V‐ATPase Domain Assembly is Increased in Ncoa7 KO Mice
Authors: Eaton Amity F. , Merkulova Maria , Brown Dennis 
Published: May 2022

The V‐ATPase is a multisubunit, transmembrane proton pump that generates electrochemical gradients across membranes driving essential cell biological processes. Critically, for proton pumping activity, the two distinct domains of the V‐ATPase (V1 and VO) must assemble into a functional holoenzyme. The kidney‐specific V‐ATPase is expressed in intercalated cells (ICs), which regulate acid‐base homeostasis by adjusting the number of apical V‐ATPase holoenzymes via regulated trafficking. Thus, V‐ATPase dysfunction in ICs leads to distal renal tubular acidosis (dRTA), which is characterized by an accumulation of acid in the blood. Despite this, little is known about the molecular regulation of the kidney V‐ATPase.We recently identified a new class of V‐ATPase interacting proteins defined by their TLDc domain which protect against oxidative stress (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2). We showed that the purified TLDc domains of Ncoa7, Oxr1, and Tldc2 interact with the IC‐specific B1 subunit of the V‐ATPase. Furthermore, endogenous Ncoa7, Oxr1, and Tbc1d24 interacted with the B1 subunit. Endogenous Tldc1 or Tldc2 were expressed at low levels in the kidney, but proteins overexpressed in cell cultures interacted with the B1 V‐ATPase from kidney lysates. Of importance, Ncoa7 knockout (KO) mice had decreased V‐ATPase expression in ICs and developed dRTA, which led us to hypothesize that the TLDc proteins regulate the kidney V‐ATPase.Here, we asked whether Ncoa7 deletion affected apical trafficking of the V‐ATPase or assembly of the V1 and VO domains into a holoenzyme in renal ICs after stimulation of proton secretion. Wild type (WT) and Ncoa7 KO mice were injected with CPT‐cAMP, to stimulate proton secretion, or saline as a control. Localization of the B1 and a4 subunits was visualized in ICs by immunofluorescence using specific antibodies, and apical accumulation was quantified by line intensity scanning. Our results show that cAMP significantly increased apical accumulation of B1 and a4 in WT mice from 55% to 78% of the cellular total, as expected. However, ICs in the Ncoa7 KO mice were already highly activated at baseline, with only a slight trend towards increased apical accumulation of the V‐ATPase after cAMP treatment (79% to 82%). A Pearson’s correlation analysis of the colocalization of B1 and a4, as a measure of V‐ATPase assembly, revealed a significant increase in assembly in Ncoa7 KO mice, relative to control animals, after both saline and cAMP treatment. Furthermore, a Proximity Ligation Assay also showed a marked increase in assembly of the V‐ATPase in untreated Ncoa7 KO ICs compared to control WT mice.These data suggest that loss of Ncoa7 does not affect apical accumulation of the V‐ATPase in ICs. Ncoa7 KO mice ICs are already highly activated in untreated mice. This is probably a compensatory response to the prevailing acidosis observed in these mice, which may be due to decreased total V‐ATPase protein expression in their ICs. Our data shows, however, increased assembly of the remaining V‐ATPase in Ncoa7 KO mice. Thus, we conclude that Ncoa7 is part of a regulatory mechanism that inhibits assembly of the V‐ATPase into a holoenzyme in WT mice, while not affecting trafficking of V‐ATPase subunits to the apical surface. How this negative effect is balanced in vivo to determine the overall degree of V‐ATPase holoenzyme assembly remains to be determined.