ABSTRACT
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.
Subject(s)
Calcium Channels/metabolism , Enterochromaffin Cells/metabolism , Gastrointestinal Motility , Nerve Tissue Proteins/metabolism , Serotonin/metabolism , Transient Receptor Potential Channels/metabolism , Animals , Ankyrins , Calcium/metabolism , Calcium Channels/genetics , Cell Line , Gene Expression Regulation/genetics , Guinea Pigs , Humans , Male , Mice , Nerve Tissue Proteins/genetics , Rats , TRPA1 Cation Channel , TRPC Cation Channels , Transient Receptor Potential Channels/geneticsABSTRACT
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.
Subject(s)
Potassium Channels/biosynthesis , Potassium Channels/physiology , Amino Acid Sequence , Analgesics, Non-Narcotic/pharmacology , Animals , Anti-Arrhythmia Agents/pharmacology , Arachidonic Acid/pharmacology , Barium/pharmacology , Cell Line , Cloning, Molecular , Docosahexaenoic Acids/pharmacology , Electrophysiology , Ethanolamines/pharmacology , Fatty Acids/metabolism , Fatty Acids, Nonesterified/metabolism , Glyburide/pharmacology , Humans , Hydrogen-Ion Concentration , Lidocaine/pharmacology , Mice , Models, Biological , Molecular Sequence Data , Patch-Clamp Techniques , Phylogeny , Potassium Channels/chemistry , Propafenone/pharmacology , Protein Structure, Tertiary , Quinidine/pharmacology , Quinine/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/metabolism , Tissue Distribution , TransfectionABSTRACT
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.
