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【关键词】 Novel
The 7 nicotinic acetylcholine receptor subunit (CHRNA7) gene harbors a high degree of polymorphism. In this study, we found a novel variant (1267 G to A) in exon 10 of the CHRNA7 gene in a Japanese population. This variant results in glycine-to-serine substitution at position 423 (G423S) located in the large cytoplasmic loop of the protein. To clarify the possibility that the G423S mutation alters the pharmacological properties of 7 receptors, acetylcholine (ACh)-elicited current through 7-G423S mutant receptors expressed in Xenopus laevis oocytes was measured using the two-electrode voltage-clamp technique. We found that the current elicited by ACh (1 mM, 5 s) through 7-G423S receptors, but not through 7 receptors, was significantly decreased by treatment with a protein kinase C activator, phorbol-12-myristate-13-acetate (PMA, 10-30 nM). In addition, PMA (10 nM) selectively promoted a progressive decrease in 7-G423S current induced by repetitive application of ACh pulses (1 mM, 0.1 s, 0.17-0.33 Hz) compared with 7 current. PMA also enhanced the inactivation of 7-G423S mutant receptors induced by a prolonged application of choline (30 µM) without affecting 7 receptor responses. Western blot analysis showed that the treatment with PMA (30 nM) increased the serine phosphorylation level of the 7-G423S mutant receptors but not that of the wild-type receptors. These findings demonstrate that the G423S mutation promotes receptor desensitization by a protein kinase C-dependent mechanism. Thus, we provide the first evidence that a variant in the human CHRNA7 gene alters the function of 7 nicotinic receptors.
Neuronal nicotinic acetylcholine receptors are ligand-gated ion channels that are widely distributed in the mammalian brain (Changeux et al., 1998; Paterson and Nordberg, 2000; Gotti and Clementi, 2004). The 7 subunit-containing receptor is one of the predominant subtypes of brain nicotinic receptors, although its distribution in the human brain has not been characterized completely. Based on the finding that 7 receptors are more diffusely distributed in the monkey brain than in the rodent brain, 7 receptors seem to play important roles in primate brain functions (Paterson and Nordberg, 2000; Gotti and Clementi, 2004). The highest expression level is detected in regions that are involved in the processing of sensory information, such as the hippocampus and thalamus (reticular, geniculate) (Gotti and Clementi, 2004). In particular, the 7 receptor-mediated release of GABA from hippocampal interneurons is responsible for the filtering or gating of auditory information passing through the hippocampus (Leonard et al., 1996).
Sensory gating deficit expressed by a poor inhibition of the P50 auditory event-evoked response is an early clinical symptom of schizophrenia and is characterized by a decrease in normal brain ability to inhibit responses to unimportant stimuli (Freedman et al., 2003). This failure of inhibition is associated with poorly sustained attention (Martin et al., 2004). A series of clinical and pathophysiological studies have indicated that reduction in 7 nicotinic receptor expression and function is involved in the sensory gating deficit (Freedman et al., 2000; Weiland et al., 2000). In addition, impaired auditory sensory gating has been linked to the human 7 nicotinic receptor subunit (CHRNA7) gene on the chromosome 15q13-q14 locus (Freedman et al., 1997), and many studies have linked schizophrenia to a locus at or near the CHRNA7 gene (Martin et al., 2004). Leonard et al. (2002) have searched for single-nucleotide polymorphisms (SNPs) in the CHRNA7 gene promoter region and found that several SNPs are more prevalent in patients with schizophrenia than in control subjects. Furthermore, these SNPs are associated with both decreased promoter activity in vitro and failure to inhibit the P50 auditory-evoked response in humans. This finding, however, is not necessarily consistent with studies of other populations (Houy et al., 2004; Li et al., 2004).
Influence of single amino acid substitution in the 7 nicotinic receptors has been intensively studied using site-directed mutagenesis (Placzek et al., 2005; McLaughlin et al., 2006). Yet, little is known about the polymorphisms located in the coding region of the human CHRNA7 gene, because the existence of a duplicated 7 receptor subunit (CHRFAM7A) gene, in which exons 5 to 10 overlap with those in the CHRNA7 gene (Gault et al., 1998), complicates polymorphism screening. Gault et al. (2003) have identified three nonsynonymous variants in the human CHRNA7 gene by a reverse transcriptase-polymerase chain reaction (PCR)-based strategy for variant mapping. However, the impact of the amino acid substitution by these variants on receptor function has not been studied. Thus, the significance of variants in the human CHRNA7 gene has long been unclarified.
In this study, we analyzed the CHRNA7 gene in a Japanese population and found a novel SNP that causes a single amino acid substitution (G423S mutation) in the 7 receptor subunit. Furthermore, we investigated the pharmacological and biochemical properties of the mutant receptors ectopically expressed in Xenopus laevis oocytes. To our knowledge, this is the first demonstration that a variant in the human CHRNA7 gene alters the function of the 7 nicotinic receptors.
Subjects. We recruited 100 unrelated Japanese subjects with schizophrenia that was diagnosed on the basis of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) and 100 Japanese control subjects at Toyama University Hospital and Hokuriku National Hospital (Toyama, Japan). The patient group comprised 58 men and 42 women (mean age ± S.D., 35 ± 11 years), whereas the control group comprised 46 men and 54 women (mean age ± S.D., 32 ± 15 years). The mean total score of the Brief Psychiatric Rating Scale, an estimated rating of psychiatric symptoms, of patients with schizophrenia included in this study was 41 ± 1. Before collecting blood samples, the purpose and risks of this study were precisely explained both orally and in writing, and written informed consent was obtained from all the subjects. The protocol of this study is in accordance with the Declaration of Helsinki and was approved by the ethics committees of the University of Toyama and Hokuriku National Hospital.
Materials. Calphostin C, methyllycaconitine (MLA), phorbol-12-myristate-13-acetate (PMA), and 4-PMA were purchased from Sigma (St. Louis, MO). Calphostin C, PMA, and 4-PMA were dissolved in dimethyl sulfoxide (DMSO; Sigma) and diluted in low-Ca2+ Ringer's solution (see below). Other standard reagents were purchased from Sigma or Wako Pure Chemicals Industries (Osaka, Japan).
Genetic Analysis. Blood samples were obtained from all the subjects, and genomic DNA was extracted from peripheral leukocytes using a Wizard Genomic DNA purification kit (Promega, Madison, WI). PCR-single-strand conformation polymorphism (SSCP) analysis and sequencing were performed to identify SNPs in the CHRNA7 and CHRFAM7A genes as described previously (Ishihara et al., 2003). Table 1 shows biotinylated primers used for amplification of the 5'-untranslated region, exons, and exon-intron boundaries of the CHRNA7 gene and for amplification of gene sequences encoding exons 5 to 10, in which 5'-end sequences are specific for either the CHRNA7 or CHRFAM7A gene. The primers were designed on the basis of sequences available in GenBank (accession number NT_010194). For the amplification of exons 1, 2, 3, and 4 of the CHRNA7 gene, PCR was carried out using a KOD-Plus DNA polymerase kit (Toyobo, Osaka, Japan) under the following conditions: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 98°C for 10 s, and annealing and elongation at 68°C for 20 to 40 s. For the amplification of exons 5 to 10 of the CHRNA7 or CHRFAM7A gene, primary long-range PCR was performed using an LA Taq DNA polymerase kit (Takara Bio, Shiga, Japan) and specific primers under the following conditions: initial denaturation at 94°C for 3 min, 30 cycles of denaturation at 98°C for 20 s, annealing at 68°C for 18 min, and final elongation at 68°C for 20 min. The second PCR was performed using the diluted primary PCR product as the template and primers to amplify each exon. The PCR conditions were the same as those used to amplify exons 1 to 4. Thereafter, PCR products were subjected to SSCP analysis. The samples were heated to 95°C for 5 min and immediately cooled on ice until loading. Single-stranded fragments were separated by electrophoresis on Mutation Detection Enhancement gel (FMC Bioproducts, Rockland, ME) at 4°C and detected with an Imaging High-Chemilumi kit (Toyobo) according to the manufacturer's instructions. All SSCP variants were directly sequenced using a BigDye terminator kit and an ABI Prism 310 sequencer (Applied Biosystems, Foster City, CA).
TABLE 1 PCR primers for amplification of the human CHRNA7 gene
Plasmid Construction. The 7 open reading frame (ORF) fragment was amplified from the human brain cDNA library (human brain, cerebellum quick-clone cDNA; Clontech, Mountain View, CA) by PCR using a KOD-plus DNA polymerase kit with the following primers: the sense primer containing a BamHI restriction site (5'-CGCGGATCCCGCTGCAGCTCCGGGACTCAACATG-3') and the antisense primer containing an AgeI restriction site (5'-GGACCGGTCGCAAAGTCTTTGGACACGGCCTCCA-3'). The primers were designed on the basis of the sequence available in GenBank (accession number NM_000746). The 7 ORF fragment was cloned into the pGEM-T Easy vector (Promega) and then subcloned into the BamHI and NotI restriction sites of the pcDNA3.1+ vector (Invitrogen, Carlsbad, CA). The original stop codon was generated by site-directed mutagenesis using the KOD-plus DNA polymerase kit with the following paired primers: 5'-CCGTGTCCAAAGACTTTGCGTAAGGTCCAATCGAATTCCCGCGG-3', and 5'-CCGCGGGAATTCGATTGGACCTTACGCAAAGTCTTTGGACACGG-3'. 7-G423S cDNA was generated by site-directed mutagenesis with the paired primers 5'-GCACCTCCTGCACAGCGGGCAACCCCCCG-3' and 5'-CGGGGGGTTGCCCGCTGTGCAGGAGGTGC-3' on the 7 pcDNA-3.1+ vector. The expression vector of the enhanced green fluorescence protein (EGFP)-7 subunit chimera was constructed by inserting the 7 ORF fragment into the BamHI and AgeI restriction sites of the pEGFP-N1 vector (Clontech). To avoid the interference between the 7 receptor and the EGFP protein, nucleotides encoding a linker consisting of four glycine residues were further inserted between the nucleotide sequences of 7 ORF and EGFP by site-directed mutagenesis with the following paired primers: 5'-GGAGGAGGAGGAACGGTCGCCACCATGGTGAGCAAGGG-3' and 5'-CGCAAAGTCTTTGGACACGGCCTCCACGAAG-3'. The nucleotide sequences of the inserts were confirmed by sequencing.
Expression in X. laevis Oocytes. X. laevis oocytes expressing recombinant nicotinic receptors were prepared as described previously (Tsuneki et al., 2004). In brief, oocytes were surgically removed from X. laevis frogs under anesthesia and enzymatically isolated. Stage V to VI oocytes were selected and nanoinjected with 23 ng of cDNAs to the nucleus. Oocytes were incubated at 19°C in 100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvic acid, 1% bovine serum albumin, and 25 µg/ml gentamicin, pH 7.5, for 3 to 7 days before electrophysiological recording was carried out. Animal experiments were performed in accordance with the guidelines approved by the University of Toyama Animal Research Committee.
Electrophysiological Recording. 7 Nicotinic current evoked by acetylcholine (ACh) or choline was recorded as described previously (Tsuneki et al., 2004). An oocyte was placed in a tube-like chamber in which low-Ca2+ Ringer's solution (82.5 mM NaCl, 2.5 mM KCl, 0.5 mM CaCl2, 2 mM MgCl2, and 5 mM HEPES, pH 7.4) was perfused by gravity (15 ml/min) to minimize the activation of endogenous Ca2+-activated chloride channels in the oocytes. Atropine (1 µM) was also present in the solution to block endogenous muscarinic receptors. Current was recorded under a two-electrode voltage clamp at a holding potential of -60 mV using a GeneClamp 500 amplifier and pCLAMP7 software (Axon Instruments, Sunnyvale, CA). The sampling rate was 20 Hz. Electrodes contained 3 M KCl and had resistances of <2M. To apply the nicotinic agonist to the oocyte, the perfusion fluid was switched to a fluid containing the agonist, using a three-way Teflon solenoid valve (Parker Hannifin Corporation, General Valve, NJ) controlled by a PC computer with pCLAMP7 software. The duration of agonist application was 5 s, excluding short pulses (0.1 or 0.2 s) of ACh as described in the legends to Figs. 4 and 5. In the case of a 5-s application of the nicotinic agonist, a 3-min wash produced reproducible control current with no obvious desensitization. To avoid artifacts due to capacitive charging of the oocyte membrane, the current elicited every 3 min by the agonist was recorded until it became stable. A selective antagonist of the 7 receptors, MLA, was applied 6 min before measuring the current elicited by ACh coapplied with MLA. In experiments examining the effects of PMA and 4-PMA, the vehicle (DMSO) concentration was kept constant at 0.05% in the bath solution throughout the experiments. In addition, a 23-nl aliquot of calphostin C solution (200 nM, an irreversible inhibitor of protein kinase C) or 0.05% DMSO was injected with an autoinjector (Nanoject; Drummond Scientific Company, Broomall, PA) into oocytes expressing nicotinic receptors at least 30 min before measuring ACh-elicited current, as described previously (Gopalakrishna et al., 1992). The effect of long-term choline treatment on the 7 nicotinic receptors was examined by applying choline 6 min before measuring the current elicited by short pulses of ACh (1 mM, 0.2 s).
Fig. 4. PMA promotes desensitization of 7-G423S nicotinic receptors induced by repetitive ACh stimulation. Oocytes expressing 7 or 7-G423S receptors were voltage-clamped at -60 mV, and the current elicited every 3 s (at 0.33 Hz) or 6 s (at 0.17 Hz) by short pulses of ACh (1 mM, 0.1 s) was recorded. Thereafter, PMA (10 nM) was superfused in the bath for 6 min, then the responses to the repetitive pulses of ACh coapplied with PMA (10 nM) were recorded in the same oocyte. A, top, typical recordings of the 7 current elicited every 6 s by the ACh pulses (arrowheads) in the absence or presence of PMA (10 nM). Horizontal open bars indicate the period of perfusion with PMA. Bottom, plot of the peak amplitudes of 7 currents elicited by repetitive ACh pulses before (, 0.17 Hz; , 0.33 Hz) and during application of 10 nM PMA (, 0.17 Hz; , 0.33 Hz). Each response was normalized to the current elicited by the initial ACh pulse. B, top, typical recordings of 7-G423S receptor-mediated current elicited every 6 s by repetitive pulses of ACh in the absence or presence of PMA (10 nM). Bottom, plot of peak amplitudes of 7-G423S current elicited by ACh pulses before (, 0.17 Hz; , 0.33 Hz) and during application of 10 nM PMA (, 0.17 Hz; , 0.33 Hz). Values represent the mean ± S.E.M. of 10 to 11 separate experiments. *, P < 0.05 compared with responses in the absence of PMA at each frequency.
Fig. 5. PMA enhances the desensitization of 7-G423S nicotinic receptors induced by prolonged exposure to choline. Current elicited by short pulses of ACh (1 mM, 0.2 s) was recorded every 3 min in the voltage-clamp mode at -60 mV. A, curves showing normalized response in terms of ACh-elicited current through 7() and 7-G423S receptors () expressed in X. laevis oocytes against choline concentration. Oocytes were preincubated with choline for 6 min and then stimulated with ACh pulses. Each response was normalized to the current recorded before the application of choline in the same oocyte. Values represent the mean ± S.E.M. of five to seven separate experiments. B, time course of desensitization of 7() and 7-G423S receptors () induced by long-term exposure to choline (30 µM) in the presence of PMA (10 nM). Oocytes were preincubated with PMA and then treated with choline plus PMA for 6 min. Current elicited by ACh coapplied with PMA and choline in each oocyte was normalized to the current recorded before application of PMA (control responses) in the same oocyte. Values represent the mean ± S.E.M. of 10 separate experiments. *, P < 0.05 compared with the responses to ACh coapplied with PMA (in the absence of choline) at 0 min.
Concentration-response curves for agonists were fitted by the equation I = Imax/[1 + (EC50/A)nH], whereas concentration-inhibition curves for antagonists were fitted by the equation I = Imax - Imax/[1 + (IC50/An)nH], using Prism (GraphPad Software, Inc., San Diego, CA), in which I is the percentage amplitude, Imax is the maximal response normalized to the current evoked by 1 mM ACh or 10 mM choline (in the absence of the antagonist for inhibitory curves), A is the agonist concentration, An is the antagonist concentration, EC50 is the agonist concentration eliciting the half-maximum response, IC50 is the antagonist concentration eliciting the half-maximal negative response, and nH is the Hill coefficient. In the present analyses, Imax was constrained to 100% and Imin was constrained to 0%.
Immunoprecipitation and Western Blotting. After measuring the ACh (1 mM, 5 s)-elicited current in oocytes expressing EGFP-conjugated 7 nicotinic receptors in the presence of PMA, 4-PMA, or vehicle (0.05% DMSO) for 6 min, the oocytes were immediately homogenized in homogenizing buffer (50 mM Na2HPO4, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM benzamidine, 15 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride, 1 µM leupeptin, 100 µg/ml aprotinin, and 1 mM Na3VO4). Three oocyte homogenates were mixed in a tube, and membrane-rich fractions were collected by centrifugation (700g, 5 min, 4°C) and then solubilized by pipetting in lysis buffer (homogenizing buffer containing 15% SDS). The obtained cell lysates were incubated with an anti-EGFP antibody (Living Colors Full-Length A.v. polyclonal antibody; Clontech) for 18 h at 4°C and then centrifuged for 20 min 12,000g at 18°C, and the supernatant was collected to remove cellular debris and mixed with protein G Sepharose (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 2 h at 18°C. Sepharose resin was collected and then washed three times with lysis buffer. After mixing with Laemmli solution (0.01% bromphenol blue, 50 mM sodium phosphate, 50% glycerol, and 10% SDS) plus 7.5% (±)-dithiothreitol, proteins were denatured in boiling water for 5 min, separated by SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The PVDF membrane was blocked for 1 h with 5% nonfat milk in Tris-buffered saline with Tween 20 (10 mM Tris, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) and then incubated with an anti-phosphoserine antibody (QIAGEN, Valencia, CA) or an anti-EGFP antibody for 2 h at 37°C. The PVDF membrane was washed and incubated with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare) at 25°C for 1 h followed by chemiluminescence detection using enhanced chemiluminescence Western blotting detection reagents according to the manufacturer's instructions (GE Healthcare). The relative serine phosphorylation level of 7 nicotinic receptor subunit was calculated as the ratio of the intensity of the band corresponding to phosphorylated 7 to that of the band corresponding to total 7 in each sample.
Statistical Analysis. Data are represented as mean ± S.E.M. unless otherwise indicated. Nonparametric data were analyzed by Fisher's exact test. For parametric data, the significance of differences between two groups was assessed by Student's t test, and the significance of differences between multiple groups were assessed by one-way analysis of variance followed by Scheffé's multiple comparison test. Values of P less than 0.05 were considered to be significant.
Identification of Nonsynonymous Variant in Human CHRNA7 Gene. We performed polymorphism screening of the CHRNA7 gene in 100 patients with schizophrenia and 100 control subjects by PCR-SSCP and sequence analyses. In the 231-bp core promoter region of the CHRNA7 gene, only one SNP was identified at 194 bp upstream from the ATG start codon (-194 G to C; Table 2). Sixteen of the 100 patients with schizophrenia and 20 of the 100 control subjects had this polymorphism, and allele frequencies were not significantly different between the two groups.
TABLE 2 Allele frequencies of variants identified in the human CHRNA7 gene
Numbering for exons begins with the A of the ATG start codon as nucleotide +1. Numbering in 5'-UTR and intronic positions is relative either to the 5' or 3' boundary of the nearest exon.
Next, we searched for polymorphisms at the coding region and exon-intron boundaries of the CHRNA7 gene. From exon 1 to exon 4, no SNP was detected in either group. CHRNA7 exons 5 to 10 are duplicated in the CHRFAM7A gene (Gault et al., 1998). To selectively analyze the CHRNA7 exons 5 to 10, the primary PCR run was performed before PCR-SSCP analysis on the basis of the difference in the 5' sequence upstream of exon 5 between the two genes. A single band was observed for every sample, and each PCR product including CHRNA7 exons 5 to 10 was observed as an expected band of 17.4 kilobase pairs (data not shown). In addition, each PCR product, including CHRFAM7A exons 5 to 10, showed the predicted band size of 16.5 kilobase pairs (data not shown). These indicate that the DNA fragments were specifically amplified from either the CHRNA7 or CHRFAM7A gene without producing artifacts or cross-contamination during the PCR. In this region of the CHRNA7 gene, we identified 12 SNPs (namely, 4 silent SNPs, 1 nonsynonymous SNP, and 7 intronic SNPs) as shown in Table 2. Allele frequencies of these variants were not statistically significantly different between patients with schizophrenia and control subjects. The G-to-A variant at 1267 bp in exon 10 is a nonsynonymous variant with a change of a glycine to a serine at amino acid 423 of the 7 receptor protein, and it was not found in the Single-Nucleotide Polymorphism database (http://www.ncbi.nlm.nih.gov/projects/SNP/). Of 100 patients, 1 was heterozygous for this variant, but none of 100 control subjects possesses the variant (Table 2). In the patient with this nonsynonymous variant, no SNP was found at the same position duplicated in exon 10 of the CHRFAM7A gene (data not shown). A variant in intron 8 in the CHRNA7 gene is also identified as a novel SNP in one of 100 patients with schizophrenia (Table 2).
Effects of Nicotinic Ligands at 7-G423S Mutant Receptors Expressed in X. laevis Oocytes. To determine the impact of G423S mutation on the 7 nicotinic receptor function, electrophysiological studies were performed on X. laevis oocytes expressing the mutant or wild-type 7 nicotinic receptors. Figure 1A shows current through 7 receptors that decayed very rapidly during application of ACh (0.1-1 mM), as reported previously (Chavez-Noriega et al., 1997). Similar currents were recorded from oocytes expressing 7-G423S receptors. Current in both types of receptor was elicited in the same range of ACh concentrations, and the concentration-response curve for ACh in oocytes expressing 7-G423S receptors (EC50 = 0.19 mM) was almost identical with that in oocytes expressing wild-type 7 receptors (EC50 = 0.20 mM) (Fig. 1, A and B). In addition, the application of an 7 receptor agonist, choline, also produced inward current through the receptors, and no marked differences in the concentration-response curves for choline were observed between the 7-G423S (EC50 = 2.5 mM) and wild-type 7 receptors (EC50 = 2.0 mM) (Fig. 1B). When the voltage-dependence of 7-G423S receptor-mediated current elicited by ACh (1 mM) was examined at different holding potentials (from -120 to +20 mV) in 20-mV steps, the voltage-current relationships were identical with those of wild-type receptor-mediated current (Fig. 1C). Furthermore, a selective 7 receptor antagonist, MLA, equally blocked the responses mediated by 7-G423S (IC50 = 0.21 nM) and wild-type 7 receptors (IC50 = 0.22 nM). These results demonstrate that the G423S mutation does not affect the responses of 7 receptors to nicotinic ligands.
Fig. 1. Comparison of responses to nicotinic ligands between 7 and 7-G423S nicotinic receptors expressed in X. laevis oocytes. Current was recorded in the voltage-clamp mode at -60 mV unless otherwise indicated. A, traces showing typical recordings of 7 and 7-G423S receptor-mediated current elicited by ACh. Horizontal bars above the traces indicate the period (5 s) of perfusion with ACh at concentrations indicated. B, concentration-response curves for ACh in oocytes expressing 7 () or 7-G423S receptors () and for choline in oocytes expressing 7 () or 7-G423S receptors (). Each current response was normalized to the current evoked by 1 mM ACh or 10 mM choline recorded in the same oocyte. C, current-voltage relationship in oocytes expressing 7() and 7-G423S receptors (). Oocytes were voltage-clamped at various membrane potentials (from -120 to +20 mV) in 20-mV steps. Current was elicited by ACh (1 mM, 5 s) at different holding potentials. D, curves showing normalized response in terms of ACh (1 mM, 5 s)-induced current in oocytes expressing 7() and 7-G423S receptors () against MLA concentration. For test responses, oocytes were preincubated with MLA for 6 min and then exposed to ACh with MLA. The test responses were normalized to the current recorded before the application of MLA (control responses) in the same oocytes. Values represent the mean ± S.E.M. of 5 to 10 separate experiments.
Decrease in 7-G423S Nicotinic Receptor Activity by Protein Kinase C Activator. Because the G423S mutation of the 7 receptors is located in the second intracellular loop between the third (M3) and the fourth transmembrane segment (M4), it might change the mode of interaction between the 7 receptors and certain intracellular molecules such as serine/threonine protein kinases. To investigate this hypothesis, we measured ACh (1 mM)-elicited current through the wild-type and mutant 7 receptors in the presence of a protein kinase C activator, PMA. After 6 min of treatment with PMA (10 nM), which by itself did not evoke current, the peak amplitude of 7-G423S receptor-mediated current significantly decreased, whereas no obvious change in wild-type 7 receptor-mediated current was observed following the treatment with PMA (Fig. 2A). This effect of PMA on 7-G423S current persisted during 21 min of observation (Fig. 2A). Similar effects were observed at a higher concentration of PMA (30 nM): after the treatment with PMA (30 nM) for 6 min, the peak amplitude of 7-G423S current decreased to 71 ± 7% (n = 4), whereas that of 7 current remained unaltered (102 ± 7%, n = 5). In contrast, 4-PMA (10 nM), an inactive enantiomer of PMA, did not affect 7 or 7-G423S receptor responses (Fig. 2B). After 6 min of treatment with 4-PMA (30 nM), the peak amplitudes of 7 current and 7-G423S current were 100 ± 13% (n = 6) and 97 ± 4% (n = 4), respectively.
Fig. 2. Attenuation of 7-G423S nicotinic receptor responses after treatment with protein kinase C activator, PMA. Current elicited by ACh (1 mM, 5 s) was recorded every 3 min in the voltage-clamp mode at -60 mV. A, top, typical recordings of ACh-elicited current in oocytes expressing either 7 or 7-G423S receptors before and 6 min after treatment with PMA (10 nM). Horizontal open bars and solid bars indicate the periods of perfusion with PMA and ACh, respectively. Bottom, plots of the peak amplitudes of ACh-elicited currents in oocytes expressing 7() and 7-G423S receptors () before and during application of PMA (10 nM). Each current response was normalized to the current recorded 3 min before application of PMA in the same oocyte. B, top, typical recordings of 7 and 7-G423S receptor-mediated current before and 6 min after treatment with 4-PMA (10 nM), an inactive analog of PMA. Bottom, plots of peak amplitudes of 7() and 7-G423S receptor-mediated current () before and during application of 4-PMA (10 nM). Each response was normalized to the current recorded 3 min before application of 4-PMA in the same oocyte. Values represent the mean ± S.E.M. of four to seven separate experiments. *, P < 0.05 compared with the current recorded in the absence of PMA at 0 min.
When an irreversible inhibitor of protein kinase C, calphostin C, was injected into oocytes expressing 7-G423S receptors, the subsequent treatment with PMA (10 nM) did not inhibit 7-G423S current (Fig. 3, A and B). When the vehicle (0.05% DMSO) was injected instead of calphostin C, treatment with PMA (10 nM) decreased the peak amplitude of 7-G423S current (Fig. 3, A and B), the extent of which was similar to that shown in Fig. 2A. These results demonstrate that PMA-induced inhibition of 7-G423S receptor-mediated responses was blocked by calphostin C.
Fig. 3. Protein kinase C inhibitor calphostin C blocks PMA-induced attenuation of 7-G423S receptor responses in X. laevis oocytes. Calphostin C (200 nM) or vehicle (0.05% DMSO) was injected into oocytes expressing 7-G423S receptors. Current elicited by ACh (1 mM, 5 s) was recorded every 3 min in the voltage-clamp mode at -60 mV. A, typical traces showing the effect of PMA (10 nM) on 7-G423S receptor-mediated current in oocytes preinjected with calphostin C or vehicle (DMSO). Horizontal open bars and solid bars indicate the periods of perfusion with PMA and ACh, respectively. B, plots of the peak amplitude of 7-G423S receptor-mediated current before and during application of PMA (10 nM) in oocytes preinjected with calphostin C () or vehicle (). Each response was normalized to the current recorded 3 min before application of PMA in the same oocyte. Values represent the mean ± S.E.M. of 7 to 10 separate experiments. *, P < 0.05 compared with the current recorded in the absence of PMA at 0 min.
Enhancement of 7-G423S Receptor Desensitization by Protein Kinase C Activator. It was shown previously that nicotinic acetylcholine receptors undergo desensitization during repeated or continuous exposure to an agonist (Quick and Lester, 2002; Giniatullin et al., 2005). To determine the desensitization characteristics of 7-G423S receptors, the effect of repetitive ACh stimulation was examined. When oocytes expressing 7 receptors were stimulated with short pulses of ACh (1 mM, 0.1 s) at 0.17 or 0.33 Hz, the current amplitude progressively declined in a frequency-dependent manner: current amplitudes decreased by 12 ± 3% and 74 ± 5% after 30 s of low- and high-frequency stimulations, respectively (Fig. 4A). The same oocytes were subsequently treated with PMA (10 nM) for 6 min, and then current in response to the repetitive pulses of ACh coapplied with PMA was measured. In this case, 7 current gradually decreased as similarly observed in the absence of PMA (Fig. 4A). When oocytes expressing 7-G423S receptors were stimulated with ACh pulses at 0.17 or 0.33 Hz, a progressive rundown of current was induced, comparable with that observed for 7 current: the current amplitudes decreased by 12 ± 3% and 72 ± 3% after 30 s of low- and high-frequency stimulations, respectively (Fig. 4B). In the presence of PMA (10 nM), however, the rundown of 7-G423S receptor-mediated current was significantly more pronounced, and the peak amplitudes of current elicited by ACh pulses at 0.17 and 0.33 Hz decreased by 43 ± 7% and 83 ± 3%, respectively, after 30 s of stimulation (Fig. 4B).
Long-term exposure to low concentrations of nicotinic agonists, including choline, promotes desensitization of 7 receptors (Papke et al., 1996). To determine whether choline desensitizes 7-G423S receptors and 7 receptors, we compared the effect of long-term exposure to various concentrations of choline between these two types of receptor. The activities of the receptors were assessed by short pulses of ACh (1 mM, 0.2 s) to avoid the desensitization caused by the ACh challenge. As shown in Fig. 5A, continuous exposure to choline for 6 min resulted in a concentration-dependent inhibition of ACh-elicited current through 7 and 7-G423S receptors. No obvious differences in concentration-inhibitory response curves for choline were observed between them (IC50 = 83.6 µMat 7; IC50 = 82.5 µMat 7-G423S). Furthermore, to compare the effect of PMA on the long-term choline-induced inhibition of the receptors, oocytes expressing 7 or 7-G423S receptors were preincubated with PMA (10 nM) for 6 min and then incubated with a low concentration (30 µM) of choline plus PMA (10 nM) for another 6 min. Under these conditions, the 7-G423S receptor-mediated current in response to ACh decreased by 22% after the 6-min treatment with choline, whereas the wild-type receptor-mediated current was not significantly affected by this treatment (Fig. 5B). These results demonstrate that the treatment with PMA selectively altered the desensitization properties of 7-G423S receptors.
Phosphorylation of 7-G423S Receptors by Protein Kinase C-Dependent Mechanism. We further investigated whether 7-G423S nicotinic receptors are phosphorylated after treatment with PMA. To this end, we prepared oocytes expressing EGFP-fused 7(7EGFP) receptors and EGFP-fused 7-G423S (7EGFP-G423S) receptors to specifically detect the 7 subunit by Western blotting (see Discussion). At first, we examined the electrophysiological properties of 7EGFP and 7EGFP-G423S receptors by measuring ACh (1 mM, 5 s)-elicited current. We observed no marked changes in the fast-decaying property and absolute peak amplitude of current compared with untagged receptors (data not shown). Moreover, the inhibitory effect of PMA (30 nM) was also observed on 7EGFP-G423S current but not on 7EGFP current (Fig. 6, A and B): by the treatment with PMA (30 nM) for 6 min, the peak amplitude of 7EGFP-G423S currents decreased to 70 ± 8% (n = 7), whereas that of 7EGFP current was unaffected (101 ± 3%, n = 3). In contrast, 4-PMA (30 nM) had no effect on 7EGFP or 7EGFP-G423S current: 6 min after treatment with 4-PMA (30 nM), the peak amplitudes of 7EGFP current and 7EGFP-G423S current were 102 ± 5% (n = 3) and 105 ± 2% (n = 7), respectively.
Fig. 6. PMA increases serine phosphorylation level of 7-G423S nicotinic receptors in X. laevis oocytes. The oocytes expressing EGFP-fused 7 (7EGFP) receptors and EGFP-fused 7-G423S (7EGFP-G423S) receptors were treated with PMA (30 nM) or 4-PMA (30 nM) for 6 min, and ACh (1 mM, 5 s)-elicited currents were recorded in the voltage-clamp mode at -60 mV. Subsequently, the oocytes were lysed, and EGFP-fused receptor proteins (84 kDa) were immunoprecipitated with the anti-EGFP antibody and analyzed by Western blotting. Traces show typical recordings of ACh (1 mM, 5 s)-elicited current in oocytes expressing either 7EGFP (A) or 7EGFP-G423S receptors (B) at -60 mV before and 6 min after treatment with PMA or 4-PMA. Horizontal open bars indicate the presence of PMA or 4-PMA, and horizontal solid bars indicate the period of perfusion with ACh. Immunoblots show bands corresponding to the 84-kDa protein recognized by the anti-phosphoserine antibody or anti-EGFP antibody in samples from oocytes expressing 7EGFP (C) and 7EGFP-G423S receptors (D). Similar results were observed in three separate experiments.
We further investigated the phosphorylation levels in 7EGFP and 7EGFP-G423S proteins, which were purified by immunoprecipitation with the anti-EGFP antibody and labeled with either the anti-phosphoserine antibody or the anti-EGFP antibody. As expected from the molecular size of the 7EGFP receptor subunit (57 kDa 7 subunit plus 27 kDa EGFP), the 7EGFP protein was recognized by the anti-EGFP antibody and observed as a major band of 84 kDa, but no band was detected in the lane of 7EGFP receptors treated with vehicle, PMA (30 nM), or 4-PMA (30 nM) and labeled with the anti-phosphoserine antibody (Fig. 6C). It is interesting that the band corresponding to an 84-kDa protein was observed in the lane of 7EGFP-G423S receptors treated with PMA (30 nM) and labeled with the anti-phosphoserine antibody, whereas no such band was observed in the lane of 7EGFP-G423S receptors treated with vehicle or 4-PMA (30 nM) (Fig. 6D). Relative phosphorylation levels in the presence of PMA were significantly higher than the control levels (data not shown). These results indicate that treatment with PMA selectively increased the serine phosphorylation level in 7EGFP-G423S receptors.
Site-directed mutagenesis studies have shown that single amino acid substitutions in drug receptors, including the 7 nicotinic acetylcholine receptors, are often sufficient to cause marked changes in receptor properties (Galzi and Changeux, 1995). To date, a limited number of nonsynonymous variants have been identified in the human CHRNA7 gene (Gault et al., 2003), and there is no report evaluating the functional significance of the variants. In the present polymorphism screening, we identified 13 SNPs in the CHRNA7 gene, including 2 novel SNPs: one was a novel SNP (1267 G to A) within exon 10, and the other was intronic. In particular, the novel coding SNP results in an amino acid substitution from glycine to serine at position 423 located in the large intracellular loop of the 7 receptor subunit. This glycine residue is conserved across several species, namely, human (GenPept accession number NP_000737), rhesus monkey (Macaca mulatta, NP_001028055), mouse (NP_031416), and rat (NP_036964), suggesting that there is evolutionary pressure for the protein sequences to maintain normal receptor function. Because statistical analyses could not reveal the direct association of the SNPs identified in this study with schizophrenia, the neurological significance of the nonsynonymous 1267A variant remains to be determined. Hence, additional studies in other cohorts with large-scale populations are needed to clarify whether this rare nonsynonymous variant is, in fact, associated with some central cholinergic disorder.
To evaluate functional consequences of the G423S mutation, we first investigated the effects of nicotinic ligands on mutant-receptor-mediated current. Compared with the responses of wild-type 7 receptors, 7-G423S receptors exhibited normal responses to the nicotinic agonists ACh and choline and to the 7 antagonist MLA. In addition, the 7 and 7-G423S receptors showed a nearly identical current-voltage relationship. These results suggest that the G423S mutation does not affect the receptor-channel opening upon agonist binding, probably because this mutant amino acid is located in an intracellular region that is not functionally related to the ACh-binding site in the extracellular N-terminal domain and to the M2 transmembrane segment that lines the channel pore.
Protein phosphorylation is considered an important mechanism of regulating the 7 nicotinic receptor function, because the tyrosine phosphorylation of 7 receptors by Src-family kinases negatively regulates receptor activity in SH-SY5Y neuroblastoma cells, X. laevis oocytes, and rat hippocampal interneurons (Charpantier et al., 2005). In addition, cAMP-dependent protein kinase (protein kinase A) directly phosphorylates the serine 342 residue of rat and chick 7 receptors (Moss et al., 1996), which is also conserved in human 7 receptors (Wecker et al., 2001), and treatment with a cAMP analog enhances calcium influx through rat 7 receptors stably expressed in GH4C1 pituitary cells (Quik et al., 1997). Neither protein kinase C nor calcium/calmodulin-dependent protein kinase II can directly phosphorylate rat and chick 7 receptors (Moss et al., 1996). Therefore, we next investigated the possibility that the G423S mutation alters the mode of interaction between the 7 receptors and certain serine/threonine protein kinases. We were surprised to find that 7-G423S receptor-mediated current markedly decreased in the presence of a phorbol ester, PMA, whereas 7 current was unaffected, consistent with previous reports (Quik et al., 1997; Filippova et al., 2000). The observations that the inhibitory effect of PMA on 7-G423S current was reversed by a protein kinase C inhibitor, calphostin C, and that 4-PMA caused no such inhibitory effect, strongly suggest that PMA causes the decrease via protein kinase C activation. Thus, the G423S mutation may provide a new site of interaction between the 7 receptors and protein kinase C, although we cannot rule out the possibility that some downstream kinases in the protein kinase C-signaling pathway mediate the effect of PMA. Further studies would be required to determine whether the mutant receptors interact with other serine/threonine protein kinases.
To elucidate whether the phosphorylation of the mutant 7 nicotinic receptors at serine 423 underlies the altered effect of PMA, serine phosphorylation was assessed by Western blotting. Because it has been shown that the most commonly used antibodies to the 7 receptor subunit unexpectedly exhibit equal immunoreactivity in tissues of both wild-type and 7-knockout mice, the labeling of epitope-tagged 7 receptors is now considered to be the most reliable method of identifying 7 immunoreactivity (Jones and Wonnacott, 2005). We therefore prepared EGFP-fused wild-type or mutant 7 receptors expressed in X. laevis oocytes. Similar EGFP-fused 7 receptors were reported previously to exhibit normal ACh sensitivity and normal single-channel conductance, although the fusion of EGFP to the receptor lengthens the channel open time (Fucile et al., 2002; Palma et al., 2002). In our present electrophysiological recordings, we observed that the fusion of EGFP to the wild-type and mutant 7 receptors did not change their responses to PMA and 4-PMA. It is noteworthy that under the condition that 7-G423S current was selectively decreased by PMA, serine phosphorylation level significantly increased in the mutant receptors but not in the wild-type receptors. These results suggest that the mutant 7 receptors are directly phosphorylated at serine 423 by a protein kinase C-dependent mechanism. In the hippocampus, high-frequency stimulation induces long-term potentiation of synaptic transmission, which underlies learning and memory formation, and the induction of long-term potentiation generates a prolonged activation of protein kinase C (Soderling and Derkach, 2000). Therefore, we suggest that the 7-G423S mutant receptors may be phosphorylated via protein kinase C under certain physiological conditions at hippocampal excitatory synapses.
The role of phosphorylation in desensitization of muscle nicotinic receptors has been studied intensively (Swope et al., 1992). For example, the direct phosphorylation of the subunit by protein kinase C, or the and subunits by protein kinase A, is associated with an increase in the rate of nicotinic receptor desensitization. Likewise, the onset of desensitization of ganglionic nicotinic receptors is promoted by phorbol ester activation of protein kinase C (Quick and Lester, 2002). However, there is no evidence at present that phosphorylation modulates the kinetics of desensitization of 7 nicotinic receptors. In this study, we found that the treatment with PMA promoted the onset of desensitization of the 7-G423S mutant receptors when there was repetitive stimulation with ACh pulses or prolonged exposure to choline. In contrast, we observed that the onset of desensitization of wild-type 7 receptors was unaffected by treatment with PMA and was equivalent to that of the 7-G423S mutant receptors in the absence of PMA. Together with the present analyses of protein phosphorylation, it is possible that phosphorylation of serine 423 residue of the mutant receptors plays an important role in enhancing the receptor desensitization, whereas undetectable levels of serine phosphorylation of wild-type receptors in the absence and presence of PMA and the mutant receptors in the absence of PMA, if any, do not contribute to the enhancement of desensitization. Thus, it is likely that 7 nicotinic receptors bearing G423S substitution acquire a new desensitization mechanism through protein kinase C.
The 7 nicotinic receptors have unique pharmacological properties, compared with other neuronal nicotinic receptors in the mammalian brain, including high Ca2+ permeability comparable with that of the N-methyl-D-aspartate (NMDA) receptors (Gotti and Clementi, 2004). In addition, current through nicotinic receptors exhibits voltage-dependence, which differs from that of voltage-operated Ca2+ channels and NMDA receptors. At hyperpolarized potentials, nicotinic receptors pass current effectively, whereas voltage-operated Ca2+ channels and NMDA receptors are inactivated (Dani, 2001). Consequently, Ca2+ influx through 7 receptors exerts unique effects, such as the promotion of neuronal survival (Gotti and Clementi, 2004). If the 7 receptors are intensively desensitized because of the G423S mutation, the loss of function may not be fully compensated by other Ca2+ channels coexpressed in neurons. The degree and types of changes in neural functions by possessing the 7-G423S mutant receptors need to be clarified in a future study.
In summary, we found a novel SNP in the CHRNA7 gene in a Japanese population, which results in G423S mutation of the 7 nicotinic receptors. We also demonstrated that the G423S mutation promotes receptor desensitization by a protein kinase C-dependent mechanism. To the best of our knowledge, this is the first study demonstrating that human gene mutation contributes to the dysfunction of the 7 nicotinic receptors.
Acknowledgements
We are grateful to Dr. Hideki Sakai (University of Toyama, Toyama, Japan) and Dr. John A. Dani (Baylor College of Medicine, Houston, TX) for support on electrophysiological data acquisition and Dr. Michio Suzuki (University of Toyama) for helpful advice.
ABBREVIATIONS: SNP, single nucleotide polymorphism; ACh, acetylcholine; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescence protein; MLA, methyllycaconitine; NMDA, N-methyl-D-aspartate; ORF, open reading frame; PMA, phorbol-12-myristate-13-acetate; PVDF, polyvinylidene fluoride; PCR, polymerase chain reaction; SSCP, single-strand conformation polymorphism; bp, base pair(s).
【参考文献】
Changeux JP, Bertrand D, Corringer PJ, Dehaene S, Edelstein S, Léna C, Le Novere N, Marubio L, Picciotto M, and Zoli M (1998) Brain nicotinic receptors: structure and regulation, role in learning and reinforcement. Brain Res Rev 26: 198-216.
Charpantier E, Wiesner A, Huh KH, Ogier R, Hoda JC, Allaman G, Raggenbass M, Feuerbach D, Bertrand D, and Fuhrer C (2005) 7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases. J Neurosci 25: 9836-9849.[Abstract/Free Full Text]
Chavez-Noriega LE, Crona JH, Washburn MS, Urrutia A, Elliott KJ, and Johnson EC (1997) Pharmacological characterization of recombinant human neuronal nicotinic acetylcholine receptors h22, h24, h32, h34, h42, h44 and h7 expressed in Xenopus oocytes. J Pharmacol Exp Ther 280: 346-356.[Abstract/Free Full Text]
Dani JA (2001) Overview of nicotinic receptors and their roles in the central nervous system. Biol Psychiatry 49: 166-174.
Filippova N, Sedelnikova A, Zong Y, Fortinberry H, and Weiss DS (2000) Regulation of recombinant gamma-aminobutyric acid (GABA)A and GABAC receptors by protein kinase C. Mol Pharmacol 57: 847-856.[Abstract/Free Full Text]
Freedman R, Adams CE, and Leonard S (2000) The 7-nicotinic acetylcholine receptor and the pathology of hippocampal interneurons in schizophrenia. J Chem Neuroanat 20: 299-306.
Freedman R, Coon H, Myles-Worsley M, Orr-Urtreger A, Olincy A, Davis A, Polymeropoulos M, Holik J, Hopkins J, Hoff M, et al. (1997) Linkage of a neurophysiological deficit in schizophrenia to a chromosome 15 locus. Proc Natl Acad Sci USA 94: 587-592.[Abstract/Free Full Text]
Freedman R, Olincy A, Ross RG, Waldo MC, Stevens KE, Adler LE, and Leonard S (2003) The genetics of sensory gating deficits in schizophrenia. Curr Psychiatry Rep 5: 155-161.
Fucile S, Palma E, Martinez-Torres A, Miledi R, and Eusebi F (2002) The single-channel properties of human acetylcholine 7 receptors are altered by fusing 7 to the green fluorescent protein. Proc Natl Acad Sci USA 99: 3956-3961.[Abstract/Free Full Text]
Galzi JL and Changeux JP (1995) Neuronal nicotinic receptors: molecular organization and regulations. Neuropharmacology 34: 563-582.
Gault J, Hopkins J, Berger R, Drebing C, Logel J, Walton C, Short M, Vianzon R, Olincy A, Ross RG, et al. (2003) Comparison of polymorphisms in the 7 nicotinic receptor gene and its partial duplication in schizophrenic and control subjects. Am J Med Genet B Neuropsychiatr Genet 123: 39-49.
Gault J, Robinson M, Berger R, Drebing C, Logel J, Hopkins J, Moore T, Jacobs S, Meriwether J, Choi MJ, et al. (1998) Genomic organization and partial duplication of the human 7 neuronal nicotinic acetylcholine receptor gene (CHRNA7). Genomics 52: 173-185.
Giniatullin R, Nistri A, and Yakel JL (2005) Desensitization of nicotinic ACh receptors: shaping cholinergic signaling. Trends Neurosci 28: 371-378.
Gopalakrishna R, Chen ZH, and Gundimeda U (1992) Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C. FEBS Lett 314: 149-154.
Gotti C and Clementi F (2004) Neuronal nicotinic receptors: from structure to pathology. Prog Neurobiol 74: 363-396.
Houy E, Raux G, Thibaut F, Belmont A, Demily C, Allio G, Haouzir S, Fouldrin G, Petit M, Frebourg T, et al. (2004) The promoter 194 C polymorphism of the nicotinic alpha 7 receptor gene has a protective effect against the P50 sensory gating deficit. Mol Psychiatry 9: 320-322.
Ishihara H, Sasaoka T, Kagawa S, Murakami S, Fukui K, Kawagishi Y, Yamazaki K, Sato A, Iwata M, Urakaze M, et al. (2003) Association of the polymorphisms in the 5'-untranslated region of PTEN gene with type 2 diabetes in a Japanese population. FEBS Lett 554: 450-454.
Jones IW and Wonnacott S (2005) Why doesn't nicotinic ACh receptor immunoreactivity knock out. Trends Neurosci 28: 343-345.
Leonard S, Adams C, Breese CR, Adler LE, Bickford P, Byerley W, Coon H, Griffith JM, Miller C, Myles-Worsley M, et al. (1996) Nicotinic receptor function in schizophrenia. Schizophr Bull 22: 431-445.[Abstract/Free Full Text]
Leonard S, Gault J, Hopkins J, Logel J, Vianzon R, Short M, Drebing C, Berger R, Venn D, Sirota P, et al. (2002) Association of promoter variants in the 7 nicotinic acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia. Arch Gen Psychiatry 59: 1085-1096.[Abstract/Free Full Text]
Li CH, Liao HM, and Chen CH (2004) Identification of molecular variants at the promoter region of the human 7 neuronal nicotinic acetylcholine receptor subunit gene but lack of association with schizophrenia. Neurosci Lett 372: 1-5.
Martin LF, Kem WR, and Freedman R (2004) Alpha-7 nicotinic receptor agonists: potential new candidates for the treatment of schizophrenia. Psychopharmacology (Berl) 174: 54-64.
McLaughlin JT, Fu J, Sproul AD, and Rosenberg RL (2006) Role of the outer -sheet in divalent cation modulation of 7 nicotinic receptors. Mol Pharmacol 70: 16-22.[Abstract/Free Full Text]
Moss SJ, McDonald BJ, Rudhard Y, and Schoepfer R (1996) Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor 7 subunit by cAMP-dependent protein kinase. Neuropharmacology 35: 1023-1028.
Palma E, Mileo AM, Martinez-Torres A, Eusebi F, and Miledi R (2002) Some properties of human neuronal 7 nicotinic acetylcholine receptors fused to the green fluorescent protein. Proc Natl Acad Sci USA 99: 3950-3955.[Abstract/Free Full Text]
Papke RL, Bencherif M, and Lippiello P (1996) An evaluation of neuronal nicotinic acetylcholine receptor activation by quaternary nitrogen compounds indicates that choline is selective for the 7 subtype. Neurosci Lett 213: 201-204.
Paterson D and Nordberg A (2000) Neuronal nicotinic receptors in the human brain. Prog Neurobiol 61: 75-111.
Placzek AN, Grassi F, Meyer EM, and Papke RL (2005) An 7 nicotinic acetylcholine receptor gain-of-function mutant that retains pharmacological fidelity. Mol Pharmacol 68: 1863-1876.[Abstract/Free Full Text]
Quick MW and Lester RA (2002) Desensitization of neuronal nicotinic receptors. J Neurobiol 53: 457-478.
Quik M, Philie J, and Choremis J (1997) Modulation of 7 nicotinic receptor-mediated calcium influx by nicotinic agonists. Mol Pharmacol 51: 499-506.[Abstract/Free Full Text]
Soderling TR and Derkach VA (2000) Postsynaptic protein phosphorylation and LTP. Trends Neurosci 23: 75-80.
Swope SL, Moss SJ, Blackstone CD, and Huganir RL (1992) Phosphorylation of ligand-gated ion channels: a possible mode of synaptic plasticity. FASEB J 6: 2514-2523.
Tsuneki H, You Y, Toyooka N, Kagawa S, Kobayashi S, Sasaoka T, Nemoto H, Kimura I, and Dani JA (2004) Alkaloids indolizidine 235B', quinolizidine 1-epi-207I, and the tricyclic 205B are potent and selective noncompetitive inhibitors of nicotinic acetylcholine receptors. Mol Pharmacol 66: 1061-1069.[Abstract/Free Full Text]
Wecker L, Guo X, Rycerz AM, and Edwards SC (2001) 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 4 neuronal nicotinic receptor subunits. J Neurochem 76: 711-720.
Weiland S, Bertrand D, and Leonard S (2000) Neuronal nicotinic acetylcholine receptors: from the gene to the disease. Behav Brain Res 113: 43-56.
作者单位：Departments of Clinical Pharmacology (H.T., S.K., K.T., S.K., T.W., I.K., T.S.) and Neuropsychiatry (M.T., T.M., M.K.), University of Toyama, Toyama, Japan; and Hokuriku National Hospital, Toyama, Japan (M.M.)