We have reported that isolated rat skeletal muscles can be preserved safely for a long period when soaked in hypotonic rat Ringer solution (HypoR) containing xanthine oxidase inhibitor allopurinol (APN) at low temperatures. The osmolality of HypoR was adjusted to 220–230 mOsm, which contained 100 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 2 mM MgCl₂, 10 mM HEPES, 10 mM glucose, 0.02 mg/ml penicillin, and 2 IU/ml heparin. Hypotonic APN Ringer solution (APN-HypoR) contained furthermore 1.5 mM APN. Hypotonic recovery solution contained (mM) : 110 NaCl, 2.5 KCl, 2 CaCl₂, 4 HEPES. Freshly isolated extensor hallucis longus muscles (EHLs) were soaked in the recovery solution and control tetanus tensions (P₀) were measured at room temperatures. Thereafter these EHLs were preserved at low temperatures (3–6 °C). Test tetanus tensions (P_T) compared with P₀ in the recovery solution after the preservation for 40 hrs using isotonic rat Ringer solution (IsoR), HypoR, and APN-HypoR were 12%, 28%, and 74%, respectively. Similarly, P_T after the preservation for 60 hrs using IsoR and HypoR showed a trace, while those using APN-HypoR remained about 30%. Mechanism of long-term survival of the muscles soaked in APN-HypoR is unclear, but production of some harmful substances may be prevented and/or their excretion may be accelerated. [J Physiol Sci. 2008;58 Suppl:S66]

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Skinned cardiac fibers exhibit SPontaneous Oscillatory Contraction (SPOC) over a broad range of intermediate activating conditions, namely, at pCa 6.0-5.0 (Ca-SPOC), or at the coexistence of MgADP and Pi under the relaxing conditions (ADP-SPOC) (Fukuda et al., 1996, 1998, 1999). We recently reported that the period of sarcomeric oscillations correlates with that of heartbeat in various animal species (Sasaki et al., 2005, 2006). We also confirmed that the period of SPOC in single cardiac myofibrils is similar to that observed in fibers (unpublished data). To further elucidate the physiological significance of SPOC, experiments need to be done with cardiomyocytes, because the excitation-contraction coupling occurs at the cellular level in vivo. The present study has therefore been undertaken to analyze SPOC in single cardiomyocytes that were enzymatically isolated from the rat ventricle. For the analysis of the waveform of sarcomeric oscillations, we used quantum dots conjugated with antibody to visualize the Z-line position in sarcomeres. We found that in skinned single myocytes the period of sarcomeric oscillations during SPOC is almost the same as that observed in fibers or myofibrils. We will report and discuss these findings at the meeting. [J Physiol Sci. 2008;58 Suppl:S66]

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Organization of myosin filaments in living smooth muscle cells is thought to dynamically change during relaxation-contraction cycles. New experimental methods with tracking movement of single smooth muscle myosin (SMM) molecules in isolated muscle tissues have been requested to evaluate dynamics of filamentous structure in vivo. In the present study, we tested whether Qdots (quantum dots) conjugated with anti-SMM monoclonal antibody is capable to track single SMM molecules movement in smooth muscles from guinea pig taenia caeci. Qdots are inorganic nanocrystals that possess unique luminescent properties; their fluorescence emission is stable and tuned by varying the particle size or composition. Using laser fluorescence microscope and high-sensitivity camera, we observed luminescence of single Qdots particles. In Triton X-100 skinned preparations, we detected number of Qdots particles not only around the cell surface but also inside of the cell. When Qdots was not conjugated with anti with anti-SMM antibody, the Qdots particles in Triton X-100 skinned preparations were detected only around the cell surface. These results suggested that we really detected luminescence originated from Qdots particles which bound with single SMM molecules. [J Physiol Sci. 2008;58 Suppl:S66]

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Many of the troponin mutants which can cause familial cardiomyopathy have been reported so far. To elucidate the molecular mechanism of the cardiomyopathy related to troponin, we performed molecular dynamics study on the structure of troponin mutants which relate to familial cardiomyopathy. Two different troponin T mutants related to hypertrophic cardiomyopathy, Glu244Asp and Lys247Arg, and a troponin C mutant related to dilated cardiomyopathy, Gly159Asp were studied. Dynamics was calculated by the use of software Amber (vers. 9). Iteration was done in TIP3 water sphere with 0.5 or 1 fs time step in periodic condition at constant temperature (310 K) and pressure. Model structures of troponin mutants were constructed by introducing the mutation to the crystal structure of human cardiac troponin (core region of TIC complex) obtained from Protein Data Bank (ID number 1J1E). More than 9 trajectories of 1ns were obtained for wild and mutant structure. It was observed that electrostatic interaction between troponin I and troponin T which linked alfa helix of troponin T and troponin I in the wild was lost in the two troponin T mutants. While in Gly159Asp mutant of troponin C, abnormal electrostatic interaction within Troponin C were formed. The involvement of these differences between the wild and mutant in the tension regulation will be discussed. [J Physiol Sci. 2008;58 Suppl:S66]

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The Frank-Starling mechanism of the heart is based on the intrinsic ability of cardiac muscle that produces higher active force at a longer length (i.e., length-dependent activation). We have reported that the giant elastic protein titin (also known as connectin) performs as one of the triggering factors in length-dependent activation. In the present study, we tested whether length-dependent activation is regulated at the thin filament level by using our quasi-complete troponin (Tn) exchange technique. Reconstitution of thin filaments with fast skeletal troponin (sTn; prepared form rabbit psoas muscle) attenuated length-dependent activation in skinned porcine left ventricular muscle. Then, to investigate the effect of troponin exchange on cross-bridge kinetics, we measured the rate of force redevelopment (k_tr) in both control and sTn-reconstituted cardiac muscles. k_tr increased upon sTn reconstitution at submaximal levels, suggesting the acceleration of cross-bridge formation and, accordingly, a reduction in the fraction of resting cross-bridges that can potentially produce additional active force upon attachment to the thin filament. An increase in titin-based passive force, induced by manipulating the pre-history of stretch, enhanced length-dependent activation, in both control and sTn-reconstituted muscles. These results favor the interpretation that Tn plays an important role in the Frank-Starling mechanism of the heart via on-off switching of the thin filament state, in concert with titin-based regulation. [J Physiol Sci. 2008;58 Suppl:S67]

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It is known that number of the thick and thin filaments in tracheal smooth muscles increases by activation with Ca²⁺ (Seow, 2005), while the content of contractile filaments does not change during relaxation-contraction cycles in smooth muscles of taenia caeci from guinea pig (e.g. Watanabe et al., 1993). Therefore, organization of the contractile filaments in taenia caeci and trachea might be regulated by different mechanisms. To test this possibility, we studied the effects of blebbistain, which suppressed contraction and disrupted thick and thin filaments organization of skinned (cell membrane permeabilizsed) taenia caeci (Watanabe et al., 2007), on the mechanical responses of the skinned preparations of tracheal smooth muscles from guinea pig. Compared with the blebbistatin effects on the skinned taenia caeci, much lower concentrations of the agent induced inhibition of contraction in skinned trachea. These results indicated that blebbistatin might accelerate myosin filaments dissociation in tracheal smooth muscles. Contribution of myosin ATPase activity to myosin filaments assembly and disassembly in smooth muscle will be discussed. [J Physiol Sci. 2008;58 Suppl:S67]

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We investigated the effects of gravitational unloading on the regeneration of injured mouse soleus muscle by using proteome analysis. Male adult mice (C57BL/6J) were subjected to unloading by hindlimb suspension. Following 2-week-suspension, cardiotoxin (CTX) was injected into soleus muscles to initiate the necrosis-regeneration cycle. Ambulation in cage was allowed for one group of mice and suspension was continued for additional 4 weeks in the remaining group. Unloading, as well as CTX-injection, resulted in a rapid loss of soleus protein contents. Recovery of muscle protein content was observed in CTX-injected soleus after 4 weeks in ambulatory control group. However, there was no gain of protein content in muscle with both CTX-injection and continuous unloading. Profiles of proteins were analyzed by two-dimensional gel electrophoresis using two immobilized pH gradient strips with overlapping pH range covering a pH 3-8 range. Twenty five spots were significantly altered in muscle with CTX-injection and suspension compared with the group with CTX-injection alone. This study was supported, in part, by Grant-in-Aid for Scientific Research (C, 17500444, KG; A, 18200042, TY; S, 19100009, YO) from Japan Society for the Promotion of Science, Ground-based Research Program for Space Utilization from Japan Space Forum (KG). [J Physiol Sci. 2008;58 Suppl:S67]

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Influence of muscle inactivity on the regenerative process of injured skeletal muscle was studied. Male mice (C57BL/6J) were randomly divided into 4 groups; normal cage control (CC), cardiotoxin (CTX)-injected (CX), hindlimb suspension (HS), and HS+CX (SX) groups. HS was performed for 2 weeks in group HS and SX. And then, CTX was injected into soleus muscles bilaterally in CX and SX groups. HS was continued for additional 6 weeks in group HS and SX. CTX-injection caused a decrease in wet weight and protein content of soleus. Both muscle weight and protein content in group CX recovered to the control level following 4 weeks. On the contrary, both parameters in group SX did not recover during the experimental period. Percentage of fibers with central nuclei, relative to total muscle fibers, in CX group at 42 days was higher than in SX group. It was suggested that loading plays a key role for the activation of regenerating potential of injured skeletal muscle. (Supported, in part, by Grant-in-Aid for Scientific Research (C, 17500444; A, 18200042; S, 19100009) from Japan Society for the Promotion of Science, Ground-based Research Program for Space Utilization from Japan Space Forum). [J Physiol Sci. 2008;58 Suppl:S67]

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Effects of hindlimb immobilization (HI) on the expression of Akt and GSK-3β, a downstream of Akt, during the regenerating process were investigated in mouse skeletal muscle. Male mice (C57BL/6J) were divided randomly into 4 groups: normal control, and with cardiotoxin (CTX)-injection, HI, and both CTX-injection and HI. CTX (0.1 ml, 0.1 mM) was injected into the left tibialis anterior (TA) muscle. The immobilization of left ankle joint at a plantar-flexed position using plaster cast in mice, with or without CTX injection, was also performed. TA muscles were dissected after 7, 14, and 28 days. Regeneration-related increases of wet weight and protein content in TA and muscle satellite cells were significantly inhibited by HI. The activation of Akt and the inactivation of GSK-3β were observed during regenerative process. The inactivation of GSK-3β was not observed in the HI group. It is suggested that the inactivation of GSK-3β may be a key signal for the regeneration of injured skeletal muscle. This study was supported, in part, by Grant-in-Aid for Scientific Research (C, 17500444; A, 18200042; S, 19100009) from Japan Society for the Promotion of Science, Ground-based Research Program for Space Utilization from Japan Space Forum. [J Physiol Sci. 2008;58 Suppl:S68]

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We recently reported the effects of long-term hindlimb immobilization (6 weeks) on the contractile properties of the soleus muscle in the rat (Annual meeting of the Physiological Society of Japan, 2007). We found that long-term hindlimb immobilization lowers the expression of the giant protein titin in the soleus muscle, resulting in reduced active force production via abnormal sarcomeric organization. In the present study, we investigated how immobilization affected fatigability by using single skinned fibers taken from the same animal model. Hydrogen ion (H⁺) and inorganic phosphate (Pi) are the well-known major causes of muscular fatigue. The intracellular concentrations of Pi and H⁺ are known to increase in skeletal muscle during intense exercise, resulting in a fall in active force production. Accordingly, we tested the effects of changes in pH and the Pi concentration on maximal Ca2⁺-activated force production in control vs. immobilized fibers. We found that lowering pH from 7.0 to 6.2 decreased maximal force in both muscles, with a greater magnitude in immobilized fibers. Likewise, the inhibitory effect of Pi up to 20 mM was more pronounced in immobilized fibers. These results suggest that fatigability is enhanced in disused muscle and that the mechanism includes a decrease in the fraction of force-generating cross-bridges coupled with abnormal sarcomeric organization. [J Physiol Sci. 2008;58 Suppl:S68]

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It is well known that exercise may cause stiffness of muscles by the study with questionnaire or palpation. However the reports concerned with objective measurement of muscle stiffness are a few. We already reported that the soleus muscle hardness raised after exercise loading and gradually lowered to the initial level using newly devised muscle hardness meter in healthy human volunteers. This devise has three foots. Both side-foots are fixed, a center foot is movable and 6.3mm longer than both side-foots. When both side-foots touch the skin surface covering the object muscle, the pushed back length of center foot indicates the muscle hardness. If the muscle were harder, push backed length would be longer. Stretching is well known to soften muscles, however the reports concerned with objective measurement of stretching effects are also a few. We reported that stretching in state with knee extension accelerated the restoration of soleus muscle hardness after exercise loading, but did not affect exercise-unloaded muscle hardness objectively. We investigate the effect of stretching in state with knee flexion on exercise-unladed soleus muscle in the present study. The stretching in state with knee flexion lowered the hardness of exercise-unloaded soleus muscle with statistical significance (p=0.009) by analysis of variance. These results indicate that the stretching in state with knee flexion may be more effective on the soleus muscle hardness than in state with knee extension in human. Furthermore the usefulness of newly devised muscle hardness meter was indicated. [J Physiol Sci. 2008;58 Suppl:S68]

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We attributed the altered contractile properties of vascular and non-vascular muscle strips in Tris containing medium to experimental variables such as sodium concentration and pH change. Imidazole is also used in various biomedical experiments. Effects of imidazole on contractile response to KCl as well as on the levels of phosphate creatine (PCr) and ATP in taenia coli isolated from guinea-pig were investigated. Methods: The cleaned preparations were placed in a chamber containing bicarbonate buffer (BB) gassed with 95% O₂-5% CO₂, or imidazol buffer (IB) gassed with 100% O₂ at 37±0.5^oC. Isometric tension was recorded via computer-based analysis using Mac-Lab. The levels of PCr and ATP in taenia coli were measured by the luciferin-luciferae method. Results: The level of PCr was gradually decreased after switching the buffer from BB to IB, and it was nearly exhausted 30 min after the exposure to IB. This alteration of the level of PCr was recovered by putting back the preparation to the BB. On the other hand, the ATP level and KCl-induced contraction in IB were not significantly different from those in BB. Effects of Tris buffer exposure were similar to those of IB exposure. Results may be related to an alteration of creatine kinase activity in the taenia coli. [J Physiol Sci. 2008;58 Suppl:S68]

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β₂-agonist, clenbuterol (CLE) is used as a non-steroidal anabolic drug in sports doping and selectively binds to β₂ adrenoceptor (AR). Although many studies have been reported on post-translational modification of β₂ AR in muscles of CLE-administered animals, little is known about transcriptional control of them. Therefore, the effects of CLE-administration (dose = 1.0 mg/kg BW/day, sc) for 10 days on the mRNA expression of β ARs (β₁, β₂ and β₃ ARs) in the fast-twitch extensor digitorum longus (EDL), the slow-twitch soleus (SOL), mixed-type gastrocnemius (GAS) and left ventricle (LV) muscles were studied in rats. Adult male Sprague Dawley rats were randomly divided into CLE-administered and control (CON) groups. The expression of β ARs was analyzed by real-time RT-PCR. EDL and GAS weights in CLE group were significantly higher than those in CON group. However, SOL and LV weights were not significantly different between both groups. The β₂ AR mRNA levels of EDL and LV in CLE group were significantly lower than those in CON group. The β₁ AR mRNA level of LV in CLE group was also significantly lower than that in CON group. From these results, CLE induced down-regulation of β₁ and β₂ ARs in LV without hypertrophy and down-regulation of β₂ AR in hypertrophied EDL, suggesting that the effects of CLE on the mRNA expression of β ARs and muscle hypertrophy are dependent upon each muscle type. [J Physiol Sci. 2008;58 Suppl:S69]

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Endothelin-1 (ET-1), a potent vasoconstrictor peptide, induces a long-lasting contraction mediated by G protein-coupled endothelin_A (ET_A) receptor in aortic smooth muscle. In isolated rat aorta without endothelium, the ET-1 (100 nM)-induced contraction was very slowly relaxed by perfusion of the aorta with physiological salt solution without ET-1, and it was incompletely inhibited by application of the ET_A receptor antagonist BQ-123 (10 μM) during the plateau tension. These relaxations were enhanced and completely performed by addition of the Rho-associated kinase inhibitor Y-27632 (10 μM) to the perfusion solution or by the combined application of BQ-123 (10 μM) and Y-27632 (10 μM). The enhanced relaxation rates were much higher than the relaxation rate for the application of Y-27632 (10 μM) alone which completely inhibited the ET-1 (100 nM)-induced contraction. In Ca²⁺-free solution or in the presence of the myosin light chain kinase inhibitor wortmannin (10 μM), ET-1 (100 nM) evoked a contraction which was completely inhibited by Y-27632. This result shows that the contraction in the Ca²⁺-free solution or insensitive to wortmannin is induced through myosin light chain phosphorylation by Rho-associated kinase. The contraction through the activation of Rho-associated kinase was fairly resistant to BQ-123. The present results would suggest that the small GTPase Rho and its effector, Rho-kinase, pathway is involved in the binding affinity of ET-1 to the ET_A receptor in rat aorta. [J Physiol Sci. 2008;58 Suppl:S69]

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Genomic and non-genomic effects of estrogen and their crosstalk in the skeletal muscle are still poorly understood. We investigated the effects of estrogen on the proliferation of mouse myoblast cells and the properties of the membrane estrogen receptor (ER). The effect of estrogen on the proliferation did not change regardless of the culture medium and serum concentration. The ER protein in the post-nuclear fraction was dose-dependently increased after treatment with 17β-estradiol and BSA-17β-estradiol-conjugate, which was attenuated by high concentrations of tamoxifen and ICI 182-780. The amount of ER was also reduced by the presence of an ERK1/2 inhibitor, PD 98059 or a specific p38 inhibitor, SB 203580 in both presence and absence of estradiol. The newly-synthesized ER was increased by 17β-estradiol dose- and time-dependently. The time course of the increase of mRNA was almost paralleled by newly-synthesized ER. The newly-synthesized ER by estrogen was further increased by the protein kinase C (PKC) activator, TPA. These results suggest that ERs located at plasma membrane are the target of estrogen action in mouse skeletal myoblast cells. The estrogen-stimulated ER synthesis was induced via both genomic and non-genomic pathways involving the PKC/MAPK signaling system. [J Physiol Sci. 2008;58 Suppl:S69]

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Some of heavy-metal ions are known to increase [Ca²⁺]_i in many kinds of cells including vascular endothelial cells. To investigate the effects of Zn²⁺, a sheet of endothelial cells was isolated from a guinea-pig mesentericartery and conventional whole-cell clamp experiments were performed. Zn²⁺ (<=20 μM) decreased the resting membrane conductance which was thought to be due to nonselective cation channels. Inward rectifier K⁺ channels also contributed to the resting conductance, however, these channels were not blocked by Zn²⁺. These concentrations of Zn²⁺ seemed also to inhibit Ca²⁺ influx, because the membrane currents induced by repeated applications of agonist such as ACh decreased. Zn²⁺ at higher concentrations additionally activated Ca²⁺-activated K⁺ and Cl⁻ channels indicating that Zn²⁺ indeed increased [Ca²⁺]_i in our preparations. As Zn²⁺ could induce these currents in the absence of the external Ca²⁺, Zn²⁺ likely released Ca²⁺ from the intracellular store sites. Both ACh-induced and ATP-induced currents were suppressed in the presence of 50 μM-Zn²⁺, and Zn²⁺ seems to bind and modify the receptor proteins for such agonists to increase [Ca²⁺]_i. Although the concentrations of Zn²⁺ used in the present experiments were beyond the physiological range, Zn²⁺ may be a good tool to investigate the intracellular Ca²⁺handling in the vascular endothelial cells. [J Physiol Sci. 2008;58 Suppl:S69]

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Organic anion transporting polypeptides (OATPs/Oatps) are important transporters for uptake of bile acids, thyroid hormones, and clinical drugs into cells. However, expression and function of OATPs/Oatps in skeletal muscles have been unknown. Anti-hyperlipidemic statins adversely affect skeletal muscles. The risk of hydrophilic statins to induce muscular injury has been believed to be less than that of lipophilic statins. However, clinical data revealed that hydrophilic statins cause myopathy as frequently as did lipophilic statins in patients. In this study, we herein compared the toxic effect of hydrophilic pravastatin and the expression of Oatps between myofibers and two types of mononuclear cells (fibroblasts and L6 skeletal myoblasts). In myofibers, the median value of the concentration-viability curve of pravastatin was 8.6 μM. Conversely, the other mononuclear cells were insensitive to pravastatin. Using RT-PCR, we examined the mRNA expression levels of the transporters reported for pravastatins; Oatp1a4 and Oatp2b1. Both transporters were expressed in the myofibers but not in the mononuclear cells. The pretreatment of myofibers with estrone sulfate, a substrate of Oatp attenuated the effect of pravastatin. Our results indicate that hydrophilic statins are transported into myofibers and could therefore cause toxicity, but not in mononuclear cells, because these mononuclear cells do not express Oatps for statins. [J Physiol Sci. 2008;58 Suppl:S70]

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The biological and molecular properties of tetrodotoxin (TTX)-sensitive voltage-gated Na⁺ currents (I_Na) in murine vas deferens myocytes were investigated using patch-clamp techniques and molecular biological analyses. In whole-cell configuration, a fast, transient inward current was evoked in the presence of Cd²⁺, and was abolished by TTX (K_d = 11.2 nM), mibefradil (K_d = 3.3 μM) and external replacement of Na⁺ with monovalent cations (TEA⁺, Tris⁺ and NMDG⁺). The fast transient inward current was enhanced by veratridine, an activator of voltage-gated Na⁺ channels, suggesting that the fast transient inward current was a TTX-sensitive I_Na. The values for half-maximal (V_half) inactivation and activation of I_Na were -46.3 mV and -26.0 mV respectively. RT-PCR analysis revealed the expression of Scn1a, 2a and 8a transcripts. The Scn8a transcript and the α subunit protein of Na_V1.6 were detected in smooth muscle layers. Using Na_V1.6-null mice (NaV1.6^−/−) lacking the expression of the Na⁺ channel gene, Scn8a, I_Na were not detected in dispersed smooth muscle cells from the vas deferens, whilst TTX-sensitive I_Na were recorded in their wild-type (Na_V1.6^+/+) littermates. This study demonstrates that the molecular identity of the voltage-gated Na⁺ channels responsible for the TTX-sensitive I_Na in murine vas deferens myocytes is primarily Na_V1.6. [J Physiol Sci. 2008;58 Suppl:S70]

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It is well known that arginine vasotocin (AVT) stimulates the sodium reabsorption by increasing the number of epithelial sodium channels (ENaC) in the apical membrane of renal epithelial cells. It is also indicated that chloride ions regulate cell functions such as cell growth and neurite elongation. However, the effect of Cl⁻ on the AVT-stimulated ENaC-mediated current is unclear. In the present study, we examined effects of Cl⁻ on the AVT-stimulated benzamil-sensitive currents in renal epithelial A6 cells. AVT significantly increased the benzamil-sensitive currents in A6 cells. Lowering the Cl⁻ concentration of the apical and basolateral solutions by replacing Cl⁻ with gluconate diminished the stimulatory action of AVT on the benzamil-sensitive current, while the replacement of Cl⁻ with NO3⁻ had no significant effect on the AVT action. The diminishing effect of lowering Cl⁻ on the AVT action was observed under a low Cl⁻ condition for the apical solution, however we did not detect the diminishing effect in a low Cl⁻ basolateral solution. These results suggest that the apical chloride ion is a key molecule for AVT to show its stimulatory action on the ENaC-mediated sodium reabsorption in renal epithelial cells. [J Physiol Sci. 2008;58 Suppl:S70]

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We have shown the synthesized ciguatoxin, CTX3C exhibits multimodal actions on voltage-dependent sodium channels. In this study, we examined the effects of structurelly-modified CTX3C analogs, 51-OH CTX3C, F(8)- and F(10)-51-OH CTX3C: 9-membered F-ring is replaced either with the 8- or 10-membered one, on Na_v1.2 and Na_v1.8 channels. The maximum condactance (G_max) of Na_v1.2 channels was blocked by 21.6 ± 6.7% (n=3) and 45.0 ± 5.2% (n=3) with 100nM of CTX3C and 51-OH CTX3C, respectively. 10 μM F(8)- and F(10)-analogs suppressed G_max of Na_v1.2 by 22.5 ± 2.5% (n=3) and 24.4 ± 11.4% (n=4), respectively. Moreover, 100 nM CTX3C induced resting conductance of Na_v1.2 and Na_v1.8, whose effects were estimated as relative conductance (%) to Gmax in the absence of toxin, by 0.14 ± 0.06% (n =3) and 0.86 ± 0.58% (n=5), while 51-OH CTX3C by 0.31 ± 0.21% (n=3) and 1.09 ± 0.12% (n=2), respectively. On the other hand, both 10 μM F(8)- and F(10)-analogs could not induce significant fraction of resting conductance of either of Na_v1.2 or Na_v1.8 channels. It is crucial to have a 9-membered ring in the middle of the semi-rigid structure comprised of 13 ether rings for CTX analogs to maintain its high biological potency. [J Physiol Sci. 2008;58 Suppl:S70]

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Noise analysis of the membrane current has been used for long period of time as one of the most powerful tools for the study of membrane excitability. Even after the development of the patch clamp, noise analyses have been employed when the channel density is not appropriate for the patch recordings. Also, one of the biggest benefits is that the technique can be applicable, as a triggering survey, for the whole-cell currents whose unitary properties are totally unknown. Generally, however, the technique has been less used, mostly because of the difficulties of the logics and analyses. In the present work, we developed a computer program executing all processes for the noise analysis running on the MS-Windows .NET Framework. This Windows application can specify the time window for data analyses, calculation of the mean and variance, and least square fitting of the data to provide unitary current amplitude and the total number of the channels. As an example for application, we addressed to a quantitative analysis of voltage-gated Na⁺ channels of the olfactory receptor cell in which Na⁺ channels play a crucial role for the spike generation. We confirmed that the non-stationary noise analysis was a valuable tool for estimating the quantum features of Na⁺ channels. Together, properties of the voltage-gated Na⁺ currents will be discussed in relation to the neuronal signal encoding. [J Physiol Sci. 2008;58 Suppl:S71]

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Hyperpolarization-activated cyclic nucleotide-gated (HCN) currents are essential regulators in rhythmic generation, membrane excitability, and synaptic transmission. Four subtypes of HCN channels (HCN1-4) have been cloned so far in mammals. HCN channels form tetramers and each subunit has 6 transmembrane domains and one pore region, an architecture shared with voltage-gated potassium channels. We previously demonstrated that the first transmembrane segment (S1) and the S1-S2 loop endow different activation kinetics between HCN1 and HCN4. We investigated 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. Presence or absence of current was consistent with the predicted α-helical structure of the S1 transmembrane segment. Two Trp mutations introduced in the middle of S1 prevented normal channel closure. These results suggest that S1 might interact with the mobile part of the voltage sensor (S4). Here, we introduced multiple cysteine mutations into the voltage sensor domain and investigated mutual interaction among transmembrane segments. [J Physiol Sci. 2008;58 Suppl:S71]

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Hyperpolarization- activated cyclic nucleotide-modulated (HCN) channels play fundamental roles in excitable cells, including central neurons and cardiac myocytes. However, whether HCN functions in non-excitable cells remains to be solved. In this study, we investigated localization of and roles of HCN in murine bone cells in vitro and in vivo. All four HCN subtypes (HCN1-4) were found in bone tissue, but their expression altered among different cell types. Immunoreactivity for HCN1 was abundant in osteoclasts, specifically at the sealing zone and ruffled border that are faced to the bone surface and responsible for acid secretion. HCN2 and HCN3 were expressed in bone marrow cells and osteoblasts respectively. The whole-cell clamp recordings revealed HCN-like currents (Ih), featured by activation at hyperpolarization and slow gating kinetics, in cultured osteoclasts. Extracellular acidification shifted the activation voltage towards more positive voltages, indicating that HCN channels would respond to changes in local pH caused by proton secretion from osteoclasts during bone resorption. An HCN blocker (ZD7288) inhibited differentiation of osteoclasts and osteoblasts from their precursors of the bone marrow. HCN2 knockout mice had low bone mass, fragile bone structure and weak bone strength. These findings suggest that HCN channels are essential in regulation of both bone formation and resorption. [J Physiol Sci. 2008;58 Suppl:S71]

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To find the different role of β subunit isoforms (β_2a and β₃) in the regulation of L-type Ca²⁺ channel by intracellular cAMP, we measured the Ca²⁺ channel current (I_Ca(L)) in wild type, β_2a- and β₃-overexpressed vascular smooth muscle (VSM) cell lines (A7r5, β_2a/A7r5 and β₃/A7r5). I_Ca(L) was recorded by whole-cell voltage clamp using a patch-clamp technique. 5 mM BaCl₂ was used as a charge carrier to record I_Ca(L). The β subunits and phosphodiesterase (PDEs) mRNA expressions were quantitated by the comparative RT-PCR method. Intracellular cAMP concentration was measured by chemiluminescent ELISA assay. Current-voltage relationships revealed that the current density of I_Ca(L) did not differ among the three groups. After 3 min-perfusion of 3 mM dibutyryl-cAMP (db-cAMP), the peak amplitude of I_Ca(L) was significantly decreased in A7r5, but was significantly increased in β_2a/A7r5. There was no change in β₃/A7r5. As compared with A7r5 cell line, type 3B and 4D PDE mRNA expressions were increased in β_2a/A7r5 cell lines and basal intracellular cAMP concentrations were decreased in β_2a/A7r5 and β₃/A7r5cell lines. Forskolin (100 μM)-induced elevation of intracellular cAMP was only seen in β_2a/A7r5 cell line. We further tested the responsiveness of I_Ca(L) to db-cAMP in β_2a and/or β₃-knock down A7r5 cells using shRNA expression vectors. [J Physiol Sci. 2008;58 Suppl:S71]

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L-type Ca²⁺ channels are up-modulated with phosphorylation mediated by protein kinase A (PKA). Consensus phosphorylation sites in the carboxyl-terminal tail of guinea-pig Cav1.2 channel, which are conserved in other species, are four (Ser1574, Ser1626, Ser1699, Ser1927). Ser1927 is reported as the functional phosphorylation site to up-modulate the channel. In this study, we investigated possible roles of other phosphorylation sites. Mutations from Ser to Ala in one of consensus phosphorylation sites or in combinations were introduced, and expressed channels were investigated for the effects of PKA activator (5 μM forskolin) using patch-clamp technique in BHK cells. Expressed wild-type (WT) channels responded to forskolin with 4-fold increase in channel activity. Responses of mutant channels (S[1574]A, S[1626]A, S[1927]A) are significantly suppressed compared with that of WT channels (1 to 2-fold). Combined mutants containing S[1574]A showed low sensitivity to forskolin. These results suggest not only Ser1927 but also Ser1574 and Ser1626 are functional sites for PKA phosphorylation. We will also discuss phosphorylation of channel fragments containing these consensus phosphorylation sites. [J Physiol Sci. 2008;58 Suppl:S72]

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In cardiomyocytes, protein kinase D1 (PKD1) plays a central role in the response to stress signal. Neuro-humoral stimuli activate PKD1 that localizes at the Z-discs and phosphorylates several myofibril proteins, regulating the cardiac contraction. In the nucleus, PKD1 promotes the activation of gene transcription. We aimed to identify novel protein-PKD1 interactions. PKD1 interacting proteins were searched by yeast-two hybrid screening. We identified Enigma homolog 1 (ENH1) as a new binding partner of PKD1. Both ENH1 and PKD1 interact with α1C, the pore-forming subunit of the L-type voltage-gated calcium channels in neonatal rat cardiomyocytes. Silencing of ENH1, using RNA interference, prevented the binding of PKD1 to α1C. Moreover, a dominant negative mutant of PKD1 or the silencing of ENH1 inhibited the α-adrenergic-induced increase of L-type calcium currents. We found a new binding partner, ENH1, and a new target, α1C, for PKD1 in neonatal rat cardiomyocytes. Upon α-adrenergic stimulation, ENH1 scaffolds PKD1 to α1C to form a signaling complex which regulates the activity of cardiac L-type voltage-gated calcium channels. [J Physiol Sci. 2008;58 Suppl:S72]

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Calmodulin (CaM) regulation of Ca²⁺ channels is a core paradigm for neurobiological Ca²⁺ signaling. CaV1 versus CaV2 classes of these channels exhibit divergent forms of regulation, despite suggestions that all such modulation involves Ca²⁺/CaM binding to an IQ domain on channels. Understanding this diversity thus promises valuable mechanistic insight. Based on the structure of the Ca²⁺/CaM-CaV1.2 (L-type)IQ domain, it has been suggested that CaV1/2 regulatory diversity might reflect isoform-dependent differences in Ca²⁺/CaM IQ conformation. Here, we report the high-resolution structures for the Ca²⁺/CaM-IQ domains of both CaV2.1(P/Q-type) and CaV2.3(R-type) channels. In these highly similar structures, CaM embraces a helical IQ domain with a parallel orientation. Only minor differences from the CaV1.2 structure are present, insufficient to predict regulatory differences. To explore alternate explanations, we combined alanine scanning of the IQ domain in CaV2.1, with CaV2 structure-based molecular simulation of CaM/IQ binding energetics. These approaches raise the possibility that the C-lobe of CaM partially dislodges from the IQ element during functional regulation, allowing exposed residues to trigger regulation via isoform-specific interactions with other regions. [J Physiol Sci. 2008;58 Suppl:S72]

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Calmodulin (CaM) is one of the important regulators of the Cav1.2 Ca²⁺ channel. CaM binding to the C-terminal tail of the α1-subunit of the Cav1.2 channel has been shown to be crucial for both Ca²⁺-dependent facilitation (CDF) and Ca²⁺-dependent inactivation (CDI). CaM consists of N-terminal and C-terminal lobes (N-lobe and C-lobe), each containing two Ca²⁺-binding EF-hand regions. However, it is unclear which lobe interacts with the channel during CDF and CDI. In this study, we have examined binding of N-and C-lobes of CaM to the C-terminal tail of Cav1.2 by using the pull-down method. Glutathione-S-transferase (GST)-fusion proteins of N-and C-lobes of CaM and C-terminal peptides containing CB domain (GST-CB) and IQ-motif region (GST-IQ) were prepared, and the GST regions of the fused N-and C-lobes was removed by cleavage. Both N-and C-lobes of CaM bound to GST-IQ in Ca²⁺-dependent manners. We have also examined binding of CaM lobes to GST-CB and other fragments of the C-terminal tail. Possible interactions between N-and C-lobes for the binding to the sites in the channel, and hypothetical conformation model for CDF and CDI will be discussed. [J Physiol Sci. 2008;58 Suppl:S72]

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It is reported that the N-terminal tail of Cav 1.2 (L-type) Ca²⁺ channel has an inhibitory function since its removal enhances channel activity. In this study, the effects of the N-terminal peptide on activity of L-type Ca²⁺ channels were investigated in patch-clamp inside-out patch mode in guinea pig myocytes. An application of the GST-fused N-terminal peptide (GST-NT, a.a. 6-140; 0.1, 1, 5 μM) + 3 mM ATP to the intracellular solution within 1 min after run-down induced by patch excision resulted in a dose-dependent activation of channel activity. The channel activity was 8.4%, 16.3%, and 30.9% of that recorded in the cell-attached mode, respectively, while it was 3.1% for 1 μM GST alone as control. Neither GST-NT nor ATP alone induced Ca²⁺ channel activity, showing a cooperative effect of GST-NT and ATP on the Ca²⁺ channel. At the 5th and 10th minutes after patch excision, GST N-terminal peptide (1 μM) + 3mM ATP induced Ca²⁺ channel activity to only 4.5% and 3.0%, respectively, revealing a time-dependent attenuation of the GST-NT effect on the Ca²⁺ channel. These results suggest that the N-terminal tail of Ca 1.2 Ca²⁺ channel plays a regulatory role in Ca²⁺ channel activity. [J Physiol Sci. 2008;58 Suppl:S73]

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Although volatile anaesthetics have moderate negative inotropic effects, they potentiate the positive inotropic action of β-adrenoceptor stimulation in isolated myocardium. The objective of the present study was to investigate the effects of sevoflurane (sevo) and halothane (halo) on β-adrenoceptor-mediated increase in L-type Ca²⁺ current (I_Ca,L). I_Ca,L of isolated rabbit ventricular myocytes was measured with whole-cell patch clamp technique. The β-adrenoceptor stimulation was induced by 30 nM isoproterenol (ISO) and similar experiments were performed using 1μM forskolin (adenylate cyclase activator, FSK) to bypass the β-adrenoceptor. Sevo (1.7%) or halo (0.74%) was bubbled in experimental solution and air was bubbled in control group. Sevo and halo decreased I_Ca,L by 19.8±2.4% and 31.7±3.1%, respectively. In control group, ISO and FSK increased I_Ca,L to 153±7.0% and 240.3±12.8% ,respectively. While ISO increased I_Ca,L to 237.2±25.4% and 247.6±16.3% in the presence of sevo and halo, respectively. FSK increased I_Ca,L to 328.5±36.3% in the presence of sevo, while FSK-induced increase I_Ca,L was not altered by halo. This study suggests that halo and sevo enhance the effect of β-adrenoceptor stimulation on I_Ca,L. However the modulations of site are different between halo and sevo. Halo may modulate signal transduction between β-adrenergic stimulation and Ca²⁺ channel but sevo may enhance the phosphorylation of cAMP-dependent PKA to increase I_Ca,L. [J Physiol Sci. 2008;58 Suppl:S73]

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RIMs are multidomain adaptor proteins that were discovered as putative effectors for Rab3. RIM1α, 2α consist of an N-terminal Zn²⁺-finger domain, central PDZ and C₂A domains, and a C-terminal C₂B domain; RIM2β consist of an PDZ, C₂A, and C₂B domains; and RIM2γ, 3γ, and 4γ consist of only a C₂B domain. While C₂B domain is common to all RIMs, the function remains poorly understood. Moreover, the physiological roles played by RIM3, and RIM4 remain unclear.Here we show, C₂B domains of all RIMs associate with voltage-dependent Ca²⁺ channel (VDCC) β-subunits to modulate currents of the P/Q-type VDCC. The most prominent effect of RIMs on VDCC currents was observed on inactivation parameters. Inactivation kinetics was markedly decelerated, resulting in the predominance of high voltage inactivation and an inactivation-resistant current component in the 2-s prepulse protocol. Since RIM3γ is reported as a post-synaptic protein, RIM3γ may modulate VDCCs at post-synapses. The similarly modulated inactivation properties by RIMs suggest that this function is a common feature of the RIM family. [J Physiol Sci. 2008;58 Suppl:S73]

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The voltage-dependent calcium channel is a multi-subunit complex comprising of a pore-forming subunit (α₁ subunit) and regulatory/auxiliary subunits (β, α₂δ and γ subunit). Recently, we have identified a new splice variant of β₄ subunit, β_4d subunit. The β_4d subunit lacks part of the GK domain and part of the C-terminus. The β_4d subunit was expressed in the human brain. To investigate the physiological effects of the β_4d subunit upon calcium channels, we compared calcium currents in BHK cells expressing α_1C and α₂δ subunits in the presence or absence of β_4d subunits. The co-expression of β_4d with α_1C and α₂δ subunits did not result in any significant change in measured currents compared with the absence of the β subunit. A GST pull down assay revealed that β_4d subunit cannot interact with the α-interaction domain of the α₁ subunit. These results suggest that the GK domain of the β subunit is essential for the expression of the voltage-dependent calcium channel. [J Physiol Sci. 2008;58 Suppl:S73]

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Neutrophils play an important role in the progression of atherosclerosis in concert with proinflammatory cytokines. Calcium channel blockers are commonly used for treatment of hypertension, and their pleiotropic effects other than lowering blood pressure are recently recognized. We studied the effects of various calcium channel blockers (amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, azelnidipine, verapamil, and diltiazem) on superoxide (ROS) release, migration, and the signaling pathways in human neutrophils stimulated by GM-CSF or TNF. GM-CSF-induced ROS release was suppressed by amlodipine, nicardipine, and cilnidipine, whereas TNF-induced ROS release was suppressed by amlodipine, nicardipine, cilnidipine, benidipine, efonidipine, nifedipine, and azelnidipine. TNF-induced phosphorylation of ERK and Akt, but not p38 MAPK, was attenuated by nicardipine, cilnidipine, benidipine, efonidipine, and azelnidipine. By contrast, GM-CSF-induced phosphorylation of ERK, p38 MAPK, and Akt was affected by neither blockers. GM-CSF-induced neutrophil migration was also suppressed by amlodipine and nicardipine, but not by azelnidipine, when these blockers were assessed for their effect on neutrophil migration. These findings suggest that some calcium channel blockers can suppress cytokine-induced neutrophil activation, leading to possible prevention of the progression of atherosclerosis, and that activation of the ERK and PI3K/Akt pathways induced by TNF, but not by GM-CSF, is selectively affected by some blockers. [J Physiol Sci. 2008;58 Suppl:S74]

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Recent genetic analyses have revealed an important association of the gene encoding the Rab3A-interacting molecule (RIM1) with CORD7, an autosomal dominant cone-rod dystrophy. However, the effects of RIM1 mutation which associated with CORD7 have remained unclear. Recently we have revealed effects of RIM1 on VDCC currents, anchoring of neurotransmitter-containing vesicles and neurotransmitter release. In this study, we demonstrate that the mouse RIM1 arginine-to-histidine substitution R655H, which corresponds to human CORD7 mutation, impaires RIM1 function in ACh release and regulation of VDCC currents. Thus, we suggest that phenotypes of familial cone-rod dystrophy with RIM1 mutation can be at least partly attributable to defects in regulation of presynaptic VDCC currents, presumably resulting in retinal deficits. [J Physiol Sci. 2008;58 Suppl:S74]

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High-fat diet affects many steps of cell functions. To study the effect of high-fat diet feeding on skeletal muscle function, male Wistar rats were fed high-fat (41% of energy, added lard) diet for 1 year. Fragmented sarcoplasmic reticulum (FSR) prepared from fast twitch leg muscles was incorporated into planner bilayers for recording single channel activity of type 1 ryanodine receptor (RyR1)/Ca²⁺ release channel. Mean open probability (Po) of the RyR1 in high-fat group (RyR1_F) was significantly lower than that in controls (RyR1_C) (0.10±0.03, n=16 vs. 0.24±0.03, n=17, mean±SEM, p<0.001). This was primarily due to longer mean closed time in RyR1_F. When measured by fura-2 fluorescence change, amounts of Ca²⁺ released from FSR by 3 mM or 10 mM caffeine was smaller in high-fat group than that in controls, whereas Ca²⁺ release by 30 mM caffeine was almost the same between groups. [³H]Ryanodine binding to FSR demonstrated that K_D for ryanodine was higher in high-fat group than in controls, indicating lower channel activity in RyR1_F. In contrast, B_max was higher in high-fat group. Tetanic tension and twitch/tetanus ratio of EDL muscle were not different between groups. These results suggest that a long-term feeding of high-fat diet decreases Ca²⁺ release activity of the individual RyR1 channel. Increase in the number of the RyR1 might compensate for the total Ca²⁺ release activity in the high-fat diet muscles. [J Physiol Sci. 2008;58 Suppl:S74]

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Type 1 ryanodine receptor (RyR1) is a large intracellular Ca²⁺ release channels in the sarcoplasmic reticulum of skeletal muscle and plays an essential role in excitation-contraction coupling. Although the RyR1 channel is believed to be opened via some physical interaction with dihydropyridine receptor of the T-tubule, the detailed mechanism remains still unclear. Fluorescence resonance energy transfer (FRET) is a powerful tool for detection of conformational changes of the protein, and should be applicable to direct monitoring of the RyR1 channel gating. We here generated the functional fluorescent RyR1 channels by randomly inserting GFP variants using transposon-based mutagenesis. The Tn5 transposon encompassing the kanamycin-resistant gene (Kan^r) was randomly inserted into full-length RyR1 cDNA via in vitro transposon reaction. Clones with a transposon were selected by kanamycin resistance, and Kan^r was replaced by genes encoding GFP variants. The resultant clones were transfected into HEK293 cells to screen GFP fluorescence. So far, several tens of fluorescent RyR1 clones were obtained. We are currently testing the channel activity by caffeine-induced Ca²⁺ release. [J Physiol Sci. 2008;58 Suppl:S74]

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Oxytocin (OT) secretion from magnocellular neurons of the supraoptic nucleus (SON) occurs from nerve terminals in the posterior pituitary and somata and dendrites in the SON and plays critical roles in parturition, lactation and synaptic plasticity in early post-natal life. Somato-dendritic OT secretion can be primed for activity-dependent release by thapsigargin (TG) which releases calcium from intracellular stores. Regulated secretion requires an increase in intracellular calcium concentration by calcium influx via voltage operated calcium channels (VOCCs). Using fluorescent immunohistochemistry, we found α-subunits for L-, P/Q-, N- and R-type VOCCs expressed in the somata and dendrites of OT neurons. Blocking N-type VOCCs was most effective, reducing high potassium stimulated (50mM) somato-dendritic secretion in vitro to 66±11% in adult female rats and 28±23% in post-natal day 8 rats (P<0.05, vs. control). TG-induced priming of high potassium stimulated OT release (447±90%, P<0.05 vs. control) was also reduced by blocking N-type VOCCs (to 133±18%, P<0.05). Whole-cell patch clamp recordings showed that calcium current carried by N-type VOCC was increased from 8±3 to 27±8 pA/pF (P<0.05) following TG treatment while currents carried by L, P/Q and R VOCCs were unaltered or decreased. These data suggest that high potassium-stimulated and primed somato-dendritic OT release involve an increase in calcium current carried predominately by N-type VOCCs. [J Physiol Sci. 2008;58 Suppl:S75]

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The inward rectifier K⁺ channels in the Kir2 subfamily carry very small outward currents compared to the inward currents and this strong inward rectification is caused by a voltage-dependent block of the channel by intracellular cations. In this study, we show using the inside-out patch recordings that Kir2.1 channels exhibit a time- and voltage-dependent gating that depends on the internal pH in the range below 7.2. This gating was observed in the absence of polyamines, Mg²⁺, and EDTA in the internal (bath) solution and thus seemed to be unrelated to the mechanism of the channel block induced by cations. Substitution of the His residue (pKa = ∼6.5) situated on the wall of the cytoplasmic pore with the basic Lys residue (H226K) did not eliminate this pH-dependent gating. We then examined Kir2.1 mutant channels known to have reduced sensitivity to internal cationic blockers. Neutralization of the acidic Glu residues situated on the wall of the cytoplasmic pore (E299S and E224G) neither notably affected the pH-dependent gating. By contrast, neutralization of the acidic Asp residue on the wall of the transmembrane pore (D172N) abolished the gating. These findings suggest that the negative charges at site 172 of Kir2.1 (composed of four subunits), which are thought to form the high-affinity binding site for polyamines, are required for the pH-dependent gating mechanism. [J Physiol Sci. 2008;58 Suppl:S75]

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Human Kv1.5 (hKv1.5) channels conduct the ultra-rapid delayed rectifier potassium current (I_Kur) that is highly expressed in atria but scarcely in ventricle. hKv1.5 mediates action potential repolarization of human atrial myocytes and blockade of this channel has been expected to prolong the action potential duration (APD) and thereby reduce the occurrence of reentrant-based arrhythmias such as atrial fibrillation (AF). The aim of this study was to investigate the effect of nisoldipine a Ca²⁺ channel blocker used widely for the treatment of variety of cardiovascular disorders on hKv1.5 currents heterologously expressed in CHO cells. Nisoldipine, rapidly and reversibly inhibited hKv1.5 current in a concentration-dependent manner with an IC₅₀ of 3.0 μM. The block of hKv1.5 progressed with time during depolarizing voltage with a more rapid block at higher concentrations. Block increased rapidly between -40 mV and +10 mV, coincident with channel opening and suggested an open channel block mechanism. Nisoldipine markedly shifted the voltage dependence of channel opening to more hyperpolarizing potentials. In addition, mutations of T480A (near to pore helix) and I508A (located in S6 segment) significantly reduced the block action (with an IC₅₀ of 16.1 μM for T480A). The present results suggest that (i) nisoldipine inhibits hKv1.5 currents at relatively low concentration as an open channel blocker and (ii) the binding sites for the block are probably related to threonine 480 and isoleucine 508. [J Physiol Sci. 2008;58 Suppl:S75]

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An astroglial inwardly rectifying K⁺ channel, Kir4.1, is inhibited by a variety of compounds including a number of antidepressants. The mechanisms for the channel-drug interaction are however poorly understood. Here we identified electrophysiologically amino acids at the Kir4.1 pore essential for binding with antidepressants, fluoxetine, a SSRI, and nortrytyline, a tricyclic antidepressant, using the chimera and alanine-substituted mutants. Wild-type and mutant rat Kir4.1 channels were expressed in Xenopus oocytes. Mutagenesis experiments revealed that two amino acids, Thr128 and Glu158 on TM2, are indispensable for both of fluoxetine and nortriptyline to inhibit Kir4.1 channel current. Homology modeling of Kir4.1 channel pore using Shaker pore as the template suggests that both Thr128 and Glu158 face the central cavity of the channel in the open and closed states. Thr128 and Glu158 are thought to interact with respectively hydrogen bond acceptor and constitutively charged regions of drugs. Further computational analysis for common features in Kir4.1 blockers revealed that at least four pharmacophores distribute in the drugs. Two of them may function in hydrogen bonding and electrostatic interaction. The pharmacophores locate within a geometrical range between Thr128 and Glu158 to be retained in central cavity of the channel. These findings strongly support the drug binding in its position. [J Physiol Sci. 2008;58 Suppl:S75]

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Voltage-sensing domain (VSD) is crucial for the function of voltage dependent ion channels. Recent advancements of structural studies on the voltage dependent ion channels have revealed various structural features of VSD; however, the movement and conformational change of segments S1-S4 within VSD in response to membrane depolarization are still under debate. We have previously shown that KCNE1, which is an auxiliary subunit for KCNQ1 K ⁺ channel, stabilizes VSD in the down state and proposed a possible interaction between VSD and KCNE1. In the present study, we found that some of the cysteine mutants of S4 domain (I227C, R228C, G229C, I230C, R231C, F232C and L233C) were trapped in the open state after depolarization in the absence of KCNE1. There are two endogenous cysteine residues in the transmembrane region, C136 on S1 segment and C331 on S6 segment, therefore, we suspected either of them might form a disulfide bond with introduced cysteine residue on S4 segment. By substituting each cysteine with alanine, we identified that C136 made a disulfide bond with the exogenous cysteine residue on S4 segment. Interestingly, except for G229C, the disulfide bode was not formed by co-expressing KCNE1. These results suggest that S1 segment interacts with S4 segment in the absence of KCNE1 and that KCNE1 disrupts the interaction between S1 and S4. KCNE1 might change the conformation of VSD and eventually affect the equilibrium between the up state and down state of VSD. [J Physiol Sci. 2008;58 Suppl:S76]

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Large-conductance Ca²⁺-activated K⁺ (BK) channel has pivotal roles in the regulation of smooth muscle tone. Here we examined single-molecule imaging of BK channel subunits in the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. BK channel α subunit (BKα-YFP) and β1 subunit (BKβ1-CFP) were expressed transiently in cultured rat aortic myocytes or HEK cells. When aortic myocytes were transfected with BKα-YFP, the fluorescent signals of individual channel unit or their clusters were not uniformly distributed in the TIRF images and the sum of fluorescent areas occupied approximately 1% of the plasma membrane in the image. Dynamic mobilizations of BKα-YFP were detected at the levels of individual channel unit and their cluster in HEK cells. When BKα-YFP was coexpressed with BKβ1-CFP in HEK cells, the molecular mobility of the complex was reduced to half of BKα-YFP alone. In contrast, the dynamics of BKα-YFP expressed in aortic myocytes was much more restricted compared with that of BKα-YFP/BKβ1-CFP coexpressed in HEK cells. And actin disruption by 1 μM cytochalasin D in aortic myocytes caused the increase in diffusion coefficient of BKα-YFP. In aortic myocytes, BKα-YFP was co-distributed with caveolin-1 (Cv1-CFP), a marker of caveolae. These results suggest that the mobilization of BK channel α subunit in plasma membrane is strongly restricted or regulated by its accessory β subunit, the cytoskeleton, and the raft structure of membrane surface in native cells such as vascular smooth muscle cells. [J Physiol Sci. 2008;58 Suppl:S76]

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It is usually understood that Ca²⁺-activated Ca²⁺ release (CICR) is a Ca release from ryanodine receptors. However, a CICR from IP3 receptors (IP3Rs) has also been described and characterized in hippocampus (Nakamura et al., 1999; Power and Sah, 2002) and visual cortex pyramidal cells (Yamamoto et al., 2000). In this particular form of CICR, which we call IP3-assisted CICR, IP3Rs are activated beforehand by IP3 but kept closed, until opening is finally triggered by action potential-induced Ca²⁺ inflow through vlotage-dependent Ca channels (VDCCs). We have shown that IP3-assisted CICR enhances SK channel current and thereby reducing neural excitability (Yamamoto et al, 2002; Yamada et al, 2004). In these previous experiments, the IP3-mobilizing neurotransmitter that we used was either the muscarinic acetylcholine receptor agonist carbachol or the group I metabotropic glutamate receptor agonist DHPG. Both of them were effective at 10uM but ineffective at 5uM. Whether these two drugs co-applied together, each at an innefective dose by itself, result in the effective enhancement of SK current is presumable but not self-evident. We have demonstrated that this is the case, suggesting that IP3 produced by different G-protein coupled receptors is additive in the cytosol and that IP3-mobilizing ligands may be synergistic in various cellular contexts. [J Physiol Sci. 2008;58 Suppl:S76]

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Androgen receptor (AR) activation by testosterone regulates the expression of many kinds of proteins and, thereby, castration, which significantly decreases serum testosterone level, prevents the maturation of mail organs. We previously reported that the functional expression of large conductance Ca²⁺-activated K⁺ (BK) channels is markedly diminished in smooth muscle from reproductive organ of castrated-rat (CAST). The decrease in BK channel expression was limited in organs, where ARs are extensively expressed. BK channels are ubiquitously expressed and possess obligatory roles in the regulation of membrane excitability in many types of cells including CNS neurons. We examine the possibility that BK channel expression in some regions of brain may possibly regulated by plasma testosterone level. Firstly, BK channel mRNA expression was examined in four regions of brain; amygdala hypothalamus, pituitary, hippocampus. Amygdala region, in which substantial expression of AR has been reported, had relative abundant expression of BK channel mRNA. Secondly, we investigated changes in BK channels expression in amygdale region in sham-operated -rats and CAST. Western blot analyses also demonstrated marked decrease in the amount of BKα protein in amygdala from CAST. The measurement of BK channel currents in isolated neurons from amygdala region was also performed by patch-clamp techniques. Taken together, the regulation of BK channel function in amygdala by testosterone and by gender difference can be suggested. [J Physiol Sci. 2008;58 Suppl:S76]

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Follicular cells surrounding Xenopus oocyte under voltage-clamp produce K⁺ current responses to follicle stimulating hormone (FSH), Adenosine (Ade) and intracellularly applied cAMP, presumably due to the opening of K_ATP channel. To identify the K_ATP channel subtype involved in the FSH-and Ade-induced responses, we investigated effects of various K⁺ channel openers (KCOs) and sulfonylurea receptor (SUR) blockers on the agonist-induced responses. Applications of PCO400, Cro, pinacidil but not diazoxide produced K⁺ current responses similar to the FSH- and Ade-induced responses in the order of PCO400 < Cro << pinacidil in favor of SUR2A. Application of glibenclamide, phentolamine and tolbutamide suppressed all the K⁺ current responses to FSH, Ade, cAMP, and KCOs. Furthermore, the both FSH- and Ade-induced responses were markedly augmented during the KCO-induced responses or vice versa. I-V curves for the K⁺ current responses induced by Cro, Ade and FSH showed outward rectification in normal [K⁺]o but weak inward rectification in 122 mM [K⁺]o. In addition, as observed previously with FSH- and Ade-induced responses, stimulations of P2Y receptor by UTP or PKC by PDBu markedly depressed the K⁺ current response to KCO in favor of Kir6.1. These results suggest that the K⁺ current responses to FSH and Ade may be produced by opening of a novel type of K_ATP channel comprising SUR2A and Kir6.1. [J Physiol Sci. 2008;58 Suppl:S77]

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Voltage sensor domain (VSD) regulates ion permeation and enzyme activity. It remains unknown how coupling mechanisms are similar between voltage-gated channels and voltage-sensing phosphatases, VSPs. The voltage dependency of the movement of the voltage sensor of Ci-VSP is less steep than that of voltage-gated channels, and is not saturated even over 100 mV. To clarify the voltage range at which enzyme activity is saturated, kinetics of PIP2 depletion was compared with confocal microcope at distinct membrane potentials by monitoring cell surface translocation of GFP fused with a PH-domain (PHD-GFP). The change of translocation of PHD-GFP occurred at time constant of 22.8 +- 15.0 sec (n=5) at 50 mV and 7.7 +- 2.8 sec (n=5) at 100 mV, whereas no significant difference was found between 100 mV and 150 mV, possibly due to slow diffusion speed of PHD-GFP protein. As alternative approach, kinetics of outward current through engineered inward-rectifier potassium channel, Kir2.1(4mut), with mutations at sites critical for inward-rectification (Fujiwara & Kubo, J. Physiol., 2002) was electrophysiologically measured. Decay of outward current through Kir2.1(4mut) became sharper as the voltage increases from 50 mV to 100 mV, and also from 100 mV to 170 mV. However, decay kinetics was similar at higher range, suggesting that enzyme activity is saturated. This provides potent evidence that voltage-dependency of downstream effecter activity is shifted rightward with reference to that of the VSD as in voltage-gated ion channels. [J Physiol Sci. 2008;58 Suppl:S77]

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Crystal structures of potassium channels in open and closed conformations have provided static pictures for the different gating status. To examine dynamic pictures of the channels in function, we recorded gating motions of single KcsA potassium channels with the diffracted X-ray tracking method. The KcsA channels were fixed on a glass plate at the extracellular side, and labelled with a gold nanocrystal at the other side. Irradiations of synchrotron white X-rays elicited diffraction spots from the attached nanocrystal. The motions of the spots, which were recorded at video rate, could be translated into the channel motions in real space. Under gating condition twisting motions around the longitudinal axis with several tens of degrees were observed. These twisting motions were detected from the channels labeled at the end of the cytoplasmic domain or at the end of M2 helices. In either case the observed motions were stopped by an open channel blocker. From these results we concluded that the KcsA channels exhibited global twisting motions during gating, which were originated from the transmembrane domain and transferred to the cytoplasmic domain. [J Physiol Sci. 2008;58 Suppl:S77]

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Potassium channels permeate K⁺ ions at rates approaching 10⁸ ions per second with strict selectivity. This process is crucial for producing proper electrical signals in biological systems. However, the mechanism of ion conduction through the selectivity filter remains unsolved. For the bacterial K⁺ channel, KcsA, high resolution crystal structures in the closed state have revealed the single-file distribution of ions and water molecules in the pore. Many simulation studies have been performed based on these static pictures but the results provided only a part of ion permeation process. Hence, the experimental data is essential to understand how water and ion cooperate to permeate through the open channel. Here we examined the streaming potential (V_stream) of K⁺ selective permeation in the KcsA channel using liposome patch-clamping technique. V_stream provides a quantitative measure of the number of water molecules accompanying a conducting ion (the water-ion coupling ratio), the knowledge of which is a prerequisite for elucidating the coordination of water and ion during permeation. Current-voltage relationship of single KcsA channel was recorded under varying osmotic pressure and the value was evaluated as a shift of the reversal potentials. Effects of concentration and species of permeating ions on the V_stream were investigated because those factors have been reported to perturb the ion coordination in the selectivity filter of crystal structure. Results will be discussed in relation to the ion conducting mechanism of K⁺ selectivity filter. [J Physiol Sci. 2008;58 Suppl:S77]

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Caffeine is known to have various pharmacological effects such as activation of ryanodine receptor, block of adenosine receptor and inhibition of phosphodiesterase. Masuho et al. (2005) reported that [Ca²⁺]_i increase was observed upon application of caffeine in STC1 enteric neuroendocrine cell and that it was inhibited by phospholipase C (PLC) inhibitor. The results cannot be explained by the known effects and imply the presence of a caffeine sensitive molecule whose activity or effect is dependent on PLC. We observed by [Ca²⁺]_i imaging that the [Ca²⁺]_i increase by caffeine in STC1 cells disappeared in the absence of Ca²⁺_o, showing that a caffeine sensitive Ca²⁺ permeable channel with PLC dependency is involved. We also observed the response was inhibited by blockers of Ca²⁺ permeable Transient Receptor Potential (TRP) channel, Gd³⁺ and ruthenium red. As TRPA1 channel is known to require basal PLC activity to maintain its function, we speculated TRPA1 channel is the molecule which provides caffeine sensitivity to STC1 cells. cDNA encoding TRPA1 was successfully isolated from STC1 cells by PCR. In HEK293 cells transfected with TRPA1 channel, [Ca²⁺]_i increase upon application of caffeine and its suppression by Gd³⁺ or ruthenium red were confirmed. TRPA1 channel current evoked by caffeine was also observed electrophysiologically in Xenopus oocytes. In summary, we showed for the first time that caffeine has a new pharmacological effect to increase [Ca²⁺]_i by activating TRPA1 channel on the cell surface membrane. [J Physiol Sci. 2008;58 Suppl:S78]

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TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels, and is expressed in sensory nerve endings of the pain pathway. It has been reported that TRPA1 is activated by a variety of noxious stimuli including pungent natural compounds and environmental irritants. However, it has been poorly reported that high-affinity endogenous ligands directly activate TRPA1. Here, we show that 15-deoxy-delta ^12,14-Prostaglandin J₂ (15d-PGJ₂), an endogenous α, β-unsaturated ketone that is produced in inflammation, activates TRPA1. Ca²⁺ imaging showed that 15d-PGJ₂ activates heterologously expressed TRPA1 in HEK293 cells with an apparent EC₅₀ of 890 nM. Site-directed mutagenesis studies and binding assay indicate that cytoplasmic N-terminal cysteines of the channel is involved in 15d-PGJ₂ activity on TRPA1. Our results indicate that 15d-PGJ₂, an inflammatory compound, may directly activate TRPA1 in nervous system. [J Physiol Sci. 2008;58 Suppl:S78]

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