhanced colony forming efficiency of the cells was observed. To understand the mechanism of arsenic-induced transformation, the genetic effects of the two arsenicals were examined over the same doses that induced transformation. No arsenic-induced gene mutations were detected at two genetic loci. However, cell transformation and cytogenetic effects, including endoreduplication, chromosome aberrations, and sister chromatid exchanges, were induced by the arsenicals with similar dose responses. These results support a possible role for chromosomal changes in arsenic-induced transformation. The two arsenic salts also induced another form of mutation-gene amplification. Both sodium arsenite and sodium arsenate induced a high frequency of methotrexate-resistant 3T6 cells, which were shown to have amplified copies of the dihydrofolate reductase gene. The ability of arsenic to induce gene amplification may relate to its carcinogenic effects in humans since amplification of oncogenes is observed in many human tumors. Epidemiological studies suggest that arsenic acts late in the carcinogenic process in humans and oncogene amplification correlates with the progression of tumors. These observations lead us to propose the hypothesis that arsenic acts as a tumor progressor, rather than a tumor initiator or tumor promoter. Arsenic-induced chromosome aberrations or gene amplifications may play a role in tumor progression.

Inhibition of neuronal nicotinic acetylcholine receptor channels expressed in Xenopus oocytes by β-amyloid1–42 peptide

Neuronal nicotinic acetylcholine receptors (nAChRs) are involved in a variety of physiological processes, including cognition and development. Dysfunctions in nAChRs have been linked to Alzheimer’s disease (AD), a human neurological disorder that is the leading cause of dementia. AD is characterized by an increasing loss of cognitive function, nAChRs, cholinergic neurons, and choline acetyltransferase activity. A major hallmark of AD is the presence of extracellular neuritic plaques composed of the β-amyloid (Aβ1–42) peptide; however, the link between Aβ1–42 and the loss of cognitive function has not been established. Many groups have shown direct interactions between Aβ1–42 and nAChR function, however, with differing results. For example, in rat hippocampal CA1 interneurons in slices, we found that Aβ1–42 inhibits nAChR channels directly, and non-α7 receptors were more sensitive to block than α7 receptors. However, some groups have found that α7 subtypes were potently blocked by Aβ1–42, whereas other groups reported that Aβ1–42 can activate nAChRs (i.e., both α7 and non-α7 subtypes). To further investigate the link between nAChR function and Aβ1–42, we expressed various subtypes of nAChRs in Xenopus oocytes (e.g., α4β2, α2β2, α4α5β2, and α7) and found that Aβ1–42 blocked these various non-α7 nAChRs, without any effect on α7 nAChRs. Furthermore, none of these channels was activated by Aβ1–42. The relative block by Aβ1–42 was dependent on the subunit makeup and apparent stoichiometry of these receptors. These data further support our previous findings that Aβ1–42 directly and preferentially inhibits non-α7 nAChRs.

Cholinergic Coordination of Presynaptic and Postsynaptic Activity Induces Timing-Dependent Hippocampal Synaptic Plasticity

Correlated presynaptic and postsynaptic activity is the key factor in inducing Hebbian plasticity and memory. However, little is known about the physiological events that could mediate such coordination. Correlated cholinergic input induces spike timing-dependent plasticity-like hippocampal synaptic plasticity. Cholinergic receptors are localized to both presynaptic and postsynaptic glutamatergic sites and thus have the potential to coordinate presynaptic and postsynaptic activity to induce plasticity. By directly monitoring presynaptic and postsynaptic activities with genetically encoded calcium indicators in mouse septohippocampal cocultures, we found interactive but independent presynaptic and postsynaptic modulations in the cholinergic-dependent synaptic plasticity. Neither presynaptic nor postsynaptic modulation alone is sufficient, but instead a coordinated modulation at both sites is required to induce the plasticity. Therefore, we propose that correlated cholinergic input can coordinate presynaptic and postsynaptic activities to induce timing-dependent synaptic plasticity, providing a novel mechanism by which neuromodulators precisely modulate network activity and plasticity with high efficiency and temporal precision.

Characterization of a nicotine-sensitive neuronal population in rat entorhinal cortex

The entorhinal cortex (EC) is a part of the hippocampal complex that is essential to learning and memory, and nicotine affects memory by activating nicotinic acetylcholine receptors (nAChRs) in the hippocampal complex. However, it is not clear what types of neurons in the EC are sensitive to nicotine and whether they play a role in nicotine-induced memory functions. Here, we have used voltage-sensitive dye imaging methods to locate the neuronal populations responsive to nicotine in entorhino-hippocampal slices and to clarify which nAChR subtypes are involved. In combination with patch-clamp methods, we found that a concentration of nicotine comparable to exposure during smoking depolarized neurons in layer VI of the EC (ECVI) by acting through the non-α7 subtype of nAChRs. Neurons in the subiculum (Sb; close to the deep EC layers) also contain nicotine-sensitive neurons, and it is known that Sb neurons project to the ECVI. When we recorded evoked EPSCs (eEPSCs) from ECVI neurons while stimulating the Sb near the CA1 region, a low dose of nicotine not only enhanced synaptic transmission (by increasing eEPSC amplitude) but also enhanced plasticity by converting tetanus stimulation-induced short-term potentiation to long-term potentiation; nicotine enhanced synaptic transmission and plasticity of ECVI synapses by acting on both the α7 and non-α7 subtypes of nAChRs. Our data suggest that ECVI neurons are important regulators of hippocampal function and plasticity during smoking.

Structural Determinates for Apolipoprotein E-Derived Peptide Interaction with the α7 Nicotinic Acetylcholine Receptor

Neuronal nicotinic acetylcholine receptor (nAChR) signaling has been implicated in a variety of normal central nervous system (CNS) functions as well as an array of neuropathologies. Previous studies have demonstrated both neurotoxic and neuroprotective actions of peptides derived from apolipoprotein E (apoE). It has been discovered that apoE-derived peptides inhibit native and recombinant α7-containing nAChRs, indicating a direct interaction between apoE peptides and nAChRs. To probe the structure/function interaction between α7 nAChRs and the apoE peptide apoE141–148, experiments were conducted in Xenopus laevis oocytes expressing wild-type and mutated nAChRs. Mutation of Trp55 to alanine blocks apoE peptide-induced inhibition of acetylcholine (ACh)-mediated α7 nAChR responses. Additional mutations at Trp55 suggest that hydrophobic interactions between the receptor and apoE141–148 are essential for inhibition of α7 nAChR function. A mutated apoE peptide also demonstrated decreased inhibition at α7-W55A nAChRs as well as activity-dependent inhibition of both wild-type α7 nAChRs and α7-W55A receptors. Finally, a three-dimensional model of the α7 nAChR was developed based on the recently refined Torpedo marmorata nACh receptor. A structural model is proposed for the binding of apoE141–148 to the α7 nAChR where the peptide binds at the interface between two subunits, near the ACh binding site. Similar to the functional data, the computational docking suggests the importance of hydrophobic interactions between the α7 nAChR and the apoE peptide for inhibition of receptor function. The current data suggest a mode for apoE peptide binding that directly blocks α7 nAChR activity and consequently may disrupt nAChR signaling.

Identification and functional characterization of a novel acetylcholine-binding protein from the marine annelid Capitella teleta.

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.

Rapid desensitization of the rat α7 nAChR is facilitated by the presence of a proline residue in the outer β‐sheet

The rat α7 nicotinic acetylcholine receptor (nAChR) has a proline residue near the middle of the β9 strand. The replacement of this proline residue at position 180 (P180) by either threonine (α7‐P180T) or serine (α7‐P180S) slowed the onset of desensitization dramatically, with half‐times of ∼930 and 700 ms, respectively, compared to 90 ms for the wild‐type receptor. To investigate the importance of the hydroxyl group on the position 180 side‐chains, the mutant receptors α7‐P180Y and α7‐P180F were studied and showed half‐times of desensitization of 650 and 160 ms, respectively. While a position 180 side‐chain OH group may contribute to the slow desensitization rates, α7‐P180S and α7‐P180V resulted in receptors with similar desensitization rates, suggesting that increased backbone to backbone H bonding expected in the absence of proline at position 180 would likely exert a great effect on desensitization. Single channel recordings indicated that for the α7‐P180T receptor there was a significantly reduced closed time without any change in single channel conductance (as compared to wild‐type). Kinetic simulations indicated that all changes observed for the mutant channel behaviour were reproduced by decreasing the rate of desensitization, and increasing the microscopic affinity to resting receptors. Molecular dynamics (MD) simulations on a homology model were used to provide insight into likely H bond interactions within the outer β‐sheet that occur when the P180 residue is mutated. All mutations analysed increased about twofold the predicted number of H bonds between the residue at position 180 and the backbone of the β10 strand. Moreover, the α7‐P180T and α7‐P180S mutations also formed some intrastrand H bonds along the β9 strand, although H bonding of the OH groups of the threonine or serine side‐chains was predicted to be infrequent. Our results indicate that rapid desensitization of the wild‐type rat α7 nAChR is facilitated by the presence of the proline residue within the β9 strand.

Expression of KAI1 in paraffin‐embedded normal, hyperplastic and neoplastic prostate and prostate carcinoma cell lines

Expression of KAI1, a tumor metastasis suppressor gene, was studied with different fixatives in frozen and paraffin‐embedded sections of human and rat prostate carcinoma cell lines and human prostate lesions by Immunohisto‐chemistry. Immunoreactivity of the membrane antigen in cell lines was associated with known expression levels in these lines and the fixative used. Formalin and paraformaldehyde helped maintain the immunoreactivity of cells. In human prostate, frozen sections revealed diffuse reactivity of the antigen in normal and neoplastic tissues while paraffin‐embedded tissues usually showed focal reactivity, although more than 50% of cases with normal epithelium and adenocarcinomas were reactive. In some cases, pretreatment with trypsln enhanced immunoreactivity. Benign prostatic hyperplasia (BPH) showed the most intense diffuse immunoreactivity, which suggested enhanced expression. Prostatic intraepithelial neoplasia (PIN) also often expressed high levels of KAI1. Three of five metastases were reactive but two primaries and their metastases were not. Lymphocytes in primary carcinomas and lymphocytes and germinal center cells in lymph nodes were immunoreactive, while adjacent primary or metastatic prostate adenocarcinoma epithelium was not immunoreactive. Although paraffin‐embedded human tissues were not optimal for determining levels of expression of KAI1, they did show immunoreactivity that could have prognostic value and showed the specific cytoplasmlc localization of the protein in cells.

Research tool: validation of floxed α7 nicotinic acetylcholine receptor conditional knockout mice using in vitro and in vivo approaches

Currently, no animal model exists in which selective deletion of α7 nAChRs from a specific cell type or tissue is possible through genetic manipulation. We have generated mice in which the fourth exon of the α7 nAChR gene (Chrna7) is flanked by loxP sites (B6‐Chrna7LBDEx4007Ehs) which we refer to as floxed α7 nAChR conditional knockout or α7nAChRflox. In the brain, α7 nAChRs are expressed both on neurons and astrocytes. Proper Cre recombinase excision of the targeted gene was verified with a combination of in vitro and in vivo approaches including demonstrating that α7 nAChR binding sites were absent on glial fibrillary acidic protein (GFAP)‐positive astrocytes in hippocampal slices obtained from offspring of α7nAChRflox and GFAP‐Cre mating. This study validates the chosen approach for deletion of α7 nAChR gene expression from any tissue for which appropriate Cre constructs are available, thus providing the nicotinic receptor research field with new technical capabilities.