FRACTIONAL CALCIUM CURRENTS THROUGH RECOMBINANT GLUR CHANNELS OF THE NMDA, AMPA AND KAINATE RECEPTOR SUBTYPES
FRACTIONAL CALCIUM CURRENTS THROUGH RECOMBINANT GLUR CHANNELS OF THE NMDA, AMPA AND KAINATE RECEPTOR SUBTYPES
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DOI：
10.1113/jphysiol.1995.sp020738
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发表时间：
1995-06-01
影响因子：
5.5
通讯作者：
SAKMANN, B
中科院分区：
文献类型：
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作者：
BURNASHEV, N;ZHOU, Z;SAKMANN, B
1. 使用钙指示剂染料 fura-e 在表达重组谷氨酸受体 (GluR) 通道的 HEK 293 细胞中同时进行荧光和全细胞电流测量，并测定分数 Ca2+ 电流（Ca2+ 携带的全细胞电流的比例；P-f）。2.表达 N-甲基-D-天冬氨酸受体 (NMDAR) 通道的细胞在正常细胞外溶液中显示谷氨酸激活的 Ca2+ 流入，电压范围为 -60 至 40 mV。由于 Mg2+ 阻断，当膜电位比 -30 mV 更负时，Ca2+ 流入量以电压依赖性方式减少。与表达NR1-NR2C亚基的细胞相比，表达NR1-NR2A的细胞在负电位下阻断的电压依赖性更强。3．通过 NMDAR 的分数 Ca2+ 电流与细胞外 Mg2+ 无关，在 -60 mV 膜电位下的正常细胞外溶液 (1.8 mM Ca2+) 中变化范围为 8.2%（NR1-NR2C 亚基）和 11%（NR1-NR2A 亚基）。 P-f值随着[Ca2+](o)的增加而增加，在0.5-10 mM[Ca2+](o)范围内呈饱和状态。4．在表达 α-氨基-3-羟基-5-甲基-4-异恶唑丙酸受体 (AMPAR) 亚基（在假定的跨膜片段 TM2 的 Q/R 位点（Q 形式）处未编辑）的细胞中，或在共表达未编辑和编辑的亚基（R 形式）的细胞中，谷氨酸诱发的 Ca2+ 流入以几乎线性的方式从 20 mV 增加到 -80 mV。5。通过 AMPAR 通道的部分 Ca2+ 电流取决于亚基组成。 Q 型同聚通道在 -60 mV 和 1.8 mM [Ca2+](o) 下的 P-f 值在 3.2% 和 3.9% 之间。它们稍微依赖于电压，并且随着 [Ca2+](o) 在 1.8-10 mM 范围内增加。共表达Q型和R型亚基的细胞中的P-f值几乎小一个数量级(0.54%)。6．用于细胞转染的 Q 型和 R 型 GluR-B 亚基特异性 cDNA 的相对浓度决定了功能不同的异聚 AMPAR 的表达。 P-f 随着 R 型编码 cDNA 的相对浓度从 3.4% 增加到 1.4% 而降低，这表明编辑 GluR-B 亚基的 Q/R 位点会减少通过异聚 AMPAR 的 Ca2+ 流入。7。表达红藻氨酸受体 (KAR) 家族 GluR-B 亚基的细胞通过 P-f 值进行表征，该值取决于 TM1 和 TM2 片段的编辑。 Q 型通道的 P-f 值最大 (1.55-2.0%)，R 型通道的 P-f 值最低 (< 0.2%)，表明 Q/R 位点编辑也减少了通过 KAR 通道的 Ca2+ 流入。共表达两种亚基形式的细胞显示出中间值(0.58%).8。使用正常 [Ca2+](o) 测量的分数 Ca2+ 电流与根据在高 [Ca2+](o) 的双离子 (Ca2+-Cs+) 条件下测量的反转电势恒定场假设预测的 P-f 值不同。9。结果表明，正常细胞外溶液中重组 GluR 通道介导的 P(f) 大小在小于 0.2% 至 11% 之间变化超过 50 倍，具体取决于表达的 GluR 通道亚基的组合。不同亚基组合的组装、亚基特异性 mRNA 的相对丰度以及 mRNA 的编辑是在生理条件下通过不同版本的 GluR 通道控制大范围 Ca2+ 流入的主要机制。
1. Simultaneous fluorescence and whole-cell current measurements using the calcium indicator dye fura-e were made in HEK 293 cells expressing recombinant glutamate receptor (GluR) channels, and fractional Ca2+ currents (the proportion of whole-cell current carried by Ca2+; P-f) were determined.2. Cells expressing N-methyl-D-aspartate receptor (NMDAR) channels showed glutamate-activated Ca2+ inflow in the voltage range -60 to 40 mV in normal extracellular solution. Ca2+ inflow decreased in a voltage-dependent manner at membrane potentials more negative than -30 mV due to Mg2+ block. Voltage dependence of block at negative potentials was stronger in cells expressing the NR1-NR2A as compared with cells expressing NR1-NR2C subunits.3. Fractional Ca2+ currents through NMDARs were independent of extracellular Mg2+ and varied between 8.2% (NR1-NR2C subunits) and 11% (NR1-NR2A subunits) in normal extracellular solution (1.8 mM Ca2+) at -60 mV membrane potential. P-f values increased with increasing [Ca2+](o) in the range of 0.5-10 mM [Ca2+](o) in a saturating fashion.4. In cells expressing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) subunits which were unedited at the Q/R site of the putative transmembrane segment TM2 (Q-form), or in cells coexpressing unedited and edited subunits (R-form), the glutamate-evoked Ca2+ inflow increased from 20 to -80 mV in an almost linear way.5. Fractional Ca2+ currents through AMPAR channels depended on subunit composition. P-f values of Q-form homomeric channels at -60 mV and 1.8 mM [Ca2+](o) were between 3.2 and 3.9%. They were slightly voltage dependent and increased with [Ca2+](o) in the range 1.8-10 mM. P-f values in cells co-expressing Q- and R-form subunits were almost one order of magnitude smaller (0.54%).6. Relative concentrations of Q-form and R-form GluR-B subunit-specific cDNAs used for cell transfection determined the expression of functionally different heteromeric AMPARs. P-f decreased with increasing relative concentration of R-form encoding cDNAs from 3.4 to 1.4%, demonstrating that editing of the Q/R site of GluR-B subunits decreases Ca2+ inflow through heteromeric AMPARs.7. Cells expressing the GluR-B subunit of the kainate receptor (KAR) family were characterized by P-f values which depended on the editing in the TM1 and TM2 segments. P-f values were largest for the Q-form (1.55-2.0%) and lowest for R-form channels (< 0.2%), suggesting that Q/R site editing also decreases Ca2+ inflow through KAR channels. Cells co-expressing both subunit forms showed an intermediate value (0.58%).8. Fractional Ca2+ currents measured with normal [Ca2+](o) were different from P-f values predicted with constant field assumptions from reversal potentials measured in bi-ionic (Ca2+-Cs+) conditions with high [Ca2+](o).9. The results indicate that the size of P(f)s mediated by recombinant GluR channels in normal extracellular solution varies over a 50-fold range between less than 0.2 and 11%, depending on the combination of GluR channel subunits expressed. Assembly of different subunit combinations, relative abundance of subunit specific mRNAs and editing of mRNA are major mechanisms which control this wide range of Ca2+ inflow through different versions of GluR channels under physiological conditions.