Schuhmann K, Romanin C, Baumgartner W, Groschner K
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, A-8010, Graz, Austria.
J Gen Physiol. 1997 Nov;110(5):503-13. doi: 10.1085/jgp.110.5.503.
Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.
在人脐静脉平滑肌的完整细胞和内向外膜片中，研究了细胞质Ca2+的持续升高对L型Ca2+通道的调节作用。以Ba2+作为电荷载体，用钙蛋白酶抑制剂加ATP防止内向外膜片中Ca2+通道活性的衰减。在离子载体A23187存在的情况下，通过提高细胞外Ca2+浓度来增加完整细胞中的细胞质Ca2+，可抑制细胞贴附膜片中L型Ca2+通道的活性。用fura-2测量细胞内游离Ca2+的实际水平，结果显示Ca2+依赖性抑制的50%抑制浓度（IC50）为260 nM，Hill系数接近4。Ca2+诱导的完整细胞中Ca2+通道活性的抑制是由于通道开放概率和可利用性的降低。Ca2+诱导的抑制不受蛋白激酶抑制剂H-7（10 microM）或细胞骨架破坏剂细胞松弛素B（20 microM）的影响，但被蛋白磷酸酶2B（钙调神经磷酸酶）的抑制剂环孢素A（1 microg/ml）所阻断。内向外膜片细胞质侧的Ca2+升高可抑制Ca2+通道，IC50为2 microM，Hill系数接近1。无细胞膜片中直接的Ca2+依赖性抑制是由于开放概率的降低，而可利用性几乎不受影响。在游离Ca2+浓度为1 microM时，将纯化的蛋白磷酸酶2B（12 U/ml）应用于内向外膜片的细胞质侧，可抑制Ca2+通道的开放概率和可利用性。在PP2B存在的情况下，细胞质Ca2+的升高可抑制内向外膜片中的通道活性，IC50约为380 nM，Hill系数约为3；即，这些特征让人想起完整细胞中Ca2+通道的Ca2+敏感性。我们的结果表明，平滑肌的L型Ca2+通道受两种Ca2+依赖性负反馈机制的控制。这些机制基于：（a）蛋白磷酸酶2B介导的去磷酸化过程，以及（b）细胞内Ca2+与可能位于通道蛋白本身的单个膜相关位点的相互作用。
