Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake
Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake
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DOI：
10.1523/jneurosci.18-23-09620.1998
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发表时间：
1998-12-01
影响因子：
5.3
通讯作者：
Attwell, D
中科院分区：
文献类型：
--
作者：
Levy, LM;Warr, O;Attwell, D
谷氨酸在神经元和神经胶质质膜上的转运是由钠、钾和pH的跨膜电化学梯度驱动的，但对与谷氨酸共转运的Na（+）的数量存在争议。谷氨酸转运体的化学计量学很重要，因为它决定了正常和病理条件下细胞外谷氨酸浓度[glu](o)的下限。我们使用全细胞夹紧技术研究了GLT-1的化学计量学，GLT-1是大脑中最丰富的谷氨酸转运蛋白，在Tet-On系统的控制下，在中国仓鼠卵巢（CHO）细胞系中表达，选择用于低内源性谷氨酸转运。多西环素诱导GLT-1表达后，谷氨酸通过GLT-1的电压依赖性和药理学作用，诱发Na（+）依赖性内向电流，使细胞质酸化。在含有钠和谷氨酸的电极夹住的细胞周围升高[K(+)](o)引起向外的反向摄取电流。特异性GLT-1阻滞剂二氢盐（DHK）可降低这些反应。DHK诱发了以NO(3)（-）为主要细胞内阴离子的向外电流，而不是以Cl（-）为主要阴离子的向外电流，这表明即使没有外部谷氨酸，转运体的阴离子传导也很活跃，但在没有NO(3)（-）等高渗透性阴离子的情况下，产生的电流很少。对不同离子条件下转运体电流的逆转电位的测量表明，一个谷氨酸阴离子的输运与三个Na（+）和一个H（+）的共输运以及一个K（+）的反输运耦合。这表明在缺血时，当[K(+)](o)升高至60 mM时，谷氨酸转运体的逆转将使[glu](o)升高至50 μ M。
Glutamate transport across the plasma membrane of neurons and glia is powered by the transmembrane electrochemical gradients for sodium, potassium, and pH, but there is controversy over the number of Na(+) cotransported with glutamate. The stoichiometry of glutamate transporters is important because it determines a lower limit to the extracellular glutamate concentration, [glu](o), in both normal and pathological conditions. We used whole-cell clamping to study the stoichiometry of the glial transporter GLT-1, the most abundant glutamate transporter in the brain, expressed under control of the Tet-On system in a Chinese hamster ovary (CHO) cell line selected for low endogenous glutamate transport. After the induction of GLT-1 expression with doxycycline, glutamate evoked a Na(+)- dependent inward current with the voltage dependence and pharmacology of GLT-1 and acidified the cell cytoplasm. Raising [K(+)](o) around cells clamped with electrodes containing sodium and glutamate evoked an outward reversed uptake current. These responses were reduced by the specific GLT-1 blocker dihydrokainate (DHK). DHK evoked an outward current with NO(3)(-), but not with Cl(-), as the main intracellular anion, suggesting that the anion conductance of the transporter is active even without external glutamate but generates little current in the absence of highly permeable anions like NO(3)(-). Measuring the reversal potential of the transporter current in various ionic conditions suggested that the transport of one glutamate anion is coupled to the cotransport of three Na(+) and one H(+) and to the countertransport of one K(+). This suggests that in ischemia, when [K(+)](o) rises to 60 mM, the reversal of glutamate transporters will raise [glu](o) to >50 mu M.