The NH₃/H⁺ Transporter Slc4a11 Facilitates Glutamine-dependent Mitochondrial Function and ROS Prevention by Mitochondrial Uncoupling

Abstract

Purpose : SLC4A11 is an electrogenic NH₃-sensitive H⁺ transporter. Slc4a11^-/^- (KO) mice show significant corneal endothelial (CE) cell oxidative damage and alteration of glutamine metabolism. Here, we tested the hypothesis that Slc4a11 protects corneal endothelial cells from glutamine (Gln) induced oxidative stress by mitochondrial uncoupling.

Methods : In WT and KO CE tissue and cell lines (MCEC) oxidative stress was evaluated by MitoSOX, protein nitrosylation and OHdGuanine, mitochondrial morphology by MitoTracker Green and autophagy by LC3B staining. Cell death was analyzed by AnV-FITC + PI, mitochondrial membrane potential (MMP) by TMRE, ATP by luciferin-luciferase assay and NH₃ production and NAD⁺/NADH ratio by colorimetric assays. Oxygen consumption rate (OCR) was measured with Seahorse XFp.

Results : KO CE from 12 and 40 week old mice show greater mitochondrial O₂^-, oxidized DNA and protein, autophagy, and altered mitochondrial morphology vs WT. In WT MCEC, Gln (0.5 mM) increases [ATP], mitochondrial O₂^-, and apoptosis vs Gluc only. In contrast, in KO Gln decreases [ATP], increases O₂^-, and apoptosis (vs WT) that can be rescued by MitoQ. Slc4a11 facilitates glutaminolysis as NH₃ production is 50% higher in WT. Apoptosis due to NH₃ alone was 100% greater in KO MCEC. Gln derived NH₃ toxicity is partially reduced by GLS1 inhibitors BPTES or CB839 and totally by α-Ketoglutarate. Even though Gln induces a higher net MMP depolarization in KO (vs WT) over time, the remaining TMRE⁺ KO cells were hyperpolarized relative to WT. Cyclosporin A (inhibitor of mPTP opening) prevents cell-death, suggesting that higher O₂^- production leads to mitochondrial damage and mPTP opening in KO. SLC4A11 colocalized with mitochondria. We hypothesized that Slc4a11 protects cells from Gln-induced mitochondrial oxidative stress by mildly depolarizing the MMP through NH₃-facilitated H⁺ transport. In agreement, BAM15 (mitochondrial uncoupler) inhibits Gln induced apoptosis in KO. Perfusion with NH₄Acetate induces fast MMP depolarization in WT but not KO. In presence of Gln, WT have higher OCR, mitochondrial proton leak and NAD⁺/NADH ratio than KO.

Conclusions : Slc4a11 protects cells from Gln-induced oxidative stress by mitochondrial uncoupling through NH₃-facilitated H⁺ transport.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.