Functional coupling of calcineurin and,protein kinase A in mouse ventricular myocytes
Functional coupling of calcineurin and,protein kinase A in mouse ventricular myocytes
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DOI：
10.1113/jphysiol.2002.020552
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发表时间：
2002-10-01
影响因子：
5.5
通讯作者：
Greven, R
中科院分区：
文献类型：
--
作者：
Santana, LF;Chase, EG;Greven, R
我们研究了钙调节蛋白磷酸酶钙调神经磷酸酶在小鼠心室肌细胞中控制钙信号的作用。采用膜片钳技术测量单个心肌细胞的膜电流和电压。使用共聚焦显微镜或落射荧光显微镜测量加载有荧光Ca 2+指示剂fluo-4或fura-2的细胞中的细胞质Ca 2+浓度（[Ca 2 +]（i））。用环孢菌素A（CsA，100 nm）或钙调磷酸酶自身抑制肽（CiP，100 μ m）抑制钙调磷酸酶，增加诱发的[Ca 2 +]（i）瞬变的幅度和衰减速率，并且也延长心室肌细胞的动作电位（AP）至类似程度。CsA（100 nm）和100 mum CiP对[Ca 2 +]（i）瞬变和AP的影响不加和。钙调磷酸酶抑制没有修改负责小鼠心室复极的K+电流。相反，钙调磷酸酶的抑制增加了钙电流（I-Ca）的幅度和诱发的钙瞬变归一化的I-Ca。作为[Ca 2 +]（i）瞬变基础的钙火花具有较高的频率和振幅，表明SR钙负荷升高。蛋白激酶A（PKA）的抑制阻止了钙调磷酸酶抑制的作用，表明钙调磷酸酶对抗PKA的作用。最后，免疫荧光图像表明，钙调神经磷酸酶和PKA共定位在心室肌细胞的T-小管附近。我们建议，钙调神经磷酸酶和PKA的共定位，以控制通过钙通道的钙离子内流和钙释放通过兰尼碱受体。
We examined the role of the Ca2+-regulated protein phosphatase calcineurin in controlling Ca2+ signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch-clamp technique. Cytoplasmic Ca2+ concentration ([Ca2+](i)) was measured in cells loaded with the fluorescent Ca2+ indicators fluo-4 or fura-2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nm) or the calcineurin auto-inhibitory peptide (CiP, 100 mum), increased the amplitude and rate of decay of the evoked [Ca2+](i) transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nm) and 100 mum CiP on the [Ca2+](i) transient and AP were not additive. Calcineurin inhibition did not modify the K+ currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca2+ current (I-Ca) and the evoked calcium transient normalized to the I-Ca. Calcium sparks, which underlie the [Ca2+](i) transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co-localize near the T-tubules of ventricular myocytes. We propose that calcineurin and PKA are co-localized to control Ca2+ influx through calcium channels and calcium release through ryanodine receptors.