Ca2+-dependent inhibition of inwardly rectifying K+ channel in opossum kidney cells
Ca2+-dependent inhibition of inwardly rectifying K+ channel in opossum kidney cells
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DOI：
10.2170/jjphysiol.51.371
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发表时间：
2001-06-01
期刊：
影响因子：
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通讯作者：
Kubota, T
中科院分区：
文献类型：
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作者：
Mori, Y;Kawasaki, A;Kubota, T
利用膜片钳技术在负鼠肾近端小管（OKP）细胞中检测了细胞内Ca2+对向内整流atp调节的K+通道活性的影响，该通道的向内电导约为90 pS。在细胞贴片中存在10(-6)M Ca2+的情况下，应用离子霉素（1mum），内整流K+通道的活性迅速下降。10(-6) M Ca2+和10(-6)M phor -12-肉豆蔻酸酯（PMA）的施用降低了K+通道活性。虽然通道活性不受钙离子从10(-7.5)M增加到10(-6)M的影响，但活性被蛋白激酶C （PKC, 1 U/ml）与10(-6)M Ca2+在内向外斑块中抑制。用特异性PKC抑制剂GF 109203X （5mum）预处理细胞贴片，Ca2+与离子霉素对通道活性的抑制作用减弱。相比之下，Ca2+/钙调蛋白激酶II （CaMK II, 300 pM）的应用显著增加了该通道在内向外斑块中的活性。在细胞贴片中，加入GF 109203X和环孢子素A (5mum)，一种有效的蛋白磷酸酶2B（钙调磷酸酶）抑制剂，取而代之的是离子霉素和10(-6)M Ca2+刺激K+通道活性。在钙调素（1 muM）和Ni2+ (10 muM)溶液中加入蛋白磷酸酶2B（钙调磷酸酶）（2 U/ml）来刺激钙调磷酸酶，可以抑制内向外斑块的通道活性。此外，通过去除浴液中的Ca2+， PKC或钙调磷酸酶对该通道活性的抑制作用被消除。这些结果表明，Ca2+依赖性对OKP细胞内纠偏K+通道的抑制作用主要是由Ca2+- pkc介导的磷酸化介导的，Ca2+-钙调素依赖性磷酸化过程可能被Ca2+-钙调素依赖性去磷酸化过程所抵消。
The effect of intracellular Ca2+ on the activity of the inwardly rectifying ATP-regulated K+ channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K+ channel rapidly declined with an application of ionomycin (1 muM) in the presence of 10(-6) M Ca2+ in cell-attached patches. The application of 10 muM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca2+ reduced the K+ channel activity. Although the channel activity was not influenced by an increase of bath Ca2+ from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca2+ in inside-out patches. The inhibitory effect of Ca2+ with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 muM), in cell-attached patches. By contrast, the application of Ca2+/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 muM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K+ channel activity with ionomycin and 10(-6) M Ca2+. The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 muM) and Ni2+ (10 muM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca2+ from bath solution. These results suggest that Ca2+-dependent inhibitory effect on the inwardly rectifying K+ channel in OKP cells was mainly mediated by Ca2+-PKC-mediated phosphorylation, and that the Ca2+ -calmodulin-dependent phosphorylation process may be counterbalanced by the Ca2+ -calmodulin-dependent dephosphorylation process.