More than 70 different K⁺ channel subunits have been identified and their molecular structure and diversity were investigated over the past 15 years, though K⁺ channel subtypes less than 10 are known as native functional ones. Furthermore, the study of K⁺ channels on the presynaptic nerve endings had been quite limited in the a few huge nerve endings such as basket cell terminals and Calyx of Held on the hearing pathway where direct approach of patch pipette is possible. Therefore, we investigated the functional K⁺ channels on the small nerve endings having less than 1µm diameter of glycinergic interneurons projecting to spinal sacral dorsal commissural nucleus (SDCN) neurons. In the presence of TTX, whole-cell patch recording of mIPSCs was made using SDCN ‘synaptic bouton ’preparation attached with functional glycinergic nerve at a holding potential of 0mV. Glutamatergic and GABAergic inputs were inhibited by CNQX, AP-5 and bicuculline. The K⁺ channel blockers, 4-AP, TEA, δ-Dendrotoxin, Iberiotoxin, Charybdotoxin and Apamin increased considerably mIPSCs frequency without affecting the amplitude.Consequently, the existence of following K⁺ channel subtypes on nerve endings was estimated: i.e., KA > KIR > IKCa > BKCa. As Ca²⁺ channels on glycinergic nerve endings, L-, N-, P/Q- and R types were recognized using respective channel blockers: i.e., nifedipine, ω-CgTX, AgTX, Cd²⁺, though heterogeneous distribution of these Ca²⁺ channel subtypes was found among preparations tested. [Jpn J Physiol 55 Suppl:S123 (2005)]

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It is recognized that brain contains all the components of the renin-angiotensin systems (RAS). The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca²⁺ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca²⁺-dependent functions such as neurotransmitter release, enzyme activity and gene expression. The purpose of this study was to investigate the effects of Angiotensin II (Ang II) on VDCCs currents (I_Ca) in the NTS using patch-clamp recording methods. An application of Ang IIcaused facilitation of L-type I_Ca. AT₁ receptors antagonist, Losartan antagonized the Ang II-induced facilitation of I_Ca. Intracellular dialysis of the G_i-protein antibody attenuated the Ang II-induced facilitation of I_Ca. Both Src tyrosine kinase inhibitor and mitogen activated protein kinase (MAPK) inhibitor attenuated the Ang II-induced facilitation of I_Ca. These results indicate that Ang II facilitates L-type VDCCs via G_i-proteins involving Src tyrosine kinase and MAPK kinase mediated by AT₁ receptors in NTS. [Jpn J Physiol 55 Suppl:S123 (2005)]

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In olfactory receptor cells, it is well established that cAMP acts as a main second messenger during odor responses. Biochemical experiments, however, showed failure of accumulation of cAMP in olfactory cilia by odorants of 40% examined (Sklar et al., 1996). We have shown that application of water soluble odorants and volatile odorants induced inward currents in olfactory cells of the Xenopus water nose but dialysis with cAMP did not (Iida and Kashiwayanagi, 1999). Breer and Boekhoff showed that odorants, which did not induce cAMP accumulation in olfactory cilia, induced IP3 accumulation (1990). Dialysis of olfactory cells in Xenopus water nose with IP3 induced inward currents. In addition, the turtle olfactory cells responded to dialysis with cyclic ADP-ribose (cADPR) with an inward current (Sekimoto and Kashiwayanagi, 2003). The magnitudes of the inward current responses to cAMP-increasing odorants were greatly reduced by prior dialyses of a high concentration of cADPR or 8-Br-cADPR, an antagonist. It is possible that IP3 and cADPR play as second messengers during the olfactory transduction. At present, channel molecules which are activated by these second messenger candidates have not been identified in olfactory cells. Transient receptor potential (TRP) channels, which are expressed in many tissues and cell types in vertebrates, are activated by various stimulations. In the present study, we explore roles of TRP channels in the olfactory system by RT-PCR and immunohistochemical techniques. [Jpn J Physiol 55 Suppl:S123 (2005)]

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TrkB receptors, whose ligands are brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-4/5, are known to be essential for neuronal proliferation and differentiation during development, but growing evidence suggests that they are crucial modulators for synaptic plasticity in adults as well. Although TrkB receptors are expressed at both pre- and postsynaptic sites in the hippocampal CA1 region, their precise roles are not well understood. Thus, we have generated conditionally gene-targeted mice in which the knockout of the trkB gene is restricted to the CA3 region from which presynaptic afferents in the CA1 region originate. Adult mutant mice show impairment of short-term presynaptic plasticity and long-term potentiation induced by high-frequency conditioning stimulation. Our results suggest that the presynaptic TrkB receptor plays significant roles in the modulation of hippocampal synaptic plasticity. We will discuss possible mechanisms by which presynaptic TrkB receptors regulate hippocampal synaptic plasticity. [Jpn J Physiol 55 Suppl:S124 (2005)]

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We previously reported an ascidian protein Ci-VSP which has a transmembrane voltage sensor motif with significant homology to voltage-gated channels and a phosphatase domain just downstream of the transmembrane region. We showed that the voltage sensor functionally couples with the phosphatase domain, and that the proximal 8 amino acid in the linker region between the voltage sensor domain and phosphatase domain plays an important role in this coupling. In this report, we further examined this coupling by functional expression of Ci-VSP in Xenopus oocyte. First, we deleted the most proximal or distal half of the 8 amino acid linker segment and also made some point mutants. cRNAs encoding these mutants were coinjected with GIRK2 phosphoinositide-sensitive ion channels into Xenopus oocyte and the two-electrode voltage clamp recording was performed. Changes of the phosphatase activity with membrane potentials were detected by monitoring changes of ion currents through GIRK2 channels. Some mutants showed no significant change of the amplitude of GIRK currents. Secondly, we analyzed the voltage dependency of the coupling with mutant constructs in which properties of voltage sensor movement are altered. Some mutants showed shift of the Q-V curve of gating current. We are currently testing whether voltage-dependency of phosphatase activity is similarly shifted in those mutants. [Jpn J Physiol 55 Suppl:S124 (2005)]

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We previously reported that many of area postrema (AP) neurons co-express nAChR and P2X receptor (P2XR) in immature rat brain. Since the negative interaction between nAChR and P2XR has been reported in some neurons, and Xenopus oocytes expressing P2X2 and α3β4 channels, we investigated whether the same interaction operates between two receptors on AP neurons. The amplitude of the ACh(100μM)-induced current in the presence of 2, 10, 20, 50, or 100μM ATP was 95 ±1% (N=20), 84 ±2% (N=24), 60±4% (N=15), 48±3% (N=30), or 44±7% (N=6), respectively, of the mean of the preceding and following responses to ACh alone. Also, the current caused by combined application of 100μM ACh and 20, 50, or 100μM of ATP was only 82±2% (N=16), 72±2% (N=18), or 75±2% (N=14) of the predicted sum of the individual responses. Unexpectedly, we also found the similar negative interactions between nAChR and kainate receptor (KAR), and between P2XR and KAR. These results suggest that the negative interactions between the different receptors modify the strength of excitatory neurotransmission when plural transmitters are simultaneously released. [Jpn J Physiol 55 Suppl:S124 (2005)]

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Bone marrow stromal cells have hardly been studied physiologically, although they are given attention to as well as hematopoietic stem cells. We investigated intracellular calcium signals in rat bone marrow stromal cells using fura-2 AM. UTP, an agonist of P2Y₂ receptor (G protein-coupled purinergic receptor) induced an increase in intracellular calcium concentration. An immunohistochemical study showed the expression of P2Y₂ receptors in these cells. Depletion of intracellular calcium stores with thapsigargin induced store-operated calcium entry (SOC). These calcium responses changed depending on the cell density. The SOC-induced calcium increase was largest at low density, whereas UTP-induced calcium increase was largest at high density. Releasable calcium contents were constant regardless of cell density. UTP induced calcium oscillation, of which frequency was highest at medium density. These cell density-dependent differences in calcium responses may suggest cell growth-dependent changes in calcium signaling mechanism. [Jpn J Physiol 55 Suppl:S124 (2005)]

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Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. In these processes, intracellular Ca (Ca_i)plays important roles for the cellular functions. Recently, we have found that hMSCs demonstrate spontaneous Ca_i oscillations without agonist stimulations, which result primarily from the release of Ca from intracellular stores via InsP₃ receptors. The precise mechanisms for its generation are not clear. In this study, we investigated the underlying mechanisms of spontaneous Ca_i oscillation using by conforcal Ca imaging. By the application of blockers for P₂Y receptor (PPADS) and P₂Y₁ receptor (A3P5PS and BzATP), Ca_i oscillations were completely blocked. The application of hexokinase, an ATP scavenger, completely blocked Ca_i oscillations, suggested that ATP stimulates P₂Y₁ receptors to produce InsP₃. To confirm this, we directly measured ATP in the supernatant by luciferin-luciferase assay and found the increase of ATP in the medium with hMSCs, indicating the existence of an autocrine purinergic signaling loop. Ca_i oscillations were completely blocked by connexin blockers but not Cl channel blockers. From these results, we conclude that spontaneous Ca_i oscillations are generated by an ATP autocrine signaling in hMSCs, which is composed primarily by hemi-gap-junction channels, P₂Y₁ receptors, PLC-β and InsP₃ induced Ca release. [Jpn J Physiol 55 Suppl:S125 (2005)]

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According to β-switch hypothesis (Findlay 2002), inactivation of I_Ca,L is physiologically dominated by a rapid voltage-dependent process. During β-sympathetic-stimulation, however, this process is replaced by a Ca²⁺-dependent inactivation. Based on his experimental data, we provided two sets of formulation for voltage-dependent inactivation of I_Ca,L (control: steady-state inactivation(f_inf) 0.2 with time constant(τ) 25msec, β-stimulated-state: f_inf=0.55 and τ=500msec at +50mV). They were incorporated into Luo-Rudy model, and our model equipped with Ca²⁺-entry dependent inactivation (Biophys.J.2003), to compare effects on action potentials. Introduction of β-type voltage-inactivation into Luo-Rudy model produced failure of repolarization, in spite of increased Ca²⁺-dependent inactivation due to elevated [Ca²⁺]_i. Repolarization was achieved by the augmented I_Ks, another important factor to modulate action potential duration (APD) during β-stimulation. When I_Ks was doubled in amplitude, however, APD was still highly prolonged. On the other hand, β-type voltage-inactivation could be incorporated into our new model without prominent effects in APDs. This was because Ca²⁺ entered through the channel efficiently produced Ca²⁺-dependent inactivation. Fine-tuning of gating parameters to produce best action potentials, however, was not fully consistent with conditions to simulate voltage-clamp experiments (current decay during square pulses). Further experimental as well as computational studies are required to clarify the highly complicated mechanisms of I_Ca,L inactivation. [Jpn J Physiol 55 Suppl:S125 (2005)]

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Recently HL-1 cells became an important tool in cardiac research due to their unique origin. However, the characteristic physiology of HL-1 cells is still not completely described. Therefore, this study is designed to elucidate the electrophysiological properties of one of the basic transporters: Na/K pump in HL-1 cells by means of whole-cell patch-clamp method. A pipette- and bath- solution content was made in order to minimize the rest of transmembrane conductance. Na/K pump current (I_p) was elicited by the use of two different voltage clamp protocols–square pulses between −120 and 40 mV with 10 mV increment from holding potential −40 mV and ramp pulses with the same voltage range (dV/dt = 0.1 V/s). I_p was identified as ouabain-sensitive time-independent current which is inhibited by ouabain with IC₅₀ = 131.7 μmol/L. Through the applied voltage range I_p was outward current showing similar to I_p of other mammalian cells current/voltage relationship: I_p increase with more depolarizing steps and reach plateau at around 0 mV. Current density obtained from 12 cells varied from 0.18±0.12 pA/pF for −120 mV to 1.70±0.45 pA/pF for depolarization 0 mV (cell capacitance was 39.75±8.26 pF).Based on experimental data it is concluded that electrophysiological properties of Na/K pump in HL-1 culture cells is qualitatively comparable to that in native mammalian cells. [Jpn J Physiol 55 Suppl:S125 (2005)]

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G protein-gated inwardly rectifying K⁺ (GIRK) channels are known to be activated by direct binding of Gβγ released from Gi/o protein, and inhibited by stimulation of coexpressed Gq-coupled receptors. Inhibitory mechanisms of the GIRK channels are thought to be due to phosphorylation by PKC and/or depeletion of PIP₂. To better understand the inhibitory mechanisms, we analysed the GIRK current expressed in Xenopus oocytes under two-electrode voltage clamp, focusing on the turning -on and -off phases upon application or removal of the ligands of the coexpressed Gi/o-coupled receptors. The time constants, τ_on and τ_off, obtained by fitting the -on and -off phases with single exponential functions, are parameters which reflect Gβγ binding to or dissociation from the GIRK channels. By pre-stimulating Gq-coupled receptors coexpressed with wild-type GIRK channels, we observed reductions of the basal and ligand induced current amplitudes, an increase in τ_on and a decrease in τ_off. As PIP₂ is abundant in oocytes, we expected the effect of PIP₂ depletion to be minor, and focused on the phosphorylation by PKC. When PMA, a direct activator of PKC, was applied in advance, similar changes were observed. These changes were not observed in the GIRK1(S185A)/GIRK2(S196A) mutant whose PKC-phosphorylation motifs are mutated. These results suggest that the affinity of GIRK channel to Gβγ is decreased by phosphorylation of these sites by PKC, resulting in speed-up of turning on, slow down of turning off and reduction of current amplitude. [Jpn J Physiol 55 Suppl:S125 (2005)]

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The protein kinase Cβ inhibitor LY333531 has been shown to be effective at alleviating diabetic hyperalgesia in experimental animals. However, it still remains unknown as to whether LY333531 affects the excitability of small dorsal root ganglion (DRG) neurons, which primarily transmit nociceptive information. The present study was undertaken to examine the effect of LY333531 on the tetrodotoxin (TTX)-resistant Na+ current in small (≤25 μm in soma diameter) DRG neurons in streptozocin (STZ)-induced diabetic rats, using the whole-cell patch-clamp method. The cell membrane was initially hyperpolarized from a holding potential of -70 mV to -120 mV for 20ms and then depolarized to various test potentials ranging from -50 to +40 mV, in the presence of TTX (0.1 μM) and the appropriate blockers for Ca²⁺ and K⁺ currents. The TTX-resistant Na+ current, identified as a transient inward current during depolarization, peaked at a test potential of 0 mV and its maximal density was 44.2±12.6 pA/pF (n = 8). Bath application of LY333531 (≥0.1 μM) acutely inhibited the TTX-resistant Na+ current, which was partially reversed upon washout of the drug. Maximal effect (34.8±5.5% inhibition, n = 8) was obtained with 1 μM LY333531. Thus, LY333531 substantially inhibits the TTX-resistant Na⁺+current in diabetic rats, which appears to mediate at least partly the anti-hyperalgesic actions of LY333531 in diabetes. [Jpn J Physiol 55 Suppl:S126 (2005)]

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We previously observed that the channel pore properties such as rectification and ionic selectivity of ATP receptor P2X₂ change depending on the expression level, suggesting dynamic structural rearrangements. However, the structure, even the stoichiometry, remains unknown. Towards the structure analysis, we aimed to purify recombinant P2X₂ protein. We first made constructs of P2X₂ tagged with FLAG at either N-or C-terminus, and confirmed that they have intact function when expressed in Xenopus oocytes. We then made them express in insect Sf9 cells using baculovirus expression system and solubilized the FLAG-tagged P2X₂ proteins of membrane fraction in a solution containing 50 mM dodecyl-β-maltoside. They were purified by FLAG affinity gel and further by gel filtration chromatography. A single major band was successfully observed by silver staining and Western blotting of SDS-PAGE. By evaluating the molecular size of the purified protein treated by 25 mM glutaraldehyde which bridges the subunits, P2X₂ protein was shown to be a trimer. The trimer band on SDS-PAGE shifted upwards by pretreating with 200 μM ATP, but the shift was not observed when ATP was applied after glutaraldehyde treatment. These results suggest that a sort of conformational changes were induced by ATP. Electron microscopic images of negative-stained proteins also supported that P2X₂ is a trimeric protein. [Jpn J Physiol 55 Suppl:S126 (2005)]

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The slow component of delayed rectifier K⁺ current (I_Ks) is essential for the repolarization process of cardiac action potential. I_Ks is also markedly potentiated by both α₁– and β–adrenergic agonists, which is assumed to reduce excess Ca²⁺ influx through the simultaneously stimulated L–type Ca²⁺ channels by facilitating the repolarization of action potential during elevated sympathetic tone. The objective of the present study was to investigate which isoform of PKC mediates the potentiation of I_Ks by α₁–adrenoceptor stimulation in guinea–pig atrial myocytes, using whole–cell patch-clamp method. I_Ks was activated by depolarizing voltage–clamp steps to various test potentials in atrial myocytes dialyzed with a control pipette solution containing 5 mM EGTA and 0.5 mM CaCl2 (free Ca²⁺ concentration, about 10⁻⁸ M). The stimulation of α₁–adrenoceptor with 30 μM phenylephrine maximally increased the amplitude of I_Ks by 77.6±8.7% (n = 14). This stimulatory action of phenylephrine (30 μM) was significantly reduced (21.2±4.6% increase, n = 6) when atrial myocyte was loaded with the PKCε–selective peptide inhibitor (PKCεV1–2, 100 nM) but was minimally affected (99.1±20.7% increase, n = 6) by loading with the selective inhibitor peptide of the classical PKC isoforms (PKCβC2–4, 100 nM). These observations suggest that the novel PKC isoform PKCε is primarily involved in the α₁–adrenergic potentiation of I_Ks in guinea–pig atrial myocytes. [Jpn J Physiol 55 Suppl:S126 (2005)]

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KCNQ3 (OMIM 602232) is a voltage-gated K-channel alpha subunit mainlyexpressed in CNS neurons. KCNQ3 forms a heterotetrameric K-channel withits homolog KCNQ2, and this KCNQ2/KCNQ3 heterotetramer provides theM-current, a delayed-rectifier type persisting K-current that regulatesneuronal excitability. A recent study has identified a point missensemutation in the pore region of KCNQ3 that leads to idiopathic neonatalepilepsy BFNC2 (OMIM 121201). However, functional evidence that linksthis mutation to epileptic neuronal hyperexcitability has so far beenlacking. In this study, we examined physiological properties of themutant KCNQ3 heterologously expressed in HEK293 cells . Our results show:1) when expressed solely, mutant KCNQ3 channels were expressed onthe plasma membrane but generated no obvious current; 2) when co-expressed with KNCQ2, KCNQ2/KCNQ3 mutant channels generated a current similar to wild-type KCNQ2/KCNQ3 channels; 3) the KCNQ2/KCNQ3 mutant current had very similar activation kinetics and voltage-dependence to the wild-type KCNQ2/KCNQ3 current, but its conductance was a way smaller than the wild-type current. We conclude that this KCNQ3 mutation reduces the conductance of M-current without changing its voltage-dependent gating, and thus causes hyperexcitability of CNS neurons that leads to the BFNC2 phenotype. [Jpn J Physiol 55 Suppl:S126 (2005)]

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The ryanodine receptor (RyR) releases Ca ions from the SR lumen to the cytoplasm as an Excitation-Contraction coupling mechanism of the cardiac myocytes. The RyR channel activities are physiologically or pathophysiologically regulated by various cytosolic biochemical molecules. Here we review the molecular mechanisms on the intracellular regulation we found thus far. (1) Spermine of a polyamine is present in cardiac myocyte. Cytoplasmic spermine formed a nonlinear current-voltage relation in the RyR channel in which the outward Ca current from the cytoplasm to the SR lumen was blocked. This inwardly-rectification could suppress a counterflow of cytoplasmic Ca ion to the SR lumen during the muscle contraction. (2) Phosphorylation of the cardiac RyR by cAMP-dependent protein kinase A (PKA) increased the channel activity. This increase in the RyR channel activities by the phosphorylation was due to the loss of sensitivity to the cytoplasmic Mg ion of the phosphorylated RyR molecule, which could contribute to the positive inotropic effect in the heart via the sympathetic nerve stimulation. (3) Sphingosylphosphatidylcholine (SPC) is metabolized from sphingomyelin of a minor sarcolemmal phospholipid during the apoptosis and the hyperlipidemia. The RyR channels were blocked by the cytoplasmic SPC. (4) Lysophosphatidylcholine (LPC) is metabolized from phosphatidylcholine of a major sarcolemmal phospholipid during the cardiac ischemia. The RyR channels were activated by LPC. [Jpn J Physiol 55 Suppl:S127 (2005)]

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M-currents are known to be inhibited by stimulating Gq-coupled receptors such as M1 receptor. M-currents can be reconstituted by expressing both KCNQ2/3 channels and M1 receptors either in mammalian cell lines or in Xenopus oocytes. However, the inhibition by stimulating co-expressing M1 receptors looks quite different in each expression system. Application of oxo-M or acetylcholine reduces KCNQ2/3 currents in HEK293T cells with no apparent change of voltage dependency while it shifts the conductance-voltage (G-V) curve of KCNQ2/3 in oocytes with no obvious reduction of maximum conductance. In this study, we aimed to know why there is a discrepancy between them. Among the signaling pathways of Gq, PIP₂ hydrolysis and/or PKC activation are supposed to be important for the inhibition of M-current. When we applied 200 nM PMA (PKC activator) to KCNQ2 channels expressed in oocytes, we observed 20 mV positive shift of G-V curve with no significant reduction of maximum conductance. Next we incubated oocytes expressing KCNQ2 channels in 10 μM wortmannin for 30 min to reduce PIP₂ and found that a minor shift of G-V curve was seen although only 20% of maximum currents were remained. These results suggest that PIP₂ breakdown and PKC contribute to M-current inhibition differently: the maximum conductance is affected by PIP₂ concentration and the voltage dependency is affected by phosphorylation by PKC. In HEK293T cells, V_1/2 of the G-V curve was around -20 mV and close to V_1/2 in PMA-treated oocytes. In addition, the currents were less sensitive to PMA. KCNQ2/3 channels may be constitutively phosphorylated in HEK293T cells. [Jpn J Physiol 55 Suppl:S127 (2005)]

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Activity regulation by phosphoinositides (PIP_ns) are known for various ion channels. In this study we aimed to examine whether the ATP-gated P2X₂ receptor channel is also regulated by PIP_ns or not, and analyzed the electrophysiological properties of P2X₂ wild type expressed in Xenopus oocytes under two-electrode voltage clamp. We observed that the preincubation in wortmannin or LY294002, PI3K inhibitors, accelerated the channel desensitization, while the preincubation in PAO, a PI4K inhibitor, decelerated it. As all PIP_ns are anionic lipids, we focused as the structural determinant on the proximal region of the C-terminus cytoplasmic domain where positively charged amino acids are clustered. We analyzed properties of several mutant P2X₂ channels, and observed that K360Q, K365Q, K365N, K369Q, R371Q or K374Q mutation accelerated the desensitization, while K365R did not. These results suggest that these positively charged amino acid residues play critical roles in preventing the channel desensitization. As a next step we purified a GST-tagged recombinant protein from L353 to S377 expressed in E. coli., and analyzed their binding to anionic lipids using a PIP_ns-coated nitrocellulose membrane. We observed that the recombinant protein including the positively charged region was bound to PIPs and PIP₂s, most strongly to PI_(3,5)P₂. Taken together, we speculate that an electrostatic binding of PI_(3,5)P₂ to the proximal C-terminus cytoplasmic domain plays a critical role in maintaing the channel activity. [Jpn J Physiol 55 Suppl:S127 (2005)]

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The synthetic ciguatoxin, CTX3C enabled us to explore the physiological effects of ciguatoxin, scarcely obtained as a natural product, on Na⁺ channels. We have previously reported, 1) the toxin speeds up time to peak, 2) activates Na⁺ channels at hyperpolarized potentials (shifts the activation curve) and 3) slows the recovery from “slow inactivation”. Site-2 toxins, such as grayanotoxin or batrachotoxin are known to similarly activate Na⁺ channels. In the present study, therefore, as a step toward identifying CTX binding sites, we tested whether the residues in Na_v1.4, providing binding sites for Site-2 toxins, also influence CTX3C affinity. In addition to Site-2 residues, we explored some residues in D4S5, which had been previously recognized as a binding region by a photo affinity labeling technique. The CTX3C sensitivity of mutants for Site2-toxins (I433A, F1579A, F1586A) or those at D4S5, (F1483A or F1492A) were similar to that of wild type channels in all respects. However, one mutant of Site2-resides located at D1S6 (N434A) was resistant to the effect of CTX3C on channel activation: CTX3C (3 µM) did not shift the activation curve in the hyperpolarizing direction, while preserving other effects, such as effects on “slow inactivation”. These results indicate the residue N434 is involved in the mechanism utilized by CTX3C to enhance activation kinetics of Na channels. [Jpn J Physiol 55 Suppl:S127 (2005)]

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Background Nifekalant, a blocker of cardiac delayed rectifier K⁺ current (I_Kr) channels encoded by a human ether-a-go-go related gene (HERG), is reported as the drug clinically effective for lethal ventricular tachyarrhythmias. But the kinetic properties of nifekalant on HERG channels are not studied sufficiently. So we attempted to analyze in detail the properties and find the characteristics. Methods and Results By using the standard two-microelectrode voltage clamp technique on HERG channels expressed in Xenopus oocytes, nifekalant blocked HERG channels in a use-dependent and non frequency-dependent manner, and caused a negative shift of the voltage-dependence of activation. Nifekalant increased HERG channel current at low voltages only in the presence of a previous strong depolarizing pulse, and so this pulse could separate the facilitation effect from the block effect. Without the facilitation effect, nifekalant blocked HERG channel current in a weak voltage-dependent manner. On the other hand, with the facilitation effect, nifekalant caused a significant negative shift in the voltage-dependence of activation. In the case of E-4031, a methanesulfonanilide anti-arrhythmic drug, the facilitation effect was not found. Conclusions The strong depolarizing prepulse allows nifekalant to increase the HERG channel currents, and the facilitation effect might be the unique characteristic of nifekalant. [Jpn J Physiol 55 Suppl:S128 (2005)]

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The strong inward rectification of the channels composed of subunits from the Kir2 subfamily is mainly caused by voltage-dependent blockade of the outward currents by the intracellular polyamines, spermine and spermidine (Nichols & Lopatin, 1997). The mechanism of the polyamine blockade of the Kir2 channels is complicated, involving more than two blocked sites or states (Yang et al. 1995; Guo & Lu, 2000). Consequently, a simple model that can describe both the voltage- and polyamine concentration-dependences of outward current amplitudes had not yet been proposed. Recently, we studied the blockade of Kir2.1 currents by spermine and spermidine and showed that the macroscopic conductances are well described by the sum of the currents through two populations of Kir2.1 channels with different susceptibilities to the polyamine blockade (Ishihara & Ehara, 2004). This finding explains well the steady-state outward current amplitude of the cardiac strong inward rectifier K⁺ current, I_K1, which plays a significant role in repolarizing the cardiac action potential. Since it has been shown that the Kir2.2 subunit co-assembles with the Kir2.1 subunit to form the I_K1 channel in some animal species, in this study, we analyzed the polyamine blockade of the Kir2.2 channels expressed in HEK 293T cells. [Jpn J Physiol 55 Suppl:S128 (2005)]

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It is controversial whether AMPA receptor (AMPAR) desensitization contributes to synaptic depression. At the calyx of Held in the rat brainstem, AMPAR-mediated EPSCs (AMPA-EPSC) show strong paired-pulse depression at postnatal day 7 (P7), but the magnitude of depression decreases as animals mature (Iwasaki & Takahashi, 2001). During postnatal development the recovery time from AMPAR desensitization, induced by paired-pulse glutamate (10 mM) applications to outside-out patches from postsynaptic neurons, also decreased. Cyclothiazide (CTZ) completely abolished AMPAR desensitization at all ages of animals examined (P7-P22) and significantly reduced paired-pulse depression (PPD) of AMPA-EPSCs at P7, but not after P14. Quantitative single-cell RT-PCR analysis showed that the transcripts for GluR1 subunits and those for the R/G-site-unedited GluR2-4 subunits decreased with development. Consistently GluR1 immunoreactivity in the medial nucleus of trapezoid body region decreased with development. Taken together with the report on recombinant AMPARs (Lomeli et al, 1994) these results suggest that the R/G-site-editing of GluR subunits underlies the developmental change of AMPAR desensitization. Our results also indicate that the contribution of AMPAR desensitization to synaptic depression decreases during postnatal development. [Jpn J Physiol 55 Suppl:S128 (2005)]

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We previously found that two types of inwardly rectifying K⁺ channels, homomeric Kir4.1 and heteromeric Kir4.1/5.1, differentially distributed in specific membrane domains of astrocytes, and were involved in K⁺ buffering. It is accompanied with Cl⁻ flux and causes osmotic gradient, resulting in movement of water. These physiological events in astrocytes are crucial for proper regulation of neural function. AQP4 is only one water channel in astrocytes and occurs together with the Kir channels on the same membrane. Although some proteins are suggested to sort these channels to the membrane surface, the mechanism coupling the K⁺ and water flux remains largely unknown. Here we report that lipid raft nanodomain may scaffold K⁺ and water channels in astrocytes. Fractionation experiments of mouse brain revealed that the two Kir channels were expressed in lipid rafts as well as in non rafts. AQP4 distributed exclusively in lipid rafts. In kidney and transfected MDCK cells, Kir4.l and Kir5.1 were localized only in non rafts, suggesting a particular system trafficking the Kir channels to lipid rafts in the astrocytes. Moreover, whereas ClC-2 chloride channel dominates in lipid rafts with its moderate expression in non rafts, Na⁺,K⁺-ATPase and Na⁺,H⁺-exchanger were enriched in non rafts. Astrocytes' membrane may therefore possess two distinct functional platforms, i.e. lipid rafts that synchronize salt and water flux, and non rafts that transports a variety of ions. [Jpn J Physiol 55 Suppl:S128 (2005)]

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The present study was designed to investigate the modulation of the HERG channel by membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) using the whole-cell patch-clamp method. Both HERG channel and M₁-muscarinic receptor (M₁R) were heterologously expressed in CHO cells either with or without phosphatidylinositol-4-phosphate 5-kinase (PI(4)P5-K) by transient transfection. The HERG current was activated by depolarizing voltage-clamp steps to various test potentials and the degree of HERG current activation was determined by the amplitude of tail current elicited on repolarization to -60 mV. The density of HERG current (36.6±6.2 pA/pF, n = 6) following a 2-s depolarizing step to +10 mV in cells heterologously expressing PI(4)P5-K was approximately two times larger than that (16.3±3.8 pA/pF, n = 6) in cells without its expression. The voltage-dependence of current activation and kinetics of current deactivation were minimally affected by heterologous expression of PI(4)P5-K. In cells without heterologous expression of PI(4)P5-K, the stimulation of M₁R with 30 μM ACh reduced the HERG current by 21.9±3.9% (n = 7), accompanied by acceleration of current deactivation, which was significantly attenuated by heterologous expression of PI(4)P5-K (10.6±2.1% decrease, n = 6). These results suggest that the HERG channel is closely dependent on changes in PIP₂ level which are regulated by PI(4)P5-K and M₁R. [Jpn J Physiol 55 Suppl:S129 (2005)]

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DRG neurons express 7 voltage-gated Na⁺ channel α-subunits, 5 of which being sensitive to tetrodotoxin (TTX) and the remaining 2 being resistant. These channels show preferential distribution within DRG depending on the type of neurons, mediating Na⁺ currents (I_Nas) with distinct kinetics. The preferential distribution of voltage-gated Na⁺ channels may bring about differential mechanisms of action potential generation. However, it is not known how each type of Na⁺ channels contributes to the generation of action potentials. Therefore, we investigated the correlation between I_Nas in voltage-clamp recordings and corresponding action potentials in current-clamp recordings to clarify action potential electrogenesis in DRG neurons. We classified I_Nas recorded in DRG neurons into four types on the basis of TTX sensitivity and kinetic properties. The action potentials in small DRG neurons were dependent on TTX-R/slow I_Na mediated by Na_V1.8, whereas action potentials in large DRG neurons were mediated by TTX-S/fast I_Na. TTX-S/fast I_Na was switched off in small DRG neurons due to a hyperpolarizing shift of the steady-state inactivation. We also found that TTX-R/persistent I_Na mediated by Na_V1.9 and TTX-S/persistent I_Na regulate subthreshold excitability in small and large DRG neurons, respectively. Thus, our results demonstrate that the action potentials in DRG neurons are generated and regulated with distinct mechanisms that may give rise to differential functional properties of DRG neurons. [Jpn J Physiol 55 Suppl:S129 (2005)]

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LPC has diverse actions on the function of various cell types including cardiac myocytes. Some of LPC actions are mediated through a G protein-coupled receptor highly selective for this phospholipid. The present study was designed to examine whether LPC receptor stimulation affects the slow component of delayed rectifier K⁺ current (I_Ks) in guinea-pig cardiac mycocytes and the KCNQ1/KCNE1 channel current heterologously expressed in HEK 293T cells, using the whole-cell patch-clamp method. Bath application of LPC (C16:0) concentration-dependently (EC50 = 1.1 μM) and reversibly increased I_Ks in atrial myocytes but produced minimal if any effect in ventricular myocytes. LPC (C6:0), LPC (C18:1) and phosphatidylcholine had almost no stimulatory effect on I_Ks. Pretreatment of atrial myocytes with an antibody against N-terminus of the LPC receptor G2A significantly reduced the LPC (C16:0)-induced potentiation of I_Ks. Immunohistochemistry confirmed that G2A-positive reaction was densely present in the plasma membrane of atrial myocytes but was much less distributed in that of ventricular myocytes. Blockers for heterotrimeric G proteins, phospholipase C (PLC) and protein kinase C (PKC) significantly attenuated the LPC (C16:0)-induced enhancement of I_Ks. LPC (C16:0) also significantly enhanced the KCNQ1/KCNE1 current when coexpressed with G2A but had little effect without coexpression of G2A. Thus, the present study provides strong evidence to suggest that stimulation of the G2A receptor coupled to G protein potentiates I_Ks via a PLC-PKC pathway. [Jpn J Physiol 55 Suppl:S129 (2005)]

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Sensory neurons in dorsal root ganglion (DRG) express two kinds of tetrodotoxin resistant isoforms of the voltage-gated sodium channel, Na_V1.8 (SNS) and Na_V1.9 (NaN). These tetrodotoxin-resistant isoforms play key roles in pathophysiology of chronic pain. Of special interest is Na_V1.9, which is preferentially expressed in small DRG neurons. Na_V1.9 mediates a persistent sodium current, which shows an extremely slow time course distinct from the sodium current mediated by Na_V1.8. Previously, we reported the unique properties of the sodium current mediated by Na_V1.9 (I_{TTX-R/persistent}) in neurons from Na_V1.8-null mutant mice, i.e., I_{TTX-R/persistent} recorded under whole-cell patch-clamp condition showed an explosive up-regulation of the peak amplitude of the current (“kindling” of I_{TTX-R/persistent}) during recording. However, the mechanism underlying the kindling of I_{TTX-R/persistent} is still unclear. Intracellular ATP (3 mM) and GTP (500 μM) significantly suppressed the kindling of I_{TTX-R/persistent}. The effect of ATP was more potent than the effect of GTP. In these cases, the shift of the steady-state inactivation was not observed. A PKC activator, PMA (10 nM) also inhibited the kindling of I_{TTX-R/persistent}. In the presence of a PKC inhibitor, Calphostin C, the effect of PMA was not observed. In addition, PMA shifted the steady-state inactivation. These results suggest that the functional expression of I_{TTX-R/persistent} may be strongly affected by a change in intracellular conditions. [Jpn J Physiol 55 Suppl:S129 (2005)]

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Humans distinguish between five primary tastes: bitter, sweet, sour, salty and umami. Bitter taste was reported in 2001 to be recognize through G-protein coupled receptors encoded by the T2R family of taste receptor genes. We proposed that sweet and umami taste might also be recognized by T2Rs, and that there is a connection between certain taste disorders and the family of T2R genes. We attempted to compare the gene expression of the T2R family between a group having known taste disorders and a group having normal taste perception. To evaluate clinical qualitative and quantitative taste perception, filter-paper disc (FPD) testing was used. Using in situ hybridization, the T2R16 taste receptor gene was observed between human tongue epithelia of normal subjects and those having disorders. Using Taste Receptor gene Expression Examination (TREE), we PCR-amplified T2R genes as well as the genes having high homology with T2R genes from human tongue epithelium mRNA. We found that T2R16 was expressed in normal subjects, but not expressed in those having disorders. In the normal taste group, some taste receptors were observed by TREE, whereas in the group having disorders, no taste receptor genes were observed. These findings indicated that TREE as well as FPD testing was useful for the assessment of human taste function. We suggested that it is possible to use the T2R genes as the biomarker for evaluating the disorder of the sense of taste. [Jpn J Physiol 55 Suppl:S130 (2005)]

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Small-conductance Ca²⁺-activated K⁺ (SK; SK1, SK2 and SK3) channels are widely distributed throughout the body. However, its role in the heart is unclear. The aim of this study was to identify and analyze SK channels expressed in rat heart. Rat heart cDNA library was screened, and nine positive phage colonies were detected. Eight of nine cDNAs were identical to reported SK3. One cDNA was similar to SK3 but its N-terminal 19 amino acid was different. We termed it SK3-b. RT-PCR experiment showed that SK3-b was found in rat heart (3/33), but not in brain, liver, lung or intestine. SK3 and SK3-b were stably expressed in HEK293. Electrophysiological experiments reveled that voltage dependency and apamin sensitivity were not different between SK3 and SK3-b. However SK3-b was less sensitive to intracellular Ca²⁺ compared to SK3. Activation of PKA, PKG or PKC did not modify SK3 or SK3-b current. In immunohistochemistry by anti-SK3 antibody, which detect both SK3 and SK3-b, strong staining has been observed in the outermost layer of blood vessel and in adjacent cardiomyocytes of heart. Summary, we cloned SK3 and novel channel SK3-b from rat heart. These channels may contribute to cardiac function and metabolism. [Jpn J Physiol 55 Suppl:S130 (2005)]

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Store-operated Ca²⁺ channels were stimulated using 4 microM thapsigargin, sarco(end)plasmic reticulum Ca²⁺ ATPase inhibitor, in Ca²⁺-free solution (no added Ca²⁺ + 1mM EGTA) followed by superfusion with control (Ca²⁺-containing) solution. This protocol resulted in a [Ca²⁺]_i increase, which was inhibited in the presence of 100 microM Gd³⁺, 100microM La³⁺ but was unaffected by addition of 1 microM Gd³⁺ or 10 microM H-89, protein kinase A inhibitor. A restoration from a high K+ solution(Ca²⁺-containing) to control solution also evoked an [Ca²⁺]_i increase during thapsigargin stimulation, and an [Ca²⁺]_i increase following reintroduction of Ca²⁺ during thapsigargin stimulation was completely inhibited by addition of 1 microM Gd³⁺ but was partially affected by addition of 10 microM H-89 . Thus, one effect of the reintroduction of Ca²⁺ is likely to be a hyperpolarized pulse. In conclusion, rat submandibular acinar cells have at least two types of Ca²⁺ channels; one is activated by the store-depletion in Ca²⁺-free solution, and the other is by the store-depletion with a hyperpolarized pulse. [Jpn J Physiol 55 Suppl:S130 (2005)]

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The voltage sensor of ion channels regulates permeation of ions. A protein recently identified from ascidian genome, named as Ci-VSP, contains voltage sensor homologous to ion-channel like voltage sensor and phosphatase domain. Ci-VSP tunes its phosphatase activity depended on membrane potential change. We cloned a similar protein from zebrafish which is homologous to Ci-VSP. Whole-mount in situ hybridization showed that zebrafish VSP (z-VSP) is expressed in gut cells during embryogenesis. The z-VSP shows moderately small amount of Off-gating current when expressed in Xenopus oocyte, whereas its On-gating current was not so robust. Half maximum activation of Off-gating current occurs around at 110 mV, 44mV higher than that of Ci-VSP. Insertion of additional positively charged residue, arginine, in the S4-like segment facilitated the movement of this domain, supporting that S4-like segment of z-VSP operates as the voltage sensor as in Ci-VSP. z-VSP shows voltage-dependent phosphatase activity, as detected by activation of co-expressed KCNQ2,3 channel which is known to be sensitive to PIP2. Despite less robust gating current, z-VSP shares the conserved nature of voltage-dependent tuning of phosphatase activity with Ci-VSP. Therefore, the nature of voltage-dependent phosphatase of VSP is not a rarity to marine invertebrate, but more widespread in the chordates. [Jpn J Physiol 55 Suppl:S130 (2005)]

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Ion channels provide the molecular bases for diverse physiological phenomena, including neuron function, respiration, absorption, secretion, and the osmotic and volume regulation of body fluids. Despite recent identification of the molecular nature and biophysical mechanisms of a wide variety of ion channels, there still remains a gap between the molecular properties of ion channels and their macroscopic physiological functions. Ascidians are marine sessile invertebrates, but the initial stage in their metamorphic life cycle, the tadpole larva, has a body form with many deep similarities to vertebrates. The larval nervous system, both dorsal and tubular, consisting of less than 100 neurons, underlies undulatory swimming. The whole genome of Ciona intestinalis, one of ascidians, has recently been sequenced. With the background provided by findings from classical physiology, when molecular tools of gene knockdown are combined with this recent flood of genomic information, C. intestinalis provides a unique opportunity to link molecular information of ion channel to physiology of whole organism. In this study, we construct a preliminary version of ion channel database of C. intestinalis based on genome information, gene expression profiles and functional expression. We summarize the repertory of the putative ion channel genes and their phylogenetic relationship with the gene sets of Chaenorhabditis elegans, Drosophila melanogaster, and the human genomes. [Jpn J Physiol 55 Suppl:S131 (2005)]

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Metabotropic glutamate receptor 1 (mGluR1) is crucial for some forms of synaptic plasticity. It is known to functionally couple to both Gq and Gs proteins, resulting in activation of phospholipase C and adenylyl cyclase, respectively. We confirmed the functional couplings of mGluR1 to different G proteins by monitoring changes in [Ca²⁺]_i (Gq pathway) and [cAMP]_i (Gs pathway) in CHO cells expressing mGluR1, by simultaneous use of Ca²⁺ indicator indo-1 and fluorescent proteins which report activation of protein kinase A. Application of glutamate transiently increased the [Ca²⁺]_i within 10 s after application, and gradually increased [cAMP]_i, which reached maximum several minutes later. The delayed increase in [cAMP]_i was not a result caused secondarily by the rapid increase in [Ca²⁺]_i, because the truncation form of mGluR1 (mGluR1β) evoked Ca²⁺ transient but failed to increase [cAMP]_i, as previously reported. In addition to the differential time course of the increase in [Ca²⁺]_i and [cAMP]_i, concentration dependency was different; increase in [Ca²⁺]_i was more sensitive to glutamate than that in [cAMP]_i. Interestingly, the functional couplings of mGluR1 to Gq and Gs pathways were ligand-type dependent: glutamate activates both Gq and Gs pathway, but Gd³⁺ activated only Gq pathway. These results suggest that the signaling pathways mediated by mGluR1 are differentially regulated by the concentration and types of ligands. [Jpn J Physiol 55 Suppl:S131 (2005)]

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Our previous study shows that calmodulin (CaM) Ca²⁺-dependently induces channel activity in the inside-out patch mode. Ca²⁺ channel activity induced by CaM increases with the increment of Ca²⁺ concentration ([Ca²⁺])in the range of 0∼300nM, but rapidly disappears at [Ca²⁺]>500nM. In the present study, we used CaM mutants, in which four Ca²⁺-binding sites were differently mutated, to investigate Ca²⁺-dependent actions of CaM on the L-type Ca²⁺channel in guinea pig ventricular myocytes. We found that increment of [Ca²⁺] from 80nM to 1μM had no effect on Ca²⁺ activity induced by CaM₁₂₃₄. However, Increment of [Ca²⁺] from <10nM to 1μM significantly increased channel activity induced by CaM₁₂. On the other hand, channel activity induced by CaM₃₄ disappeared when [Ca²⁺] was increased to 1μM. Furthermore, channel activity induced by CaM₁ and CaM ₂ was not inactivated by 1μM [Ca²⁺]. These results indicate that the Ca²⁺-dependent regulation of Ca²⁺ channel activity is mediated by CaM. CaM bifurcates the Ca²⁺-dependent facilitation and inactivation through its N and C lobes: N lobe (containing 1st and 2nd Ca²⁺ binding sites) is responsible for Ca²⁺-dependent inactivation, while C lobe (containing 3th and 5th Ca²⁺ binding sites) for Ca²⁺-dependent facilitation. [Jpn J Physiol 55 Suppl:S131 (2005)]

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Verrucotoxin (VTX) is the major component of venom from a stonefish (Synanceia Verrucosa). Stings of the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system leading sometimes to death. It is reported that VTX might modulate ATP-sensitive K⁺ (K_ATP) current in frog atrial fibres. However, the mechanism by which VTX acts on K_ATP current remains unclear. In this study, we examined whether VTX could inhibit K_ATP current in guinea-pig ventricular myocytes by the patch clamp method. VTX suppressed K_ATP current induced by pinacidil (a K_ATP channel opener) in a dose-dependent manner with a half maximum dose of 16.3 μg/ml. The effect of VTX on K_ATP current was suppressed by atropine (1 μM), a muscarinic receptor antagonist or 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, 100 nM), a M₃ muscarinic receptor antagonist. Moreover the effect of VTX on K_ATP current was attenuated by a PKC inhibitor chelerythrine (10 μM) but not by a PKA inhibitor H-89 (0.5 μM). These results suggest that VTX inhibits K_ATP current through the M₃ receptor- PKC pathway. [Jpn J Physiol 55 Suppl:S131 (2005)]

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Our previous studies suggested that mitochondria supplied ATP for activation of nonselective cation (NS) channels in response to muscarinic receptor (mAChR) stimulation in guinea-pig adrenal chromaffin cells. Thus, we investigated with fluorescent techniques which part of the cell membrane contained mAChRs and NS channels in guinea-pig chromaffin cells and whether mitochondria were present near the receptor and channel. The m4AChR was detected in the adrenal medulla and not in the cortex with the immunohistochemical technique and immunoblotting, whereas the m3AChR was not in either the adrenal medulla or the cortex. This result taken together with previous findings indicates that m4AChR is functionally associated with a NS channel in chromaffin cells. The confocal microscopic study revealed that the m4-like immunoreactivity was mainly distributed in the membrane area near the nucleus. The intracellular concentration of Na⁺, as measured with SBFI dye, increased in response to muscarine, and this increase was larger in the nuclear portion than in the non-nulcear portion. Mitochondria, as visualized with rhodamine 123, were also localized underneath the plasma membrane in the nuclear portion. The results indicate that the m4AChR and probably NS channels are distributed in the plasma membrane near the nucleus and mitochondria may be present underneath such a membrane. [Jpn J Physiol 55 Suppl:S132 (2005)]

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Conductance of myocardial gap junctions is controlled by divalent cations, such as Ca²⁺ and Mg²⁺in addition to the trans-junctional potential. Although the steady-state dose-dependency is available, kinetics of the chemical gating is not determined. Thus, it is difficult to estimate the gap junctional conductance during the Ca²⁺ transient accompanying the cardiac contraction. We applied the two-electrodes voltage clamp technique to one of the paired ventricular myocytes (cell 1) isolated from hearts, and measured clamp currents evoked by ±5 mV test pulses from a holding potential of 0 mV. After replacing the external solution to a Cs⁺-rich intracellular solution containing a given concentration of divalent cation, the membrane of the paired cell (cell 2) was instantaneously ruptured by applying a pulse of nitrogen laser near the gap junction. We first examined effects of Mg²⁺ because Mg²⁺ does not induce contraction or the Ca²⁺ release from SR, and is not influenced by the intracellular Ca²⁺ buffer. Immediately after rupturing the cell 2 membrane, the input conductance was enlarged to a peak of (220∼730) nS from a steady level of (8∼70) nS before the rupture. In the presence of divalent cations, the conductance then decreased over the following several seconds in a [Mg²⁺]-dependent manner. The conductance decay was best fit by a sum of two exponential functions with time constants of 3 and 106 sec at 10 mM [Mg²⁺]. [Jpn J Physiol 55 Suppl:S132 (2005)]

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The L-type Ca²⁺ channels have a wide tissue distribution and play essential roles in physiological responses. Reguration of L-type Ca²⁺ channels involves the assembly of macromolecular signaling complexes. To find out new molecule involved in the L-type Ca²⁺ channels modulation, we used the intracellular II-III linker of the α_1C subunit of the L-type Ca²⁺ channel as a bait in the yeast two-hybrid system to screen a human heart cDNA library. We identified clones encoding sequence of human COP9 signalosome subunit 5(CSN5)/Jun activation domain-binding protein1(Jab1). The α_1C subunit and CSN5/Jab1 were coimmunoprecipatated in rat heart and colocalaized in sarcolememal membranes and transverse tubules of cardiac myocytes. To examine the effect of CSN5/Jab1 on L-type Ca²⁺ channel activity, patch clamp experiments were performed. Inhibition of endogeneous CSN5/Jab1 expression using siRNA had no effect on voltage-dependent property of Ca²⁺ channels, although the inhibition markedly increased the peak amplitude of the Ca²⁺ current. Our results indicate that CSN5/Jab1 is a protein that plays a newly defined functional role in association with L-type Ca²⁺ channels. [Jpn J Physiol 55 Suppl:S132 (2005)]

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The lateral superior olive (LSO) is the first auditory center that processes differences in the sound level between the two ears. Here we report the developmental changes in GABA_B receptor-mediated presynaptic inhibition of GABAergic and/or glycinergic synaptic transmission onto developing rat LSO neurons. MNTB-evoked GABAergic and/or glycinergic inhibitory postsynaptic currents (IPSCs) were recorded from LSO neurons of acute brain stem slices at postnatal (P) 2 to 20 day old rats divided in three groups (P2–6, P8–12, P16–20) using conventional whole-cell patch clamp technique. The pharmacological isolation of MNTB-evoked inhibitory transmission revealed a developmental switch from GABAergic to glycinergic IPSCs during these developmental stages. Bath application of baclofen, a selective GABAB receptor agonist, greatly reduced IPSC amplitude in neonatal (< P6) with a significant change in the paired-pulse ratio, and these effects were eliminated in the presence of GABA_B receptor antagonist, suggesting that baclofen acts presynaptic GABA_B receptors to reduce the release probability of GABA and/or glycine from presynaptic nerve terminals. However, the GABA_B receptor-mediated presynaptic inhibition was gradually reduced with postnatal development so that baclofen had little effect on MNTB-evoked IPSCs recorded from P16–20 LSO neurons. Based on these results, the functional roles of presynaptic GABA_B receptors in the development of LSO neurons will be further investigated and discussed. [Jpn J Physiol 55 Suppl:S132 (2005)]

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The delta2 glutamate receptor (GluRdelta2), which is predominantly expressed at distal dendrites of cerebellar Purkinje cells, plays a crucial role in cerebellar functions; mutant mice deficient in GluRdelta2 (delta2^−/−) impair synapse formation and long-term depression (LTD) in the cerebellum, and consequently lead to motor discoordination. However, a fundamental question whether GluRdelta2 is activated by glutamate analogues has remained elusive. To address this issue, we here introduced a GluRdelta2 transgene, which had a mutation (Arg514Lys) in the putative ligand-binding motif conserved in all mammalian ionotropic glutamate receptors (iGluRs) and their ancestral bacterial periplasmic amino acid-binding proteins, into delta2^−/− mice. Surprisingly, a mutant GluRdelta2 transgene, as well as a wild-type GluRdelta2 transgene, completely rescued the abnormal phenotypes of delta2^−/− mice: motor discoordination, multiple climbing fiber innervation, parallel fiber spine morphology, and impaired LTD. Thus, these results indicate that the conserved arginine residue, which is crucial for the binding of iGluRs to glutamate analogues, is not essential for the restoration of GluRdelta2 functions in delta2^−/− mice. [Jpn J Physiol 55 Suppl:S133 (2005)]

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ATP induced ion currents observed in rat myometrial cells are thought to enhance uterine contractility. Though the properties of the currents through myometrial ATP channels were similar to those of P2X channels, the subtype of the channel has not been determined in the uterus. The purpose of this study was to determine which of P2X subtype is mainly expressed in myometrium. We have reported mRNA of P2X4 was detected as a dominant subtype in rat myometrium by quantitative PCR method. The sequence of mRNA of P2X4 was determined and the homology between myometrium and brain was higher than 99%. The cloned mRNA of P2X4 was transfected into the COS-7 cell line that has no ATP current. The whole cell patch clamp method was applied to the COS-7 cells. The expressed P2X4 channel currents were induced by external ATP application in the range between 0.01 and 0.5 mM. This channel was permeable to various monovalent ions (K⁺ = Cs⁺ > Na⁺ > Li⁺). The current was also activated by ADP, GTP, UTP, 2-methylthio ATP and αβ-methylene ATP, and was blocked by both suramin and PPADS (blockers of P2 and P2X channel). These results indicate that the expressed P2X4 channels resemble to ATP channels in native myometrial cells. However, desensitization to ATP was much faster (<60s) in expressed P2X4 channels, compared with myometrial ATP channels. The expression of P2X channels has been detected in pregnant rat myometrium. P2X4 may be the main functioning channel of myometrial ATP channel, leading to enhancement of uterine contractility. [Jpn J Physiol 55 Suppl:S133 (2005)]

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The ClC-7 chloride channel is crucial for bone resorption as it is part of osteoclast acid secretion mechanism, and the ClC-7 gene (ClCN7) KO mice shows sever osteoporosis. Heterozygous mutations in ClCN7 suggest to be responsible for autosomal osteopetorosis type II (ADOII). Although an extracellular acidification dependent Cl⁻ current was reported in ClCN7-expressing Xenopus oocytes, the characterization and structure-functional relationships of ClC-7 channel were still unknown. To clarify the future of ClC-7 channel in osteoclasts, we cloned the ClCN7 in mouse osteoclasts and investigated Cl⁻ currents in ClCN7-expressing HEK293 cells. Extracellular acidification to pH 6.0 evoked strongly Cl⁻ currents in ClCN7-expressing HEK293 cells, but slightly in mock-transfected cells. The acidification dependent Cl⁻ currents showed properties of outward rectification, Ca²⁺-independency and anion permeability order of Cl⁻≥I⁻>Br⁻>gluconate⁻, indicating that it is different from swelling-activated Cl⁻ currents. We also detected the expression of ClC-7 channel in membrane fraction of ClCN7 cDNA-transfecting HEK293 cells using Western blot analysis. A point mutation (G215R) of ClCN7 dramatically suppressed the acidification dependent Cl⁻ currents.These results suggested that the ClC-7 chloride channel in osteoclasts may only activate in acidic microenvironment during bone resorption and mutations in ClCN7 may be caused by dysfunction of osteoclasts in ADOII patients. [Jpn J Physiol 55 Suppl:S133 (2005)]

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Identification of a mechanosensing domain is central to the biophysics of stretch-activated (SA) channels. Here, we identify that Stress-axis Regulated Exon (STREX), a particular amino-acid sequence at the C-terminus of the stretch-activated, large-conductance, calcium- and voltage-activated potassium (BKCa) channels cloned from chick embryonic heart (we designated as SAKCa channels), is responsible for the stretch sensitivity through the interaction with cytoskeletal complex (EF1a/actin). Deletion of the STREX insert diminished the stretch sensitivity of the channel. Sequence analysis revealed that the ERA 672-674 sequence of the chick STREX is indispensable for channel stretch sensitivity and single amino acid substitution from Ala674 to Thr674 completely eliminated the stretch sensitivity. Co-expression of chick STREX-EGFP and SAKCa in CHO cells, induced a strong GFP signal in the cell membrane and inhibited the stretch sensitivity significantly, implying that the STREX requires binding partner(s) to contribute to the stretch sensitivity. We found that actin and elongation factor 1A (EF-1α) bind specifically to chick STREX and the knockdown of EF-1α by siRNA treatment severely inhibited the stretch sensitivity of SAKCa. These results suggest that SAKCa senses membrane tension through an interaction between STREX and cytoskeletal complex such as actin and EF-1α. [Jpn J Physiol 55 Suppl:S133 (2005)]

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It is generally considered that exocrine secretion is mediated by [Ca²⁺]_i increases in acinar cells. We investigated Ca²⁺ signaling in the exocrine tissues of the double knockout mice lacking inositol 1,4,5-trisphosphate receptors type2 (IP3R2) and type3 (IP3R3), in which exocrine dysfunctions were seen, in order to know Ca²⁺ signaling the contribution of these receptors to exocrine secretion. Muscarinic receptor (mAChR)-induced [Ca²⁺]_i increases were severely impaired in the salivary gland acinar cells prepared from the double mutants, while those in single knockout mice of IP3R2 or IP3R3 was only slightly reduced, compared to those in the wild-type mice. Next, agonist-induced [Ca²⁺]_i changes were similarly examined in pancreatic acinar cells. Pancreatic acinar cells dissociated from the double mutants failed to show the [Ca²⁺]_i increases in response to ACh or cholecystokinin. In contrast, acinar cells prepared from single knockout mice of IP3R2 or IP3R3 showed the Ca²⁺ signaling similar to that in the wild-type mice: the [Ca²⁺]_i increases initiated at the apical pole, propagated to the basal regions as Ca²⁺ waves and oscillated, suggesting the redundant role for IP3R2 and IP3R3. These results demonstrate that the severe impairment of exocrine secretion in the IP3R2/IP3R3 double knockout mice was due to the lack of Ca²⁺ signaling in these glands and that IP3R2 and IP3R3 are the major receptor subtypes responsible for the physiological function in exocrine cells. [Jpn J Physiol 55 Suppl:S134 (2005)]

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To examine the physiological roles of type 2 and type 3 inositol 1,4,5-trisphosphate receptors(IP3R2 and IP3R3) in vivo, we generated mice lacking these receptors, channels for intracellular calcium release. The double knockout caused severe hyposalivation, which resulted in difficulties in dry food intake leading to death shortly after the weaning period. A diet of wet mashed food could rescue the double mutants from the lethal phenotype. In these mice, pancreatic acinar cells were highly eosinophilic, suggesting that zymogen granules accumulated throughout the cytoplasm because of deficits in exocrine secretion. In fact, experiments using dissociated acinar cells demonstrated that secretion of digestive enzymes (such as amylase and lipase), in response to muscarinic acetylcholine receptor stimulation, was abolished in the double mutants. Despite a caloric intake no less than that of wild-type mice, the double mutants had reduced body weights and low serum glucose levels. Difficulties in food digestion caused by the failure of pancreatic exocrine secretion could account for the undernourishment phenotype seen in these mice. These abnormalities were not seen in single gene mutants for IP3R2 or IP3R3. Taken together, these results demonstrate that IP3R2 and IP3R3 play redundant but indispensable roles in exocrine secretion of the salivary glands and the pancreas, thus, in normal animal growth and survival. [Jpn J Physiol 55 Suppl:S134 (2005)]

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In bovine ciliary muscle (BCM) cells, stimulation of M₃-muscarinic receptor (MR₃) opens two types of receptor-operated non-selective cation channel (ROC) which serve as major pathways for Ca²⁺ entry during the tonic phase of contraction¹. It has been shown by RT-PCR that BCM contains mRNA for four TRP channel homologues (TRPC 1, 3, 4 and 6), commonly regarded as molecular candidates for ROCs¹. Here we tried to visually identify these TRPCs in the plasma membrane of BCM by immunofluorescence microscopy. A CCD fluorescence microimaging system (Olympus) was used. After 4-5 days culture of BCM cells on fibronectin-coated glass-bottomed dish in a serum-free HAM F12 media, the body of the cells was removed by gentle sonication under a hypotonic condition. The plasma membrane remaining attached on the glass surface was treated with polyclonal primary antibodies (2.5-5 μg/ml; Chemicon Co.) against putative cytoplasmic segments of the four TRPCs and visualized with secondary antibodies labelled with a fluorescent dye (Alexa Fluor 546; Invitrogen). The membrane preparations were also similarly immunostained with antibodies against MR₃ and α-actin. All the anti-TRPC antibodies used gave many microscopically visible spots of immunofluorescence (roughly 1 spot/μm²) in the cell-free membrane preparation which was also positively stained with antibodies against MR₃ and α-actin. The present results clearly demonstrate the existence of the four TRPC homologues in the plasma membrane of the BCM. 1. Takai et al (2004) J. Physiol. 559, 899-922 [Jpn J Physiol 55 Suppl:S134 (2005)]

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Removal of external K⁺ ions abolishes not only inward currents but also outward currents through the inwardly rectifying K⁺ channel which determines the resting potential. This suggests that external K⁺ ions are essential for the channel activation. To investigate how extracellular K⁺ ions act the channel, we made point mutants neutralized at negatively charged amino acid residues in the extracellular loops of the murine inwardly rectifying K⁺ channel (Kir2.1). cDNA was transfected into COS-1 or HEK293 cells using the liposome method, and voltage clamp experiments were done after 24-72 h. Single point mutants (D112N, D114N, E125Q, D152N and E153Q) showed functional expression, and the gating properties and the unitary conductance of the mutants (D112N, D114N and D152N) were similar to those of the wild type (WT). Double point mutation of D152N/E153Q eliminated the ionic conductance of Kir2.1. Channels from tandem tetramers of Kir2.1 with one WT and three D152N/E153Q mutant subunits (WT-(D152N/E153Q)3) showed inward rectification. However, the whole-cell current density of the mutant was significantly smaller than that of wild-type channels in Tyrode solution. It is suggested that two negative charges at the D152 and E153 sites may be required for K⁺ ions to bind to and activate the channel. [Jpn J Physiol 55 Suppl:S134 (2005)]

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Inactivation of the L-type Ca²⁺ channel regulates the amount of Ca²⁺ influx into cardiac myocytes. The inactivation can be driven not only by depolarization (voltage-dependent inactivation, VDI) but Ca²⁺ flow itself (Ca²⁺-dependent inactivation, CDI). Until now, the study of the modeling for these inactivation processes of the L-type Ca²⁺ channel has not been fully studied. In this study, we have modeled the VDI at first, and then the CDI was estimated to fit the channel current using Ca²⁺ as the charge carrier. To estimate the gating of VDI with minimizing the effect of CDI, we used measured the outward-going K⁺ current flowing through the Ca²⁺ channel in the absence of extracellular Ca²⁺ (Findlay I, J. Physiol. 2002). Thus, estimated VDI showed a biphasic decay, and were composed of fast, slow and steasy-state components. The VDI model composed of three components was developed to fit the time- and voltage-dependent behavior of each. Using the developed model, Ba²⁺ inward currents flowing through Ca²⁺ channel at various potentials were calculated with taking the surface charge into account. The simulated currents were in good agreement with the experimental results. Next, to develop CDI model, we added another inactivation parameter to fit the experimental Ca²⁺ current. In this attempt, we found it necessary to incorporate voltage-dependent nature of CDI. In conclusion, we have developed a reasonable model of Ca²⁺ channel inactivation, which may facilitate further experimental researches. [Jpn J Physiol 55 Suppl:S135 (2005)]

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BDM, a chemical phosphatase commonly used to suppress muscle contraction, is often used for isolating single heart cells as an easy relief for ischemic damages. However, BDM is also known to interfere with the intracellular protein phosphorylation, and to modify calcium signaling by suppressing Ca channels and Na/Ca exchangers in heart cells. In mouse heart cells, where BDM is most oftenly used for cell isolation, the action of BDM on intracellular signaling pathways and ion channels/transporters still remains unknown. We therefore examined the effect of BDM on calcium current in whole-cell clamped mouse heart cells to survey how BDM affects Ca channels and their regulation via the well-established signaling pathway via PKA. Our results show: 1) when applied extracellularly, BDM decreased the amplitude of calcium current in a dose-dependent manner (K1/2=7.3 mM, nH=1.33); 2) at the same time, BDM shifted the peak of the I-V relation to more depolarized potential; 3) also, BDM had no significant effect on the dose-response relation of calcium current to isoproterenol; 4) when applied intracellularly, BDM had very little effect on ICa amplitude. These lines of evidence indicate that BDM inhibits cardiac calcium current by blocking Ca channels; although involvement of its action as chemical phosphatase cannot be ruled out. BDM is reportedly useful for heart cell isolation and culture, it may alter the electrophysiological properties of the cells. [Jpn J Physiol 55 Suppl:S135 (2005)]

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The present study was designed to examine the effects of the nonsteroidal anti-inflammatory agent mefenamic acid on the native slowly activating delayed rectifier K⁺ current (I_Ks) and heterologously expressed KCNQ1/KCNE1 channels, the molecular constituents believed to underlie cardiac I_Ks. I_Ks in guinea-pig cardiac myocytes and KCNQ1/KCNE1 channel heterologously expressed in COS-7 cells were recorded using whole-cell patch-clamp method. Bath application of mefenamic acid (0.1 mM) gradually increased the amplitude of outward current jump but decreased the magnitudes of both the time-dependent developing current and deactivating tail current in the KCNQ1/KCNE1 channels during repetitively depolarization from a holding potential of –80 mV to various test potentials. These changes in the outward currents were totally sensitive to the I_Ks blocker chromanol 293B (100 μM). Thus, the KCNQ1/KCNE1 channels appear to be stabilized in an open state in the presence of mefenamic acid; the channel that is once activated by depolarization hardly deactivates upon repolarization. In contrast, mefenamic acid at the same concentration (0.1 mM) hardly affected the properties of native I_Ks with the exception that the deactivation kinetics was minimally slowed, consistent with the finding that mefenamic acid does not appreciably affect I_Ks in vivo. Thus, the present study confirms the presence of a marked differential sensitivity of native I_Ks and heterologously expressed KCNQ1/KCNE1 channel to mefenamic acid. [Jpn J Physiol 55 Suppl:S135 (2005)]

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