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1.
Acta Pharmacol Sin ; 36(7): 800-12, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25948478

RESUMO

AIM: Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca(2+)-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR. METHODS: Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B. RESULTS: Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 µmol/L. Br-IQ17B is selective over other subtypes such as α4ß2 and α3ß4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [(3)H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive. CONCLUSION: We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.


Assuntos
Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacologia , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Humanos , Masculino , Técnicas de Cultura de Órgãos , Células PC12 , Ratos , Ratos Sprague-Dawley , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/fisiologia
2.
Biophys J ; 107(5): 1090-1104, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25185545

RESUMO

A-type Kv4 potassium channels undergo a conformational change toward a nonconductive state at negative membrane potentials, a dynamic process known as pre-open closed states or closed-state inactivation (CSI). CSI causes inhibition of channel activity without the prerequisite of channel opening, thus providing a dynamic regulation of neuronal excitability, dendritic signal integration, and synaptic plasticity at resting. However, the structural determinants underlying Kv4 CSI remain largely unknown. We recently showed that the auxiliary KChIP4a subunit contains an N-terminal Kv4 inhibitory domain (KID) that directly interacts with Kv4.3 channels to enhance CSI. In this study, we utilized the KChIP4a KID to probe key structural elements underlying Kv4 CSI. Using fluorescence resonance energy transfer two-hybrid mapping and bimolecular fluorescence complementation-based screening combined with electrophysiology, we identified the intracellular tetramerization (T1) domain that functions to suppress CSI and serves as a receptor for the binding of KID. Disrupting the Kv4.3 T1-T1 interaction interface by mutating C110A within the C3H1 motif of T1 domain facilitated CSI and ablated the KID-mediated enhancement of CSI. Furthermore, replacing the Kv4.3 T1 domain with the T1 domain from Kv1.4 (without the C3H1 motif) or Kv2.1 (with the C3H1 motif) resulted in channels functioning with enhanced or suppressed CSI, respectively. Taken together, our findings reveal a novel (to our knowledge) role of the T1 domain in suppressing Kv4 CSI, and that KChIP4a KID directly interacts with the T1 domain to facilitate Kv4.3 CSI, thus leading to inhibition of channel function.


Assuntos
Proteínas Interatuantes com Canais de Kv/metabolismo , Canais de Potássio Shal/metabolismo , Animais , Biotinilação , Western Blotting , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Potenciais da Membrana/fisiologia , Microscopia Confocal , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ácidos Polimetacrílicos , Compostos de Amônio Quaternário , Canais de Potássio Shal/genética , Transfecção , Xenopus laevis
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