Peterson W M, Miller S S
Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
J Gen Physiol. 1995 Dec;106(6):1089-122. doi: 10.1085/jgp.106.6.1089.
Intracellular microelectrodes, fluorescence imaging, and radiotracer flux techniques were used to investigate the physiological response of the retinal pigment epithelium (RPE) to the major retinal inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). GABA is released tonically in the dark by amphibian horizontal cells, but is not taken up by the nearby Müller cells. Addition of GABA to the apical bath produced voltage responses in the bullfrog RPE that were not blocked nor mimicked by any of the major GABA-receptor antagonists or agonists. Nipecotic acid, a substrate for GABA transport, inhibited the voltage effects of GABA. GABA and nipecotic acid also inhibited the voltage effects of taurine, suggesting that the previously characterized beta-alanine sensitive taurine carrier also takes up GABA. The voltage responses of GABA, taurine, nipecotic acid, and beta-alanine all showed first-order saturable kinetics with the following Km's: GABA (Km = 160 microM), beta-alanine (Km = 250 microM), nipecotic acid (Km = 420 microM), and taurine (Km = 850 microM). This low affinity GABA transporter is dependent on external Na, partially dependent on external Cl, and is stimulated in low [K]o, which approximates subretinal space [K]o during light onset. Apical GABA also produced a significant conductance increase at the basolateral membrane. These GABA-induced conductance changes were blocked by basal Ba2+, suggesting that GABA decreased basolateral membrane K conductance. In addition, the apical membrane Na/K ATPase was stimulated in the presence of GABA. A model for the interaction between the GABA transporter, the Na/K ATPase, and the basolateral membrane K conductance accounts for the electrical effects of GABA. Net apical-to-basal flux of [3H]-GABA was also observed in radioactive flux experiments. The present study shows that a high capacity GABA uptake mechanism with unique pharmacological properties is located at the RPE apical membrane and could play an important role in the removal of GABA from the subretinal space (SRS). This transporter could also coordinate the activities of GABA and taurine in the SRS after transitions between light and dark.
采用细胞内微电极、荧光成像和放射性示踪剂通量技术，研究视网膜色素上皮（RPE）对主要视网膜抑制性神经递质γ-氨基丁酸（GABA）的生理反应。在黑暗中，两栖动物水平细胞持续释放GABA，但附近的米勒细胞不摄取GABA。将GABA添加到顶端浴中，会在牛蛙RPE中产生电压反应，而任何主要的GABA受体拮抗剂或激动剂均不能阻断或模拟该反应。GABA转运的底物尼克酸抑制了GABA的电压效应。GABA和尼克酸还抑制了牛磺酸的电压效应，这表明先前鉴定的β-丙氨酸敏感牛磺酸载体也摄取GABA。GABA、牛磺酸、尼克酸和β-丙氨酸的电压反应均表现出一级饱和动力学，其Km值如下：GABA（Km = 160μM）、β-丙氨酸（Km = 250μM）、尼克酸（Km = 420μM）和牛磺酸（Km = 850μM）。这种低亲和力GABA转运体依赖于细胞外Na，部分依赖于细胞外Cl，并在低[K]o（接近光开始时的视网膜下间隙[K]o）中受到刺激。顶端GABA还会使基底外侧膜的电导显著增加。这些GABA诱导的电导变化被基底Ba2+阻断，表明GABA降低了基底外侧膜的K电导。此外，在GABA存在的情况下，顶端膜Na/K ATP酶受到刺激。一个关于GABA转运体、Na/K ATP酶和基底外侧膜K电导之间相互作用的模型解释了GABA的电效应。在放射性通量实验中也观察到了[3H]-GABA从顶端到基底的净通量。本研究表明，一种具有独特药理学特性的高容量GABA摄取机制位于RPE顶端膜，可能在从视网膜下间隙（SRS）清除GABA中起重要作用。这种转运体还可以在明暗转换后协调SRS中GABA和牛磺酸的活性。
