Ncbe is the main basolateral Na ⁺ loading mechanism of the choroid plexus epithelium

Cerebrospinal fluid (CSF) provides a specialized extracellular environment for the central nervous system, which is predominantly produced by the choroid plexus, a highly vascularized epithelial structure whose ion transport processes are fundamental to CSF secretion, composition, and homeostasis. The mechanisms of Na ⁺ entry into choroid plexus epithelial cells (CPECs) from the interstitial side remain disputed. The slc4a10 gene product encoding the Na ⁺ dependent Cl ^- /HCO ₃ ^- exchanger, Ncbe, was suggested as a key transport mechanism based on its impact on the cell’s Na ⁺ -dependent regulation of intracellular pH and its basolateral membrane expression. The current study was undertaken to directly assess the contribution of Ncbe to the Na ⁺ uptake into CPECs. Intracellular Na ⁺ was recorded by fluorometry using the Na ⁺ probe Sodium Binding Fluorescent Indicator in clusters of CPECs with access to both the luminal and basolateral membranes. Removal of extracellular Na ⁺ reduced the apparent ex vivo intracellular [Na ⁺ ] to ∼5 mM from a baseline of ∼43 mM in the absence of CO ₂ /HCO ₃ ^- and ∼54 mM in the presence of CO ₂ /HCO ₃ ^- . Flame photometry estimated the intracellular [Na ⁺ ] ex vivo to ∼28 mM. The CO ₂ /HCO ₃ ^- -dependent rate of [Na ⁺ ] recovery amounted to ∼53% of the total recovery rate upon re-addition of Na ⁺ . Experiments with access to only the luminal membrane show a [Na ⁺ ] recovery of a similar rate as observed in the absence of CO ₂ /HCO ₃ ^- in the clusters. The CO ₂ /HCO ₃ ^- -independent [Na ⁺ ] recovery was inhibited to ∼50% by the NKCC1 inhibitor bumetanide and to ∼30% by the TRPv4 inhibitor RN1734. NHE contributed to a minor extent to the CO ₂ /HCO ₃ ^- -independent transport. The HCO ₃ ^- transport inhibitor DIDS, however, inhibited the total [Na ⁺ ] recovery rate to ∼50%, indicating a role for Ncbe rather than NBCn1 in the cellular [Na ⁺ ] recovery. Indeed, docking of DIDS into Ncbe and NBCn1 indicated that both proteins can accommodate the binding of DIDS. However, the orientation of the DIDS poses in Ncbe suggests a binding mode more similar to that found in the Anion Exchangers (SLC4A1-3), which seems to accommodate the covalent-type docking more than NBCn1. The Ncbe inhibition by DIDS was supported by the rate of [Na ⁺ ] recovery that was significantly higher in CPECs from Ncbe-wt than Ncbe-ko mice in the presence of CO ₂ /HCO ₃ ^- . As both NKCC1 and TRPv4 are localized to the luminal membrane, the findings collectively suggest that Ncbe is the most prominent mechanism for Na ⁺ entry into CPECs expressed at the basolateral side. We suggest Ncbe as the rate-limiting mechanism in the vectorial Na ⁺ transport driving CSF secretion.