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中国生理学会第24届全国会员代表大会暨生理学学术大会

作者：樊红琨罗天霞郑春光杨阳涂会引章茜郑州大学基础医学院生理教研室

Aim:Junctophilins（JPs）are main membrane-binding proteins that are associated with the functions of junctional membrane complexes（JMC）.Small conductance Ca2+activated K+channels（SK）profiles the shape of the action potential of the cardiac *** Rs are an intracellular Ca2+release channel existing in SR of eukaryotic *** study showed that a functional crosstalk between the SK channels and the Ry Rs Ca2+*** the present study we documented the interactions of JP2 with SK2 and Ry R2 in the cardiac ***:Solubilized proteins from mouse cardiac muscle or from HEK293-T cells were detected using anti-SK2,anti-JP2 and anti-Ry R2 antibodies by co-immunoprecipitation,*** recombinant GST fusion proteins were constructed and induced in BL21 *** pull-down was used to validate the relationship of JP2 with SK2 and Ry R2 in *** recombinants of lentiviral vectors targeting JP2 gene were constructed using RNAi technique and transduced into the culture neonatal mouse cardiac myocytes（NMCMs）.The levels of JP2 and SK2m RNA and protein were test by q RT-PCR and Western blot ***:JP2 was immunoprecipitated with an antibody against SK2 and with an anti-Ry R2 antibody,but was absent in samples incubated with an unrelated *** GST-JP2 fusion protein was able to pull down the cell lysates from HEK293/T cells transfected with p *** addition,GST-JP2 was also able to pull down endogenous Ry R2 from solubilized proteins prepared from the mouse cardiac ***,the levels of JP2 and SK2 m RNA in the NMCMs treated with the recombinants of the lentivirul-mediated si RNA targeting JP2 were significantly decreased compared with the control and scramble *** expressions of the SK2 and JP2 proteins in the NMCMs transduced with the lentivirul-mediated si RNA were obviously ***:These results suggest that the interactions of JP2 with SK2 channel and Ry R2-calcium release channel exist in the nat

关键词： JP2 SK2 RyR2 cardiac myocyte

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Isolation of Ca^(2+) Tolerant Cardiomyocytes from Aadult Rats for Patch Clamp Studies

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Journal of Nanjing Medical University 2004年第1期18卷 4-6页

作者：徐华娥陶金陈洁汪红仪李胜男南京医科大学药理学系

Objective: To investigate the factors affecting the viability and Ca 2+ tolerance of isolated rats' cardiac myocytes for patch clamp research. Methods: Hearts were firstly perfused by the Langendorff perfusion apparatus with normal Tyrode's solution, then with Ca 2+-free Tyrode's solution and subsequently with low Ca 2+ enzyme solution containing collagenase 0.1-0.2 g/L. All the solutions were saturated with oxygen and the perfusion temperature was kept at 37 ℃. Finally hearts were washed by Ca 2+-free Tyrode's solution, after which the ventricles were minced into small pieces in KB solution, dispersed and filtered. The isolated myocytes were stored in KB solution at room temperature for 1 h and recovered to normal calcium concentration before patch clamp ***: When all the factors such as water, enzyme, Ca 2+,pH, and oxygen were well controlled, the well constructed and rod-like cardiac myocytes with a yielding rate of 30%-50% came ***: All the factors should be well controlled, which ensured the isolated cells Ca 2+ tolerant and appropriate for patch clamp experiments.

关键词： isolation cardiac myocyte patch-clamp Tyrode

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