| Project/Area Number |
63570062
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Neurophysiology and muscle physiology
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| Research Institution | Nagoya City University |
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Principal Investigator |
OBA Toshiharu Nagoya City University Medical School, Department of Physiology, Associate Professor, 医学部, 助教授 (50008330)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1990: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1989: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1988: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Skeletal Muscle / Transverse Tubule / Fluorescence Dye / Ca Channel Protein / Sulfhydryl Group / Voltage Sensor / 筋小胞体 / Caチャネル蛋白質 / PH160 / 骨格筋膜電位 / 二重マニト-ル間隙法 / 蛍光強度 / RH160 / 膜電位 / 骨格筋単収縮 / Ca電極 / ETH1001 |
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| Research Abstract |
Skeletal muscle contraction is known to be regulated by cytoplasmic Ca^<2+> released from sarcoplasmic reticulum (SR). Excitation of transverse (T) tubule (action potential) triggers Ca^<2+> release. Recent studies suggest that DHP receptor alpha_1 subunit is involved in signal transmission from T tubule to SR. The present project was designed to elucidate the mechanism by which action potential of T tubule (voltage sensor activation) elicits the release of Ca^<2+> from the SR in skeletal muscle. Two experiments were performed using frog single fibers. First one is to study whether a cyanine fluorescence dye (RH 160) can monitor the T tubular membrane potential. Fiber was stained at 20 muM RH 160 for 5 min in the experimental chamber on inverted microscopic stage equipped with the fluorescence detector. Decrease in fluorescence intensity (excited at 530 nm and emitted at 685 nm) was observed after membrane depolarization upon 120 mM K^+ exposure (corresponding to F/Fo=0.125 /100 mV mem
… More
brane potential). The time course of optical actin potential monitored by fluorescence intensity change in response to electrical stimulation was very similar to that by cnventional glass microelectrode method, indicating that membrane potential change can be followed by using this fluorescent dye. Therefore, this optical method will be useful to monitor membrane potential in membranes or small cells in which glass microelectrode method can not be applicable. Second, I studied the molecular mechanism where voltage sensor in the T tubule senses membrane depolarization and triggers the Ca^<2+> release from the SR. Silver ion (Ag^+) produces a transient contraction followed by complete inhibition of excitation-contraction coupling. DHP completely inhibits Ag-contraction. Blockade of Ca^<2+> entry with cadmium does not inhibit Ag-contraction. Ag^+ binds to the partially purified skeletal muscle Ca^<2+> channel and inhibits DHP binding and SH reactivity over the concentration range where it inhibits E-C coupling. Oxidation of free SH groups by H_2O_2 or DTNB prevents Ag-contraction and DTT reduction of oxidized SH groups restored Ag-contraction. These results suggest that Ag^+ binds to critical SH groups on the DHPR Ca^<2+> channel resulting in inhibition of the channel's voltage sensor and the failure of E-C coupling. Less
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