1994 Fiscal Year Final Research Report Summary
A Novel Method to Quantify Intracellular Free Calcium Concentration in Muscle Cells with a Fluorescent Indicator.
| Project/Area Number |
05670055
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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 |
General physiology
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| Research Institution | The Jikei University School of Medicine |
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Principal Investigator |
KONISHI Masato Associate Professor, Department of Physiology, The Jikei University School of Medicine, 医学部, 助教授 (20138746)
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| Co-Investigator(Kenkyū-buntansha) |
WATANABE Masaru Instructor, Department of Physiology The Jikei University School of Medicine, 医学部, 助手 (60191798)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Muscle / Intracellular calcium concentration / Fluorescent Ca indicator / beta-escin |
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| Research Abstract |
To measure intracellular Ca^<2+> concentration ( [Ca^<2+>] _i) in skeletal muscle fibers at rest, the Ca^<2+> indicator, fura-2, conjugated to dextran (fura dextran, MW -10,000) was injected into single twitch fibers of frogs, and the indicator's Ca^<2+> -dependent fluorescence in the cytoplasm was measured at 17ﾟC.
Calibration of the indicator fluorescence (in terms of [Ca^<2+>] _i) was carried out in the muscle fibers treated with beta-escin to permeabilize the cell membrane. After the treatment with 5 muM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca^<2+>, ATP) , while the 10 kDa fura dexran only slowly leaked out of the cell. The major fraction of cytoplasmic proteins (14-80 kDa) was retained in the beta-escin-treated cells, as only a trace amount of proteins was detected in the exracellular solution samples by SDS-PAGE with silver staining. It was thus possible to estimate the calibration parameters of the indicator fluorescence in the cell (in the presence of cellular proteins) by changing the Ca^<2+> concentration in the bething solution to various levels. When [Ca^<2+>] of the bathing solution was changed to pCa>9 to higher levels (pCa7-4), the Ca^<2+> -dependent fluorescence of fura dextran changed to a new steady level within a few minutes. The indicator's dissociation constant for Ca^<2+> (K_D) estimated in the cell was 1.0 muM,which is two-fold higher than that obtained in vitro (0.52 muM). The second method to estimate the K_D, the kinetic analysis of the indicator fluorescence change following electrical stimulation in intact muscle fibers, gave an estimated value of 2.1 muM.The K_D values thus estimated in the cell interior (1.0-2.0 muM) is two- to four-fold higher than that obtained in vitro (0.52 muM). From the parameters estimated in the fibers, we conclude that the resting [Ca^<2+>]_i in frog skeletal muscle fibers is likely in the range of 55-155 nM.
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