Aldosterone Promotes Nanoscale Colocalization Of Epithelial Sodium Channel Subunit Alpha And Sodium Calcium Exchanger In Neuro2a Cells

We previously reported that hypertensive patients have a higher concentration of sodium ([Na + ]) in their cerebrospinal fluid (CSF) compared with normotensive subjects but no difference in plasma [Na + ] was observed, suggesting the potential importance of CSF [Na + ] in hypertension. Epithelial sodium channels (ENaC) in the brain have been shown to be up regulated in salt-induced hypertension (SH). However, the molecular mechanisms of how brain ENaC contributes to SH development are not yet understood. We hypothesize that ENaC forms a nanoscale colocalization with the sodium-calcium exchanger (NCX) that mediates downstream signaling pathways leading to SH. To test our hypothesis, using self-labeling proteins we generated SNAP-ENaC-α and Halo-NCX1 constructs, enabling high-resolution imaging of surface expressed proteins. Expression of ENaC-α-SNAP and NCX1.5-Halo was confirmed by western blot showing an increase in expression (fold change) of ENaC-α (7.6 ± 1.7 vs. 1 ± 0.3, p<0.02) and NCX1.5 (10.1 ± 0.8 vs. 1 ± 0.4, p<0.0006) in transfected Neuro2A cells compared with controls. Transfected cells were treated with vehicle or aldosterone (100 nM) for 2 hours (n=15 cells from 3 repeat experiments/group) and imaged using ground-state depletion super-resolution microscopy. Data were analyzed using object-based analysis (OBA). Aldosterone treatment increased the density of individual ENaC-α clusters on the cell surface (12.4 ± 1.3 vs. 6.9 ± 1.08 clusters/μm 2 , p<0.002), supporting the functional role of aldosterone on increasing ENaC-α surface abundance. Importantly, the frequency (3.1 ± 0.34% vs 1.7 ± 0.22% colocalized /total clusters, p<0.001) and the density (0.5 ± 0.1 vs 0.2 ± 0.04 clusters/μm 2 of cell surface, p<0.009) of colocalized (< 40nm) ENaC-α and NCX 1.5 clusters were significantly increased in aldosterone treated cells. In sum, these data show aldosterone increases the number of ENaC-α channels on the cell surface and promotes its nanoscale colocalization with NCX, providing evidence for a functional role of ENaC-α and NCX1 coupling. Nanoscale colocalization of ENaC and NCX is potentially significant in SH, and future studies will focus on elucidating the functional importance and mechanisms.