Naturally expressed nicotinic acetylcholine receptors composed of ␣4 and ␤2 subunits (␣4␤2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human ␣4␤2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human ␣4 and ␤2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed ␣4␤2-nAChR engage in high-affinity, specific binding of Rbϩ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-␤-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human ␣4␤2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human ␣4␤2-nAChR.

Amyloid-␤ (A␤) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human ␣4 and ␤2 subunits (h␣4␤2-nAChR). However, the relationships between these phenomena are uncertain, and effects of A␤ on h␣4␤2-nAChR function have not been investigated in detail. We first confirmed expression of h␣4 and h␤2 subunits as messenger RNA in transfected, human SH-EP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed ␣4 and ␤2 subunit protein expression and coassembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-h␣4␤2 cells of functional h␣4␤2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by A␤ 1-42 in a dosedependent manner. Functional inhibition was selective for A␤ 1-42 compared with the functionally inactive, control peptide A␤ 40 -1 . A␤ 1-42 -mediated inhibition of h␣4␤2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5-yl thiophosphate failed to prevent A␤ 1-42 -induced inhibition, suggesting that down-regulation of h␣4␤2-nAChR function by A␤ 1-42 is not mediated by nAChR internalization. Sensitivity to A␤ 1-42 antagonism at 1 nM was evident for h␣4␤2-nAChRs, but not for heterologously expressed human ␣7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nM A␤ 1-42 , with the magnitude of functional block being higher for 100 nM A␤ 1-42 acting on h␣7-nAChRs. These findings suggest that h␣4␤2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for A␤ neurotoxicity in AD.Alzheimer's disease (AD) 1 is a progressive, neurodegenerative disorder manifest as a severe impairment of learning and memory. Pathophysiological hallmarks of AD include extracellular deposits of ␤-amyloid peptide (A␤) in senile plaques, formation of intraneuronal neurofibrillary tangles, and cholinergic neuron death (1). Although the precise mechanisms of AD pathogenesis are only partially understood, it is now widely accepted that the accumulation and aggregation of A␤ 1-42 plays a key role in the disease (2). Evidence has indicated an interaction between A␤ and the cholinergic system (3). For example, very low concentrations (pico to nanomolar) of A␤ can directly induce cholinergic hypofunction (4 -6). It has been reported that solubilized A␤ inhibits several steps of acetylcholine synthesis and release (4, 7), inhibits cholinergic enzyme activity (6), impairs cholinergic metabolism and neurotransmission (8 -10), and depresses hippocampal synaptic function (11).Recent evidence suggests possible roles for nicotinic acetylcholine receptors (nAChRs) as central targets for A␤-induced neurotoxicity manifest as...

Temporal filtering is a fundamental operation of nervous systems. In peripheral sensory systems, the temporal pattern of spiking activity can encode various stimulus qualities, and temporal filtering allows postsynaptic neurons to detect behaviorally-relevant stimulus features from these spike trains. Intrinsic excitability, short-term synaptic plasticity, and voltage-dependent dendritic conductances have all been identified as mechanisms that can establish temporal filtering behavior in single neurons. Here we show that synaptic integration of temporally-summating excitation and inhibition can establish diverse temporal filters of presynaptic input. Mormyrid electric fish communicate by varying the intervals between electric organ discharges. The timing of each discharge is coded by peripheral receptors into precisely-timed spikes. Within the midbrain posterior exterolateral nucleus, temporal filtering by individual neurons results in selective responses to a particular range of presynaptic interspike intervals. These neurons are diverse in their temporal filtering properties, reflecting the wide range of intervals that must be detected during natural communication behavior. By manipulating presynaptic spike timing with high temporal resolution, we demonstrate that tuning to behaviorally-relevant patterns of presynaptic input is similar in vivo and in vitro. We reveal that GABAergic inhibition plays a critical role in establishing different temporal filtering properties. Further, our results demonstrate that temporal summation of excitation and inhibition establishes selective responses to high and low rates of synaptic input, respectively. Simple models of synaptic integration reveal that variation in these two competing influences provides a basic mechanism for generating diverse temporal filters of synaptic input.

Dopamine (DA) neurons located in the mammalian midbrain have been generally implicated in reward and drug reinforcement and more specifically in nicotine dependence. However, roles played by nicotinic acetylcholine receptors, including those composed of ␣7-subunits [␣7-nicotinic acetylcholine receptors (nAChRs)], in modulation of DA signaling and in nicotine dependence are not clearly understood. Although midbrain slice recording has been used previously to identify functional ␣7-nAChRs, these preparations are not optimally designed for extremely rapid and reproducible drug application, and rapidly desensitized, ␣7-nAChRmediated currents may have been underestimated or not detected. Here, we use patch-clamp, whole-cell current recordings from single neurons acutely dissociated from midbrain nuclei and having features of DA neurons to characterize acetylcholineinduced, inward currents that rapidly activate and desensitize, are mimicked by the ␣7-nAChR-selective agonist, choline, blocked by the ␣7-nAChR-selective antagonists, methyllycaconitine and ␣-bungarotoxin, and are similar to those of heterologously expressed, human ␣7-nAChRs. We also use reverse transcriptasepolymerase chain reaction, in situ hybridization, and immunocytochemical staining to demonstrate nAChR ␣7 subunit gene expression as message and protein in the rat substantia nigra pars compacta and ventral tegmental area. Expression of ␣7 subunit message and of ␣7-nAChR-mediated responses is developmentally regulated, with both being absent in samples taken from rats at postnatal day 7, but later becoming present and increasing over the next 2 weeks. Collectively, this electrophysiological, pharmacological, and molecular evidence indicates that nAChR ␣7 subunits and functional ␣7-nAChRs are expressed somatodendritically by midbrain DA neurons, where they may play important physiological roles and contribute to nicotine reinforcement and dependence.

We examined a 7 b 2 -nicotinic acetylcholine receptor (a 7 b 2 -nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric a 7 -nAChRs and a 7 b 2 -nAChRs. a-Bungarotoxin affinity purification or immunoprecipitation with anti-a 7 subunit antibodies (Abs) was used to isolate nAChRs containing a 7 subunits from mouse or human brain samples. a 7 b 2 -nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using b 2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (a 7 ) 5 -nAChRs or for homomeric a 7 -nAChRs constituted from unlinked a 7 subunits. Pharmacological profiles were similar for (a 7 ) 5 -, (a 7 ) 4 (b 2 ) 1 -, and (a 7 ) 3 (b 2 ) 2 -nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at a 7 b 2 -versus a 7 -nAChRs. This study represents the first direct confirmation of a 7 b 2 -nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of a 7 b 2 -nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that a 7 b 2 -nAChR subunit isoforms with different a 7 /b 2 subunit ratios have similar pharmacological profiles to each other and to a 7 homopentameric nAChRs. This supports the hypothesis that a 7 b 2 -nAChR agonist activation predominantly or entirely reflects binding to a 7 /a 7 subunit interface sites.

␣7-Nicotinic acetylcholine receptors (␣7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human ␣7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human ␣7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional ␣7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human ␣7-nAChRs. Currentvoltage relationships show inward rectification for agonist-induced currents. Human ␣7-nAChRs exhibit some sensitivity to ␣7-nAChR antagonists ␣-bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and ␣7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of ␣7-nAChR function. Whole-cell current peak amplitudes and halftimes for inactivation of ␣7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca 2ϩ , suggestive of high Ca 2ϩ permeability of the ␣7-nAChR channel. Thus, heterologously expressed human ␣7-nAChR in mammalian cells have properties of native ␣7-nAChR or of ␣7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of ␣7-nAChR function.

This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and β4-subunit-containing human nicotinic acetylcholine receptors (α3β4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3β4*-nAChR isoforms (α3β4)β4-, (α3β4)α3-, (α3β4)α5(398D)-, and (α3β4)α5(398N)-nAChR in oocytes. In the presence or absence of lynx1, α3β4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3β4)β4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3β4)α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3β4)α5-nAChR function that was mediated by lynx1. No effects were observed when α3β4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3β4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3β4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.

Background:The naturally occurring ␣5(D398N) variant alters smoking behavior, but functional differences have not been detected between ␣3␤4␣5 nAChR harboring these variants. Results: ACh-induced ␣3␤4␣5 nAChR function is lower when ␣5(Asn-398) substitutes for ␣5(Asp-398). Conclusion:The ␣5 variant-induced change in ␣3␤4␣5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. Significance: ␣3␤4␣5 nAChR function may be a useful target for smoking cessation pharmacotherapies.Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR ␣5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of ␣3␤4␣5 nAChR in Xenopus oocytes. ␣5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common ␣5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked ␣3, ␤4, and ␣5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric ␣3␤4* nAChRs. ␣5 subunit incorporation reduces ␣3␤4* nAChR function after coinjection with unlinked ␣3 and ␤4 subunits but increases that of ␣3␤4␣5 versus ␣3␤4-only concatemers. ␣5 subunit incorporation into ␣3␤4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of ␣5 subunits, free ␣3 and ␤4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit ␣3␤4-only subtypes are dissimilar both to each other and to those of ␣3␤4␣5 nAChR. The ␣5 variant-induced change in ␣3␤4␣5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism. Nicotinic acetylcholine receptors (nAChR)2 are prototypical members of the ligand-gated ion channel superfamily of neurotransmitter receptors. nAChR exist as a diverse family of molecules composed of different pentameric combinations of homologous subunits derived from at least 17 genes (␣1-␣10, ␤1-␤4, ␥, ␦, ⑀). The properties of nAChR are determined by their subunit composition, giving rise to multiple subtypes with a range of overlapping pharmacological and biophysical properties (1). It also has become apparent that different stoichiometries of the same subunits can produce subtypes with distinctly different characteristics, a phenomenon observed in both heterologous and natural expression systems (1-5).Recently, genome-wide association studies have indicated that single-nucleotide polymorphisms (SNPs) within nAChR subunits can substantially affect nAChR-mediated smoking behavior in humans. Most prominent among these single-nucleotide polymorphisms have been those located in the CHRNA5/CHRNA3/CHRNB4 locus, located on chromosome 15q25, which encodes the ␣5, ␣3 and ␤4 subunits of nicotinic recept...