pH-dependent ion- transport mechanism in the hfRPE

在视网膜中，视杆光感受器产生大量的代谢副产物，如CO2，其必须通过RPE运输到血流中。 为了有效地缓冲并将大量的CO2从光感受器输送到血流中，RPE开发了一种CO2/HCO 3运输系统，以碳酸氢盐的形式将CO2从光感受器输送到血流中。 当CO2产生增加时（在黑暗中），RPE通过增加整个RPE的HCO 3吸收来消散高CO2负荷，以防止视网膜下酸中毒。 在黑暗中，1）视网膜下腔的K+浓度从2 mM增加到5 mM，2）视网膜下腔的pCO 2增加。 该项目需要研究hfRPE中的离子转运蛋白，这些蛋白参与眼睛中的光暗转换。 我们目前的pH成像和电生理数据，以确认存在的产电Na+/HCO 3-共转运蛋白（NBC 1）在基底膜的RPE。 接下来，我们表明，碳酸酐酶II（CA II）的功能相互作用与基础NBC 1，顶端NBC 1，和，AE 1根据碳酸氢盐运输代谢。 这些实验涉及将RPE膜暴露于不同的扰动（例如，还原基础浴碳酸氢盐）以在存在和不存在特异性碳酸酐酶II抑制剂（多佐胺）的情况下探测目的蛋白。 通过电生理学实验，我们还表明，将顶部CO2从5%增加到13%会增加基础NBC 1活性。 与pH成像技术，我们发现，增加/减少顶端浴CO2引起一个更强的pH值变化相比，基础浴，从而表明，基底膜的RPE是不可渗透的CO2。 接下来，我们表明，基础AE 1活性是依赖于顶端NBC 1活性显示，顶端DIDS降低碱化率时，基础Cl-从126.1mM减少到1 mM。 此外，AE 1活性降低时，顶端浴灌注林格氏平衡与13%CO2。 这些数据证实并支持离子转运模型的有效性，该模型说明了顶部NBC 1，基底NBC 1，AE 1和基底Cl-通道在光照和黑暗条件下穿过RPE的K+，Na+，Cl-和HCO 3-转运中的作用。 上述离子是RPE中液体转运的主要介质，因此对这些蛋白质的研究将使我们能够操纵生理条件以增加穿过RPE的液体转运，从而减轻视网膜脱离的症状。

In the retina, rod photoreceptors generate large amounts of metabolic byproducts such as CO2 which must be transported to the bloodstream by the RPE. In order to efficiently buffer and deliver large amounts of CO2 from the photoreceptors to the bloodstream, the RPE developed a CO2/HCO3- transport system to deliver CO2 from the photoreceptors to the bloodstream in the form of bicarbonate. When there is an increase in CO2 generation (in the dark), the RPE dissipates the high CO2-load by increasing HCO3- absorption across the RPE in order to prevent subretinal acidosis. In the dark, 1) K+ concentration at the subretinal space increases from 2mM to 5mM, 2) there is an increase in pCO2 at the subretinal space. This project entails the study of the ion-transport proteins in the hfRPE that are involved in light-dark transition in the eye. We present pH-imaging and electrophysiology data to confirm the presence of an electrogenic Na+/HCO3- co-transporter (NBC1) at the basal membrane of the RPE. Next we show that carbonic anhydrase II (CA II) functionally interact with basal NBC1, apical NBC1, and, AE1 in accordance to the bicarbonate transport metabolon. These experiments involve exposing the RPE membrane to different perturbations (e.g., reducing basal bath bicarbonate) to probe the protein-of-interest with and without the presence of a specific carbonic anhydrase II inhibitor (dorzolamide). With electrophysiology experiments, we also show that increasing apical CO2 from 5% to 13% increases basal NBC1 activity. With pH-imaging techniques, we find that increasing/decreasing apical bath CO2 causes a much stronger pH-change compared to the basal bath, thus showing that the basal membrane of the RPE is impermeable to CO2. Next, we show that basal AE1 activity is dependent on apical NBC1 activity by showing that apical DIDS reduces the rate of alkalinization when basal Cl- is reduced from 126.1mM to 1mM. Moreover, AE1 activity is reduced when the apical bath is perfused with Ringers equilibrated with 13% CO2. These data confirm and support the validity of an ion-transport model that illustrates the role of apical NBC1, basal NBC1, AE1, and basal Cl- channels in K+, Na+, Cl- and HCO3- transport across the RPE under light and dark conditions. The abovementioned ions are the main mediators of fluid-transport in the RPE, and therefore the study of these proteins will allow us manipulate physiological conditions to increase fluid-transport across the RPE in an effort to alleviate the symptoms of retinal detachment.