Signal transduction mechanisms in negulation of Na/Ca exchanger via PI(4,5)P_2

通过 PI(4,5)P_2 调节 Na/Ca 交换器的信号转导机制

• 批准号: 14570039
• 负责人: MATSUOKA Satoshi
• 金额: $ 2.24万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570039/
• 关键词: Na^+-Ca^<2+> exchange; Inactivation; Heart; PI(4,5)P_2; Tyrosine phosphatase; PKA; Macro patch; β受容体; クロール電流; ATP; ジャイアントパッチ; リン酸化; Na^+-Ca^<2+> exchange; Inactivation; Heart; PI(4,5)P_2; Tyrosine phosphatase; PKA; Macro patch; β受容体; クロール電流; ATP; ジャイアントパッチ; リン酸化

To investigate the regulatory mechanisms of Na^+-Ca^<2+> exchange under physiological conditions, we recorded Na^+-Ca^<2+> exchange current (I_<NaCa>) in the inside-out patch excised from guinea-pig ventricular myocytes (the macro patch). Applying ATP or PI(4,5)P_2 to the cytoplasmic surface of the patch membrane augmented the I_<NaCa>. Treatment of the patch membrane with PI-specific phospholipase C, which inhibits the production of PI(4,5)P_2 from PI, attenuated the ATP effect. This result suggests that the stimulatory effect of ATP is in part due to the production of PI(4,5)P_2 from PI. However, the application of inhibitors of protein tyrosine phosphatase, vanadate or bpV(phen), significantly augmented the I_<NaCa> in the presence of ATP. This result and others suggest that protein phosphorylation, probably protein tyrosine phosphorylation, is also involved in the stimulatory ATP effect on the I_<NaCa>.To validate the report that the PKA signaling stimulates the Na^+-Ca^<2+> activity, we measured whole-cell I_<NaCa> from guinea-pig ventricular myocytes. Isoproterenol, which is an agonist of the β-adrenoceptor, activated the cAMP-dependent Cl^-current, but did not stimulate the I_<NaCa>. We concluded that the PKA signaling does not stimulate the Na^+-Ca^<2+> exchange in guinea-pig ventricular cells. The previous reports might underestimate the activation of the cAMP-dependent Cl^-current.To study the regulatory mechanisms of NCX3, which is mainly expressed in brain and skeletal muscle, we measured the I_<NaCa> in the giant membrane patch excised from oocytes expressing NCX3. It was revealed that NCX3 possesses both the Ca^<2+>-dependent activation and inactivation, and that the increase of the I_<NaCa> by ATP is less potent in NCX3 than NCX1.

为了研究在生理条件下Na^+-ca^<2+>交换的调节机制，我们记录了Na^+-ca^<2+>交换电流（I_ <naca>）在Inser-Out贴片中，来自豚鼠心室心肌细胞（宏观贴片）。将ATP或PI（4,5）P_2应用于斑块膜的细胞质表面增强I_ <NACA>。用PI特异性磷脂酶C处理贴片膜，该磷脂酶C抑制PI的PI（4,5）P_2的产生，从而减弱了ATP效应。该结果表明，ATP的刺激作用部分是由于PI从PI中产生P_2。然而，在ATP存在下，蛋白酪氨酸磷酸酶，钒酸盐或BPV（PEN）的抑制剂的应用显着增强了I_ <NACA>。该结果和其他结果表明，蛋白质磷酸化可能（可能是蛋白酪氨酸磷酸化）也参与了刺激性ATP对I_ <NACA>的影响，以验证报告的报告表明，PKA信号刺激Na^+-ca^<2+>活性，我们测量了Guineaea-pig pentullullolultol myocymocytormocytimal myocytess i_ <naca>。异丙肾上腺素是β-肾上腺素受体的激动剂，激活了cAMP依赖性的Cl^crurrent，但没有刺激I_ <NACA>。我们得出的结论是，PKA信号不会刺激豚鼠心室细胞中的Na^+-ca^<2+>交换。先前的报告可能会低估cAMP依赖性Cl^-Current的激活。要研究主要在大脑和骨骼肌中表达的NCX3的调节机制，我们在从表达Ncx3的卵母细胞中卓越的巨型膜贴片中测量了I_ <NACA>。据表明，NCX3既具有CA^<2+> - 依赖性激活和失活，并且I_ <NACA>通过ATP的增加在NCX3中比NCX1的效力较小。