Ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress

Abstract

SLC4A11 is a NH 3 sensitive membrane transporter with H + channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH 3 -sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, Slc4a11 −/− (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH 3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. Slc4a11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.

MitoROS due to loss ofSlc4a11in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

“…Studies from our lab and others have identified mitochondrial ROS to be the major cause of cell death in KO MCECs 5,6 . In addition, mitochondrial ROS can also cause lysosomal dysfunction and ER stress in some systems [25][26][27][28] .…”

“…The energy needed for this process is derived from a high density of mitochondria. Corneal endothelial diseases, such as Fuchs Corneal Endothelial Dystrophy (FCED) and Congenital Hereditary Endothelial Dystrophy (CHED), are associated with dysfunctional mitochondria, increased reactive oxygen species (ROS), alterations in cell morphology, function, and eventually cell death [3][4][5][6] . Corneal edema is the main clinical manifestation of FCED and CHED 7,8 , and corneal transplantation is the only available treatment option.…”

“…Corneal endothelial cells highly express Slc4a11, an electrogenic NH3 + /H + transporter, located in the basolateral membrane 10 as well as in the inner mitochondrial membrane 5 . Homozygous recessive mutations in this gene are linked to early-onset CHED 11 , whereas heterozygous mutations are implicated in later life FCED 12 .…”

“…Homozygous recessive mutations in this gene are linked to early-onset CHED 11 , whereas heterozygous mutations are implicated in later life FCED 12 . A mouse model in which Slc4a11 is knocked out emulates the disease progression of CHED along with increased oxidative stress in the corneal endothelium 13,14 , a well-known characteristic of corneal endothelial dystrophies 5,6,8 . Recent work from our lab demonstrated that Slc4a11 is an ammonia sensitive mitochondrial uncoupler.…”

“…Absence of Slc4a11 causes glutamine-induced mitochondrial hyperpolarization, excess ROS production, deficient ATP production, and eventual apoptosis 5 . Mitochondrial ROS can lead to increased autophagy which in certain cell types help in survival 15 .…”