Shinobu Mochizuki

TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells

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 Ca2+ 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-HT3 receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.

Molecular cloning and characterization of Kv6.3, a novel modulatory subunit for voltage‐gated K+ channel Kv2.11

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.

New Ether-à-go-go K+ Channel Family Members Localized in Human Telencephalon

A cDNA encoding a novel voltage-gated K+ channel protein was isolated from human brain. This protein, termed BEC1, is 46% identical to rat elk in theether-à-go-go K+ channel family. TheBEC1 gene maps to the 12q13 region of the human genome. Northern blot analysis indicates that BEC1 is exclusively expressed in human brain, where the expression is concentrated in the telencephalic areas such as the cerebral cortex, amygdala, hippocampus, and striatum. By in situ hybridization, BEC1 is detected in the CA1–CA3 pyramidal cell layers and the dentate gyrus granule cell layers of the hippocampus. Specific signals are also found in neocortical neurons. Transfection of mammalian L929 and Chinese hamster ovary cells with BEC1 cDNA induces a voltage-gated outward current with a fast inactivation component. This current is insensitive to tetraethylammonium and quinidine. Additionally, a second related gene BEC2 was isolated from human brain. BEC2 is also brain-specific, located in the neocortex and the striatum, and functional as a channel gene. Phylogenetic analysis indicates that BEC1 andBEC2 constitute a subfamily, together with elk, in the ether-à-go-go family. The two genes may be involved in cellular excitability of restricted neurons in the human central nervous system.

Disruption of the Ether-à-go-go K+ Channel Gene BEC1/KCNH3 Enhances Cognitive Function

The K+ channel, one of the determinants for neuronal excitability, is genetically heterogeneous, and various K+ channel genes are expressed in the CNS. The therapeutic potential of K+ channel blockers for cognitive enhancement has been discussed, but the contribution each K+ channel gene makes to cognitive function remains obscure. BEC1 (KCNH3) is a member of the K+ channel superfamily that shows forebrain-preferential distribution. Here, we show the critical involvement of BEC1 in cognitive function. BEC1 knock-out mice performed behavioral tasks related to working memory, reference memory, and attention better than their wild-type littermates. Enhanced performance was also observed in heterozygous mutants. The knock-out mice had neither the seizures nor the motor dysfunction that are often observed in K+ channel-deficient mice. In contrast to when it is disrupted, overexpression of BEC1 in the forebrain caused the impaired performance of those tasks. It was also found that altering BEC1 expression could change hippocampal neuronal excitability and synaptic plasticity. The results indicate that BEC1 may represent the first K+ channel that contributes preferentially and bidirectionally to cognitive function.

Identification of a domain affecting agonist potency of meta-chlorophenylbiguanide in 5-HT3 receptors.

The pharmacological properties of rat and human 5-HT3 receptors expressed in Xenopus oocytes were assessed using a two-electrode voltage clamp technique. Meta-chlorophenylbiguanide (mCPBG), a 5-HT3 receptor-selective agonist, elicited typical current responses in both rat and human 5-HT3 receptor-expressing oocytes. However, the EC50 value for rat 5-HT3 receptors was 13-fold lower than for human 5-HT3 receptors. Using several chimeric human-rat 5-HT3 receptors, we identified a potential domain responsible for this difference in mCPBG-response. The domain is in the N-terminal extracellular region adjacent to the first transmembrane domain of rat 5-HT3 receptors and includes a rat-specific seven amino acid sequence (Phe197, Thr198, Lys199, Gln201, Ile205, Thr207 and Ser210). Replacement of corresponding amino acids in human 5-HT3 receptors by rat receptor residues increased the potency of mCPBG on human receptors indicating these amino acids play an important role in the pharmacological response to mCPBG.

Cloning, expression, and characterization of ferret 5-HT3 receptor subunit

Abstract Ferrets ( Mustela putorius furo ) are useful animals for determining anti-emetic activity via 5-HT 3 receptors in vivo. We isolated a cDNA encoding the 5-hydroxytryptamine (5-HT) type 3A receptor subunit (5-HT 3A ) from ferret colon, expressed it in a human embryonic kidney cell line and determined its pharmacological properties. The open reading frame of the isolated cDNA encoded a 483-amino acid protein, corresponding to the shorter splice variant of 5-HT 3A receptors. Splice variants were no longer detected by reverse transcriptase-polymerase chain reaction. The ferret 5-HT 3A receptor exhibits a high degree of amino acid sequence identity (≥80%) to that of other species. Binding studies demonstrated the following rank order of potency for agonists: meta -chlorophenylbiguanide ( m CPBG)>2-methyl-5-hydroxytryptamine (2-Me-5-HT)=5-HT, and for antagonists: ondansetron=tropisetron>(+)-tubocurarine>metoclopramide. Electrophysiological studies revealed that m CPBG was a partial agonist and 2-Me-5-HT was an almost fully effective agonist compared to 5-HT.