pH-dependent ion- transport mechanism in the hfRPE

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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.