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

为探讨生理条件下Na~+-Ca~(2+)-Ca~(2+)交换的调节机制，我们记录了豚鼠心室肌细胞内向外Na~+-Ca~(2+)交换电流(I_&lt；NACA&gt；)。在膜的细胞质表面加入三磷酸腺苷或PI(4，5)P_2，可使I_lt；NACA&Gt；用PI专一性磷脂酶C处理膜，抑制PI产生PI(4，5)P_2，减弱了ATP的作用。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，应用蛋白酪氨酸磷酸酶的抑制剂，钒酸或BPV(Phen)，在有ATP存在的情况下显著增加I_&lt；NACA&gt；。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Na+-NACA的刺激作用。为了验证PKA信号刺激Na~+-Ca~(2+)活性的报道，我们测量了豚鼠心室肌细胞的全细胞I_&lt；NACA&gt；。β受体激动剂异丙肾上腺素可激活依赖cAMP的Cl~-电流，但不能兴奋I_naca&gt；结论：PKA信号不能刺激豚鼠心室肌细胞Na~(++)-Ca~(2+)和Gt~(2+)交换。以前的报道可能低估了cAMP依赖的氯离子电流的激活。为了研究主要在脑和骨骼肌中表达的NCX3的调节机制，我们测量了表达NCX3的卵母细胞切除的巨大膜片的I_&lt；NACA&gt；。结果表明，NCX3具有钙依赖的激活和失活作用，而ATP对NCX3的激活和失活作用不如NCX1。

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.