小鼠γ-氨基丁酸转运体（mGAT3）的前稳态和稳态动力学及周转率

Presteady-state and steady-state kinetics and turnover rate of the mouse gamma-aminobutyric acid transporter (mGAT3).

作者信息

Sacher A, Nelson N, Ogi J T, Wright E M, Loo D D F, Eskandari S

机构信息

Department of Biochemistry, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel.

出版信息

J Membr Biol. 2002 Nov 1;190(1):57-73. doi: 10.1007/s00232-002-1024-6.

DOI:10.1007/s00232-002-1024-6

PMID:12422272

Abstract

We expressed mouse gamma-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl(-)-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl(-) was not. At a membrane potential of -50 mV, the half-maximal concentrations for Na+, Cl(-), and GABA were 14 mM, 5 mM, and 3 microM. The Hill coefficient for GABA activation and Cl(-) enhancement of the inward current was 1, and that for Na+ activation was > or =2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 +/- 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V(0.5)) was +25 mV, and the effective valence of the moveable charge (zdelta) was 1.6. In contrast to the ON transients, which relaxed with a time constant of < or =30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl(-) ([Cl(-)]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl(-)), no mGAT3-mediated transients were observed, and at zero [Cl(-)]o (100 mM Na+), the charge movements decreased to approximately 30% of the maximal charge (Q(max)). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl(-) enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Q(max) and I(max) obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec(-1) at -50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface.

摘要

我们在非洲爪蟾卵母细胞中表达了小鼠γ-氨基丁酸（GABA）转运体（mGAT3），并运用示踪通量和电生理方法检测了其稳态和预稳态动力学以及周转率。在表达mGAT3的卵母细胞中，GABA诱发了一种依赖Na⁺且由Cl⁻促进的内向电流。对Na⁺的依赖性是绝对的，而对Cl⁻的依赖性并非如此。在膜电位为 -50 mV时，Na⁺、Cl⁻和GABA的半数最大浓度分别为14 mM、5 mM和3 μM。GABA激活和Cl⁻增强内向电流的希尔系数为1，而Na⁺激活的希尔系数≥2。GABA诱发的内向电流与GABA流入细胞的量（2.2±0.1电荷/GABA）成正比，这表明在这些条件下，转运循环中离子与GABA紧密偶联。在膜电压发生阶跃变化且无GABA的情况下，mGAT3表现出预稳态电流瞬变（电荷移动）。电荷 - 电压（Q - V）关系符合单一的玻尔兹曼函数。半数最大电荷时的电压（V(0.5)）为 +25 mV，可移动电荷的有效价（zδ）为1.6。与以≤30毫秒的时间常数松弛的开启瞬变不同，关闭瞬变的时间常数为1.1秒。降低外部Na⁺（[Na⁺]o）和Cl⁻（[Cl⁻]o）浓度会使Q - V关系向负膜电位偏移。在零[Na⁺]o（106 mM Cl⁻）时，未观察到mGAT3介导的瞬变，而在零[Cl⁻]o（100 mM Na⁺）时，电荷移动减少至最大电荷（Q(max)）的约30%。GABA导致电荷移动消失。Na⁺激活、Cl⁻增强和GABA消除电荷移动的半数最大浓度分别为48 mM、19 mM和5 mM。在同一细胞中获得的Q(max)和I(max)得出mGAT3在 -50 mV时的周转率为1.7秒⁻¹。mGAT3的低周转率可能是由于空转运体从内膜表面返回外膜表面的速度较慢。