Qiu Ling-Yu, Duan Guang-Ling, Yan Yu-Feng, Li Yuan-Yuan, Wang Huan, Xiao Ling, Liao Zhang-Ping, Chen He-Ping
The Key Laboratory of Basic Pharmacology, School of Pharmaceutical Science, Nanchang University, Nanchang, Jiangxi 330006, P.R. China.
Shanghai Public Health Clinical Center, Fudan University, Shanghai 201508, P.R. China.
Mol Med Rep. 2017 Sep;16(3):2953-2961. doi: 10.3892/mmr.2017.6882. Epub 2017 Jun 29.
Anion exchanger 3 (AE3) is known to serve crucial roles in maintaining intracellular chloride homeostasis by facilitating the reversible electroneutral exchange of Cl‑ for HCO3‑ across the plasma membrane. Our previous studies reported that sasanquasaponin (SQS) can inhibit hypoxia/reoxygenation (H/R)‑induced elevation of intracellular Cl‑ concentration ([Cl‑]i) and elicit cardioprotection by favoring Cl‑/HCO3‑ exchange of AE3. However, the molecular basis for SQS‑induced increase of Cl‑/HCO3‑ exchange of AE3 remains unclear. The present study demonstrated that SQS activates protein kinase Cε (PKCε) and stimulates the phosphorylation of AE3 in H9c2 cells. Notably, SQS‑induced AE3 phosphorylation was blocked by the PKCε selective inhibitor εV1‑2, and a S67A mutation of AE3, indicating that SQS could promote phosphorylation of Ser67 of AE3 via a PKCε‑dependent regulatory signaling pathway. Additionally, both inhibition of PKCε by εV1‑2 and S67A mutation of AE3 eradicated the SQS‑induced increase of AE3 activity, reversed the inhibitory effect of SQS on H/R‑induced elevation of [Cl‑]i, Ca2+ overload and generation of reactive oxygen species, and eliminated SQS‑induced cardioprotection. In conclusion, PKCε‑dependent phosphorylation of serine 67 on AE3 may be responsible for the increase of Cl‑/HCO3‑ exchange of AE3 and intracellular chloride efflux by SQS, and contributes to the cardioprotection of SQS against H/R in H9c2 cells.
已知阴离子交换蛋白3（AE3）通过促进氯离子（Cl⁻）与碳酸氢根离子（HCO₃⁻）在质膜上的可逆电中性交换，在维持细胞内氯离子稳态中发挥关键作用。我们之前的研究报道，山茶花皂苷（SQS）可抑制缺氧/复氧（H/R）诱导的细胞内氯离子浓度（[Cl⁻]i）升高，并通过促进AE3的Cl⁻/HCO₃⁻交换发挥心脏保护作用。然而，SQS诱导AE3的Cl⁻/HCO₃⁻交换增加的分子机制尚不清楚。本研究表明SQS可激活蛋白激酶Cε（PKCε）并刺激H9c2细胞中AE3的磷酸化。值得注意的是，PKCε选择性抑制剂εV1-2和AE3的S67A突变可阻断SQS诱导的AE3磷酸化，表明SQS可通过PKCε依赖性调节信号通路促进AE3丝氨酸67位点的磷酸化。此外，εV1-2抑制PKCε和AE3的S67A突变均消除了SQS诱导的AE3活性增加，逆转了SQS对H/R诱导的[Cl⁻]i升高、Ca²⁺超载和活性氧生成的抑制作用，并消除了SQS诱导的心脏保护作用。总之，PKCε依赖性的AE3丝氨酸67磷酸化可能是SQS增加AE3的Cl⁻/HCO₃⁻交换和细胞内氯离子外流的原因，并有助于SQS对H9c2细胞H/R损伤的心脏保护作用。
