Cheung J Y, Zhang X Q, Bokvist K, Tillotson D L, Miller B A
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, USA.
Blood. 1997 Jan 1;89(1):92-100.
Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (-50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 +/- 0.75 to 11.15 +/- 1.80 ms) and open probability (from 0.26 +/- 0.06 to 2.56 +/- 0.59%) of this Ba(2+)-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.
促红细胞生成素（Epo）可使人类成红细胞内的细胞内游离钙离子浓度（[Ca2+]i）呈剂量依赖性增加，这一过程依赖于细胞外钙离子，且会被高剂量硝苯地平或Ni2+阻断。此外，用小鼠抗人促红细胞生成素受体抗体而非小鼠免疫纯IgG预处理人类成红细胞，可阻断Epo诱导的[Ca2+]i增加，这表明Ca2+对Epo刺激的反应具有特异性。在本研究中，通过单通道记录鉴定了促红细胞生成素调节的钙通道。使用传统的全细胞膜片钳未能检测到Epo诱导的全细胞Ca2+电流。为了尽量减少细胞溶质成分的洗脱，我们接下来使用了制霉菌素穿孔膜片钳，但未发现任何Epo诱导的全细胞Ca2+电流。在细胞贴附膜片中使用Ba2+（30 mmol/L）作为电荷载体，我们检测到了单通道，其单位电导为3.2 pS，反转电位为+72 mV，且其单位电流（在+10 mV时）随Ba2+浓度增加而单调增加。在所测试的电压范围（-50至+30 mV）内，通道开放概率没有明显变化。Epo（2 U/mL）增加了该Ba(2+)通透通道的平均开放时间（从4.27 +/- 0.75毫秒增加到11.15 +/- 1.80毫秒）和开放概率（从0.26 +/- 0.06%增加到2.56 +/- 0.59%）。我们的数据有力地支持了这样的结论：Epo诱导的人类成红细胞内[Ca2+]i增加是通过电压非依赖性钙通道介导的Ca2+内流实现的。
