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

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.

为了研究生理条件下Na^+-Ca^&lt;2+&gt;交换的调节机制，我们记录了豚鼠心室肌细胞内面朝外膜片（宏膜片）的Na^+-Ca^&lt;2+&gt;交换电流（I_<NaCa>）。将ATP或PI（4，5）P_2作用于膜片膜的胞质表面，可增加I_（10）<NaCa>。用PI特异性磷脂酶C处理膜片膜，可抑制PI产生PI（4，5）P_2，从而减弱ATP效应。这一结果表明，ATP的刺激作用部分是由于PI产生PI（4，5）P_2所致。然而，蛋白酪氨酸磷酸酶抑制剂钒酸盐或bpV（phen）的应用，显着增加了在ATP存在下的I_2<NaCa>。这一结果和其他结果表明，蛋白质磷酸化，可能是蛋白质酪氨酸磷酸化，也参与了ATP对I_2的刺激作用。<NaCa>为了验证PKA信号刺激Na^+-Ca^&lt;2+&gt;活性的报道，我们测量了<NaCa>豚鼠心室肌细胞的全细胞I_2。β-肾上腺素受体激动剂异丙肾上腺素激活cAMP依赖性Cl^-电流，但不刺激I_<NaCa>。我们的结论是PKA信号不刺激豚鼠心室肌细胞的Na^+-Ca^&lt;2+&gt;交换。为了研究主要在脑和骨骼肌中表达的NCX 3的调节机制，我们测量了<NaCa>从表达NCX 3的卵母细胞中切下的巨膜斑中的I_2。结果表明，NCX 3具有Ca^&lt;2+&gt;依赖的激活和失活作用，ATP对I_2的增加作用<NaCa>在NCX 3中弱于NCX 1。

项目成果

Sarai N, Matsuoka S, Kuratomi S, Ono K, Noma A.: "Role of Individual Ionic Current Systems in the SA Node Hypothesized by a Model Study."Japanese Journal of Physiology. 53. 125-134

Sarai N、Matsuoka S、Kuratomi S、Ono K、Noma A.：“模型研究假设的个体离子电流系统在 SA 节点中的作用。”日本生理学杂志。

松岡 達: "Na/Ca交換輸送体の構造と機能"心臓HEART. 34. 296-305 (2002)

T. Matsuoka：“Na/Ca 交换转运蛋白的结构和功能”Heart HEART 34. 296-305 (2002)

Sarai N, Kihara Y, Izumi T, Mitsuiye T, Matsuoka S., Noma A.: "Nonuniformity of sarcomere shortenings in the isolated rat ventricular myocyte."Japanese Journal of Physiology. 52. 371-381 (2002)

Sarai N、Kihara Y、Izumi T、Mitsuiye T、Matsuoka S.、Noma A.：“离体大鼠心室肌细胞中肌节缩短的不均匀性。”日本生理学杂志。

Matsuoka S.: "Stoichiometry of the Na^+-Ca^<2+> exchanger : models & implications."Annals of the New York Academy of Sciences.. 976. 121-132 (2002)

Matsuoka S.：“Na^ -Ca^<2> 交换器的化学计量：模型

Powell T., Matsuoka S., Sarai N., Noma A.: "Intracellular Ca^<2+> dynamics and sarcomere length in ventricular myocytes."Cell Calcium.. 35. 535-542 (2004)

Powell T.、Matsuoka S.、Sarai N.、Noma A.：“心室肌细胞中的细胞内 Ca^<2> 动力学和肌节长度。”细胞钙.. 35. 535-542 (2004)