pH-dependent ion- transport mechanism in the hfRPE

在视网膜中，视杆感光器产生大量的代谢副产物，如二氧化碳，这些副产物必须由RPE运输到血液中。为了有效地缓冲和将大量的二氧化碳从感光器输送到血液中，RPE开发了一种CO2/HCO3运输系统，以碳酸氢盐的形式将二氧化碳从感光器输送到血液中。当二氧化碳产生增加时(在黑暗中)，RPE通过增加RPE对HCO3-的吸收来驱散高CO2负荷，以防止视网膜下酸中毒。在黑暗中，1)视网膜下间隙K+浓度从2 mm增加到5 mm，2)视网膜下间隙二氧化碳分压升高。 该项目需要研究hfRPE中参与眼睛明暗转换的离子转运蛋白。我们提供了pH成像和电生理学数据来证实RPE基底膜上存在一个生电的Na+/HCO3-共转运体(NBC1)。接下来，我们展示了碳酸酐酶II(CA II)与基础NBC1、顶端NBC1和AE1的功能相互作用，符合碳酸氢盐的转运代谢。这些实验包括将RPE膜暴露在不同的扰动下(例如，减少基础浴重碳酸盐)，以在存在和不存在特定的碳酸氢酶II抑制剂(多唑胺)的情况下探测感兴趣的蛋白质。通过电生理实验，我们还发现，心尖二氧化碳从5%增加到13%会增加基础NBC1的活性。通过pH成像技术，我们发现与基底浴相比，根尖浴CO2的增加/减少引起了更强烈的pH变化，从而表明RPE的基底膜对CO2是不通透的。接下来，我们通过证明当基础Cl-从126.1 mM减少到1 mM时，顶端DIDS降低了碱化速率，从而证明了基础AE1的活性依赖于顶端NBC1的活性。此外，当根尖浴液中灌入与13%二氧化碳平衡的Ringers时，AE1活性会降低。这些数据证实和支持了离子转运模型的有效性，该模型说明了在光暗条件下，顶端NBC1、基础NBC1、AE1和基础Cl-通道在K+、Na+、Cl-和HCO3-跨RPE运输中所起的作用。上述离子是RPE中液体运输的主要介质，因此对这些蛋白质的研究将使我们能够操纵生理条件来增加RPE中的液体运输，从而努力缓解视网膜脱离的症状。

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.