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本文旨在研究Mg2+对表达在非洲爪蟾卵母细胞上P2X4受体介导的ATP-激活电流(ATP-activated current,IATP)的调制及机理。大鼠P2X4受体野生型及突变体型cDNA在体外转录成cRNA,通过显微注射技术将该cRNA注入非洲爪蟾卵母细胞中进行表达。应用全细胞双电极电压钳技术研究Mg2+对IATP的影响。结果显示,细胞外液加MgCl2对IATP的影响如下:(1)Mg2+在0.5~10mmol/L范围内,浓度依赖性地抑制IATP,半效抑制浓度(IC50)为(1.24±0.07)mmol/L,冲洗10min后,IATP可恢复;(2)1mmol/L的Mg2+使IATP量-效曲线右下移,最大效应电流(Emax)降低了(42.0±2.1)%,但半数有效浓度(EC50)不变;(3)预加Mg2+80s后再加ATP,Mg2+对IATP抑制效应可达最大;(4)Mg2+对IATP的抑制不受膜电位的影响;(5)以100μmol/L ATP激活的电流为对照,将P2X4受体280位的天冬氨酸突变成不带电荷的谷氨酰胺后,相同浓度的ATP诱发的IATP幅值仅为对照值的(4.12±0.15)%;将280位的天冬氨酸突变成带负电荷的谷氨酸,IATP幅值和对照值相比没有显著性差异;(6)当P2X4受体280位的天冬氨酸缺失后,100μmol/L ATP诱发的IATP的幅值几乎为对照值的两倍,且0.5~10mmol/L的Mg2+对相应的IATP无明显影响。以上结果提示:Mg2+抑制P2X4受体介导的IATP是非竞争性的、可逆的、具有浓度依赖性、时间依赖性、无电压依赖性;Mg2+的这种抑制效应可能是通过作用于P2X4受体280位的天冬氨酸而实现的。
This article aims to investigate the modulation of P2X4 receptor-mediated ATP-activated current (IATP) by Mg2 + on Xenopus laevis oocytes. The rat P2X4 receptor wild type and mutant type cDNA were transcribed into cRNA in vitro and injected into Xenopus laevis oocytes by microinjection to express the cRNA. Whole cell bipolar voltage clamp technique was used to study the effect of Mg2 + on IATP. The results showed that the effects of extracellular fluid and MgCl2 on IATP were as follows: (1) Mg2 + inhibited IATP in a concentration-dependent manner from 0.5 mmol / L to 10 mmol / L, with IC50 of (1.24 ± 0.07) mmol / L , And IATP could be recovered after 10 min of washing. (2) The maximum effective current (Emax) decreased by (42.0 ± 2.1)% when Immol / L Mg2 + (3) Pretreatment with Mg2 + 80s followed by ATP increased the inhibitory effect of Mg2 + to IATP to the maximum; (4) inhibition of IATP by Mg2 + was not affected by membrane potential; (5) currents activated by 100μmol / L ATP As a control, the same concentration of ATP-induced IATP amplitude was only (4.12 ± 0.15)% of the control value after mutating aspartate at position 280 of P2X4 to uncharged glutamine; Of aspartate mutated to negatively charged glutamic acid, IATP amplitude compared with the control value was no significant difference; (6) when P2X4 receptor 280 aspartate deletion, 100μmol / L ATP The amplitude of induced IATP was almost double that of the control value, and 0.5 ~ 10mmol / L Mg2 + had no significant effect on the corresponding IATP. These results suggest that: Mg2 + inhibits P2X4 receptor mediated IATP is non-competitive, reversible, in a concentration-dependent, time-dependent, voltage-dependent; Mg2 + this inhibitory effect may be through the role of P2X4 receptor 280 Bit of aspartic acid and achieve.