173 - SLC4A1111 provides NH3 sensitive mitochondrial uncoupling and ROS prevention that facilitates glutamine catabolism

Abstract

Mutations in SLC4A11 can cause CHED (Congenital Hereditary Endothelial Dystrophy) a disorder of the Corneal Endothelium that produces corneal edema and poor vision in early childhood. Slc4a11-/- (KO) mice recapitulate CHED showing corneal edema, evidence of oxidative stress, and eventual death of corneal endothelial cells. SLC4A11, which is highly expressed in the corneal endothelium, functions as an NH3-sensitive electrogenic H+ transporter. Loss of SLC4A11 depresses glutamine (Gln) catabolism, yielding lower [ATP] and NH3 production relative to wild-type. SLC4A11 has been localized to the basolateral membrane of polarized cells and undetermined cytoplasmic locations. In the current study, we found that SLC4A11 co-localizes to mitochondria, and is associated with inner mitochondrial membrane proteins. To test if SLC4A11 is acting as an NH3-activated mitochondrial uncoupler, we used Mouse Corneal Endothelial Cell (MCEC) lines derived from WT and Slc4a11 KO mice. We found that application of NH3 depolarized WT mitochondrial membrane potential (MMP), but not KO. Relative to KO, WT cells had a higher oxygen consumption rate (OCR), greater proton leak, greater NAD/NADH ratio, and higher [ATP] in the presence of 0.5 mM Gln. Moreover, mitochondrial ROS after 24 hours Gln-incubation was significantly higher in KO with concomitant MMP depolarization, low [ATP], and mPTP opening leading to increased apoptosis. Inhibiting Gln derived NH3 production by GLS1 inhibitors BPTES or CB839 or Dimethyl-α-Ketoglutarate ameliorated these effects. NH3 (25mM) alone increased ROS, decreased [ATP], and increased apoptosis in KO without affecting WT cells, indicating that KO cells are more sensitive to NH3. Slc4a11 KO cells were rescued from the effects of Gln or NH3 by MitoQ ROS scavenging or the mitochondrial uncoupler BAM15. In summary, these results indicate that mitochondrial Slc4a11 NH3 sensitive H+ influx is providing mitochondrial uncoupling that lowers Gln-dependent ROS production, facilitating Gln catabolism and protecting mitochondria.