Biomedical subjects

Takahiro M Ishii

Publications and source records attributed to Takahiro M Ishii.

8 recordsLinked to original sources

PubMed2006

Tryptophan-scanning mutagenesis in the S1 domain of mammalian HCN channel reveals residues critical for voltage-gated activation.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are essential regulators in rhythmic activity, membrane excitability and synaptic transmission. There are four subtypes in mammals (HCN1-4); HCN4 has the slowest activation kinetics and HCN1 the fastest. Although voltage gating originates with the voltage-dependent motion of the S4 segment, the different activation kinetics between HCN1 and HCN4 are generated mainly by S1 and the S1-S2 loop. In this study, we investigate the structural basis of the ability of S1 to affect activation kinetics by replacing each individual S1 residue in HCN1 with a tryptophan (Trp) residue, a Trp perturbation scan. Robust currents were generated in 11 out of 19 Trp mutants. Hyperpolarization-activated currents were not detected in four mutants, and two other mutants generated only small currents. Presence or absence of current reflected the predicted alpha-helical structure of the S1 transmembrane segment. Tryptophan replacements of residues responsible for the different kinetics between HCN1 and HCN4 made the activation kinetics slower than the wild-type HCN1. Tryptophan mutations introduced in the middle of S1 (L139W and V143W) prevented normal channel closure. Furthermore, a negatively charged residue at position 139 (L139D) induced a positive voltage shift of activation by 125 mV. Thus, L139 and V143 probably face a mobile part of the S4 voltage sensor and may interact with it. These results suggest that the secondary structure of S1 is alpha-helical and profoundly affects the motion of the voltage sensor.

Amino Acid Sequence↗

PubMed2006

Misexpression of Sox9 in mouse limb bud mesenchyme induces polydactyly and rescues hypodactyly mice.

Our previous studies have demonstrated the essential roles of the transcription factor Sox9 in the commitment of mesenchymal cells to a chondrogenic cell lineage and in overt chondrogenesis during limb bud development. However, it remains unknown if Sox9 induces chondrogenesis in mesenchyme ectopically in vivo as a master regulator of chondrogenesis. In this study, we first generated mutant mice in which Sox9 was misexpressed in the limb bud mesenchyme. The mutant mouse embryos exhibited polydactyly in limb buds in association with ectopic expression of Sox5 and Sox6 although markers for the different axes of limb bud development showed a normal pattern of expression. Misexpression of Sox9 stimulated cell proliferation in limb bud mesenchyme, suggesting that Sox9 has a role in recruiting mesenchymal cells to mesenchymal condensation. Second, despite the facts that misexpression of Sonic hedgehog (Shh) induces polydactyly in a number of mutant mice and Shh-null mutants have severely defective cartilage elements in limb buds, misexpression of Sox9 did not restore limb bud phenotypes in Shh-null mutants. Rather, there was no expression of Sox9 in digit I of Hoxa13Hd mutant embryos, and Sox9 partially rescued hypodactyly in Hoxa13Hd mutant embryos. These results provide evidence that Sox9 induces ectopic chondrogenesis in mesenchymal cells and strongly suggest that its expression may be regulated by Hox genes during limb bud development.

Animals↗

PubMed2006

NRSF regulates the developmental and hypertrophic changes of HCN4 transcription in rat cardiac myocytes.

The HCN4 channel shows differential expression patterns during the embryonic development and hypertrophy of hearts. Briefly, HCN4 expression is maximally activated in embryonic hearts and quickly diminishes after birth. However, it is reactivated during cardiac hypertrophy. The sequence analysis of HCN4 gene revealed the presence of a conserved NRSE motif, which is known to bind the transcriptional factor neuron-restrictive silencing factor (NRSF). A promoter analysis of HCN4 with rat cardiac myocytes identified the region inducing a basal transcriptional activity. This region drove a high activity in embryonic myocytes, but not in neonatal myocytes treated with hypertrophic agents. After confirming that NRSF protein binds to the NRSE, HCN4 promoter activities modified by NRSE were evaluated. With wild-type NRSE, the promoter activity correlated well with the developmental and hypertrophic changes of HCN4 expression, whereas mutant NRSE constructs failed. We conclude that the NRSE-NRSF system was implicated in HCN4 expression in cardiac myocytes.

Animals↗

PubMed2006

Axonal site of spike initiation enhances auditory coincidence detection.

Neurons initiate spikes in the axon initial segment or at the first node in the axon. However, it is not yet understood how the site of spike initiation affects neuronal activity and function. In nucleus laminaris of birds, neurons behave as coincidence detectors for sound source localization and encode interaural time differences (ITDs) separately at each characteristic frequency (CF). Here we show, in nucleus laminaris of the chick, that the site of spike initiation in the axon is arranged at a distance from the soma, so as to achieve the highest ITD sensitivity at each CF. Na+ channels were not found in the soma of high-CF (2.5-3.3 kHz) and middle-CF (1.0-2.5 kHz) neurons but were clustered within a short segment of the axon separated by 20-50 microm from the soma; in low-CF (0.4-1.0 kHz) neurons they were clustered in a longer stretch of the axon closer to the soma. Thus, neurons initiate spikes at a more remote site as the CF of neurons increases. Consequently, the somatic amplitudes of both orthodromic and antidromic spikes were small in high-CF and middle-CF neurons and were large in low-CF neurons. Computer simulation showed that the geometry of the initiation site was optimized to reduce the threshold of spike generation and to increase the ITD sensitivity at each CF. Especially in high-CF neurons, a distant localization of the spike initiation site improved the ITD sensitivity because of electrical isolation of the initiation site from the soma and dendrites, and because of reduction of Na+-channel inactivation by attenuating the temporal summation of synaptic potentials through the low-pass filtering along the axon.

Action Potentials↗

PubMed2006

Effects of perfluorooctane sulfonate on action potentials and currents in cultured rat cerebellar Purkinje cells.

Recently, PFOS was reported to be ubiquitously detected in the environment, as well as in human serum, raising concerns regarding its health risks. We investigated the effects of PFOS on action potentials and currents in cultured rat cerebellar Purkinje cells using whole-cell patch-clamp recording. In current-clamp experiments, PFOS significantly decreased the action potential frequency during current injection, the maximum rate of fall and the threshold of action potential, and negatively shifted the resting membrane potential at doses over 30microM. In voltage-clamp experiments, PFOS shifted the half-activation and inactivation voltages of I(Ca), I(Na), and I(K) toward hyperpolarization at 30microM. I(HCN1) expressed in Xenopus oocytes was similarly affected. Incorporation of PFOS into the cell membrane probably increased the surface negative charge density, thereby reducing the transmembrane potential gradient and resulting in hyperpolarizing shifts of both the activation and inactivation of ionic channels. These findings indicate that PFOS may exhibit neurotoxicity.

Action Potentials↗

PubMed2005

A novel Ca2+ indicator protein using FRET and calpain-sensitive linker.

Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha-spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha-spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca(2+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca(2+) concentration ([Ca(2+)](i)) was raised. F2C had a Ca(2+) sensitivity with an apparent dissociation constant (K(d) for Ca(2+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca(2+)](i) was measured simultaneously. These properties of F2C are useful to be a Ca(2+) indicator.

Amino Acid Sequence↗

PubMed2005

Hyperpolarization-activated cyclic nucleotide-gated cation channels regulate auditory coincidence detection in nucleus laminaris of the chick.

Coincidence detection of bilateral acoustic signals in nucleus laminaris (NL) is the first step in azimuthal sound source localization in birds. Here, we demonstrate graded expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels along the tonotopic axis of NL and its role in the regulation of coincidence detection. Expression of HCN1 and HCN2, but not HCN3 or HCN4, was detected in NL. Based on measurement of both subtype mRNA and protein, HCN1 varied along the tonotopic axis and was minimal in high-characteristic frequency (CF) neurons. In contrast, HCN2 was evenly distributed. The resting conductance was larger and the steady-state activation curve of Ih was more positive in neurons of middle to low CF than those of high CF, consistent with the predominance of HCN1 channels in these neurons. Application of 8-Br-cAMP or noradrenaline generated a depolarizing shift of the Ih voltage activation curve. This shift was larger in neurons of high CF than in those of middle CF. The shift in the activation voltage of Ih depolarized the resting membrane, accelerated the EPSP time course, and significantly improved the coincidence detection in neurons of high CF, suggesting that Ih may improve the localization of sound sources.

Animals↗

PubMed2003

Halothane inhibits an intermediate conductance Ca2+-activated K+ channel by acting at the extracellular side of the ionic pore.

BACKGROUND: Actions of volatile anesthetics on ligand-gated ion channels, such as gamma-aminobutyric acid type A receptors, have been studied extensively. However, actions on other types of channels, such as K+ channels, are poorly understood. The authors previously showed that a Ca2+-activated K+ channel, IK, is sensitive to halothane, whereas SK1, another Ca2+-activated K+ channel, is insensitive. To explore how halothane acts on Ca2+-activated K+ channels, chimeras between IK and SK1 were constructed, and halothane sensitivity was analyzed. METHODS: IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 microm Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations. RESULTS: Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK. CONCLUSIONS: It is suggested that halothane interacts with the extracellular part of the ionic pore of IK. Whether this type of interaction is involved in the mechanism of anesthetic actions on ligand-gated ion channels warrants further investigation.

Anesthetics, Inhalation↗