Peng Fang, Zhang Yu-Qin, Zeng Yan, Zhou Yan-Ling
Department of Physiology, Medical College, Wuhan University of Science and Technology, Wuhan 430065, China.
Sheng Li Xue Bao. 2012 Feb 25;64(1):75-81.
To investigate the modulation of Mg(2+) on rat P2X4 receptors and its underlying mechanism, we transcribed cDNA coding for wild-type and mutant P2X4 receptors to cRNA in vitro, injected the cRNA to oocytes of Xenopus laevis using the microinjection technique and revealed the effect of Mg(2+) on ATP-activated currents (I(ATP)) mediated by P2X4 receptors using the two-electrode whole-cell voltage clamp technique. The effects of extracellular Mg(2+) on I(ATP) were as follows: (1) In oocytes expressing P2X4 receptors, Mg(2+) with concentration ranging from 0.5-10 mmol/L inhibited the amplitude of I(ATP) in a concentration-dependent and reversible manner, with a 50% inhibitory concentration value (IC(50)) of (1.24 ± 0.07) mmol/L for current activated by 100 μmol/L ATP. (2) Mg(2+) (1 mmol/L) shifted the dose-response curve for I(ATP) right-downward without changing the EC(50), but reduced the maximal current (E(max)) by (42.0 ± 2.1)%. (3) After being preincubated with Mg(2+) for 80 s, the inhibitory effect of the Mg(2+) on I(ATP) reached the maximum. (4) The inhibition of Mg(2+) on I(ATP) was independent of membrane potential from -120 mV to +60 mV. (5) Compared with the current activated by 100 μmol/L ATP in the wild-type P2X4 receptors, mutant P2X4 D280Q responded to the application of 100 μmol/L ATP with a smaller current. The peak current was only (4.12 ± 0.15)% of that seen in wild-type receptors. Mutant P2X4 D280E responded to ATP stimulation with a current similar to that observed in cells expressing wild-type receptors. (6) When Asp280 was removed from P2X4, the current amplitude of I(ATP) was increased almost one-fold, and Mg(2+) with concentration ranging from 0.5-10 mmol/L did not affect the I(ATP) significantly. The results suggest that Mg(2+) inhibits I(ATP) mediated by P2X4 receptors non-competitively, reversibly, concentration-dependently, time-dependently and voltage-independently. The inhibitory effect of Mg(2+) might be realized by acting on the site Asp280 of the P2X4 receptors.
为研究Mg(2+)对大鼠P2X4受体的调控作用及其潜在机制，我们在体外将编码野生型和突变型P2X4受体的cDNA转录为cRNA，采用显微注射技术将cRNA注射到非洲爪蟾卵母细胞中，并运用双电极全细胞膜片钳技术揭示Mg(2+)对P2X4受体介导的ATP激活电流（I(ATP)）的影响。细胞外Mg(2+)对I(ATP)的影响如下：（1）在表达P2X4受体的卵母细胞中，浓度范围为0.5 - 10 mmol/L的Mg(2+)以浓度依赖性和可逆的方式抑制I(ATP)的幅度，对于由100 μmol/L ATP激活的电流，其半数抑制浓度值（IC(50)）为(1.24 ± 0.07) mmol/L。（2）Mg(2+)（1 mmol/L）使I(ATP)的剂量 - 反应曲线向右下方移动，而不改变EC(50)，但使最大电流（E(max)）降低了(42.0 ± 2.1)%。（3）与Mg(2+)预孵育80 s后，Mg(2+)对I(ATP)的抑制作用达到最大。（4）Mg(2+)对I(ATP)的抑制作用在膜电位从 - 120 mV至 + 60 mV范围内与膜电位无关。（5）与野生型P2X4受体中由100 μmol/L ATP激活的电流相比，突变型P2X4 D280Q对100 μmol/L ATP的反应电流较小。峰值电流仅为野生型受体中所见电流的(4.12 ± 0.15)%。突变型P2X4 D280E对ATP刺激的反应电流与在表达野生型受体的细胞中观察到的电流相似。（6）当从P2X4中去除Asp280时，I(ATP)的电流幅度几乎增加了一倍，浓度范围为0.5 - 10 mmol/L的Mg(2+)对I(ATP)无明显影响。结果表明，Mg(2+)以非竞争性、可逆、浓度依赖性、时间依赖性和电压依赖性的方式抑制P2X4受体介导的I(ATP)。Mg(2+)的抑制作用可能是通过作用于P2X4受体的Asp280位点实现的。
