RESUMO
Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.
Assuntos
Canais de Cálcio/metabolismo , Células Enterocromafins/metabolismo , Motilidade Gastrointestinal , Proteínas do Tecido Nervoso/metabolismo , Serotonina/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Anquirinas , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Cobaias , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Ratos , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Canais de Potencial de Receptor Transitório/genéticaRESUMO
To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.
Assuntos
Canais de Potássio/biossíntese , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Analgésicos não Narcóticos/farmacologia , Animais , Antiarrítmicos/farmacologia , Ácido Araquidônico/farmacologia , Bário/farmacologia , Linhagem Celular , Clonagem Molecular , Ácidos Docosa-Hexaenoicos/farmacologia , Eletrofisiologia , Etanolaminas/farmacologia , Ácidos Graxos/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Glibureto/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Lidocaína/farmacologia , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Filogenia , Canais de Potássio/química , Propafenona/farmacologia , Estrutura Terciária de Proteína , Quinidina/farmacologia , Quinina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Medula Espinal/metabolismo , Distribuição Tecidual , TransfecçãoRESUMO
We report identification and characterization of Kv6.3, a novel member of the voltage-gated K(+) channel. Reverse transcriptase-polymerase chain reaction analysis indicated that Kv6.3 was highly expressed in the brain. Electrophysiological studies indicated that homomultimeric Kv6.3 did not yield a functional voltage-gated ion channel. When Kv6.3 and Kv2.1 were co-expressed, the heteromultimeric channels displayed the decreased rate of deactivation compared to the homomultimeric Kv2.1 channels. Immunoprecipitation studies indicated that Kv6.3 bound with Kv2.1 in co-transfected cells. These results indicate that Kv6.3 is a novel member of the voltage-gated K(+) channel which functions as a modulatory subunit.
