Loss of the V-ATPase B1 Subunit Isoform Expressed in Non-Neuronal Cells of the Mouse Olfactory Epithelium Impairs Olfactory Function

The vacuolar proton-pumping ATPase (V-ATPase) is the main mediator of intracellular organelle acidification and also regulates transmembrane proton (H+) secretion, which is necessary for an array of physiological functions fulfilled by organs such as the kidney, male reproductive tract, lung, bone, and ear. In this study we characterize expression of the V-ATPase in the main olfactory epithelium of the mouse, as well as a functional role for the V-ATPase in odor detection. We report that the V-ATPase localizes to the apical membrane microvilli of olfactory sustentacular cells and to the basolateral membrane of microvillar cells. Plasma membrane V-ATPases containing the B1 subunit isoform are not detected in olfactory sensory neurons or in the olfactory bulb. This precise localization of expression affords the opportunity to ascertain the functional relevance of V-ATPase expression upon innate, odor-evoked behaviors in B1-deficient mice. This animal model exhibits diminished innate avoidance behavior (revealed as a decrease in freezing time and an increase in the number of sniffs in the presence of trimethyl-thiazoline) and diminished innate appetitive behavior (a decrease in time spent investigating the urine of the opposite sex). We conclude that V-ATPase-mediated H+ secretion in the olfactory epithelium is required for optimal olfactory function

V-ATPase expression in the mouse olfactory epithelium

The vacuolar proton-pumping ATPase (V-ATPase) is responsible for the acidification of intracellular organelles and for the pH regulation of extracellular compartments. Because of the potential role of the latter process in olfaction, we examined the expression of V-ATPase in mouse olfactory epithelial (OE) cells. We report that V-ATPase is present in this epithelium, where we detected subunits ATP6V1A (the 70-kDa “A” subunit) and ATP6V1E1 (the ubiquitous 31-kDa “E” subunit isoform) in epithelial cells, nerve fiber cells, and Bowman's glands by immunocytochemistry. We also located both isoforms of the 56-kDa B subunit, ATP6V1B1 (“B1,” typically expressed in epithelia specialized in regulated transepithelial proton transport) and ATP6V1B2 (“B2”) in the OE. B1 localizes to the microvilli of the apical plasma membrane of sustentacular cells and to the lateral membrane in a subset of olfactory sensory cells, which also express carbonic anhydrase type IV, whereas B2 expression is stronger in the subapical domain of sustentacular cells. V-ATPase expression in mouse OE was further confirmed by immunoblotting. These findings suggest that V-ATPase may be involved in proton secretion in the OE and, as such, may be important for the pH homeostasis of the neuroepithelial mucous layer and/or for signal transduction in CO2 detection

Home-cage tests assessing behavior of wild-type and B1-deficient female mice in investigating urine of heterozygous (Atp6v1b1+/−) male mouse urine.

<p>Water presentations revealed no significant difference between wild type and B1-deficient mice or between presentations. Both urine presentations showed that wild-type mice spent significantly more time investigating the urine of the opposite sex than Atp6v1b1−/− mice (n = 6). Data are shown as mean ± SEM (**, p<0.01).</p

Dual immunofluorescence labeling for the V-ATPase B1 subunit isoform (red) and cytokeratin-18 (CK-18, green) in the olfactory mucosa of an adult male mouse decalcified with Cal-Ex.

<p>As also shown in the previous figure for the A subunit, B1 localizes to the apical membrane microvilli of SCs and to the basolateral membrane of a subset of OE cells (A and D, arrows). Interestingly, these cells also exhibit basolateral membrane staining for CK-18 (B and E, arrows). The merge panels (C and F) confirm the co-expression of the V-ATPase B1 subunit and CK-18. Bar = 20 µm.</p

Reduced odor-evoked freezing by a predator odor.

<p>(A) A schematic diagram of the behavioral arena is depicted. (B) The percentage of time freezing in adult male B1-deficient mice (“KO”, n = 7) in the presence of the predator odor trimethyl-thiazoline (TMT) is less than half the time recorded in wild-type mice (“WT”, n = 6). Data are shown as mean ± SEM (p = 0.02). (C) Increased investigation of TMT in the V-ATPase B1-deficient mice: The number of individual sniffs of TMT is increased in adult male B1-null mice (“KO”, black bar) relative to wild-type mice (“WT”, gray bar). Data is shown as mean ± SEM (p = 0.05) and confirm the blunted response to TMT in B1-deficient mice compared to their wild type counterparts.</p

Immunocytochemical localization of the V-ATPase A subunit (A, red) in the OE of an adult male mouse decalcified with ImmunoCal.

<p>The V-ATPase is expressed on the apical microvilli of SCs and on the basolateral membrane of a subpopulation of cells (arrows). Olfactory marker protein (OMP, B, green) localizes to the OSN cilia and to the basolateral membrane of OE cells (arrowheads) which, as revealed by the merge panel (C), do not coincide with the cells that express B1 in this membrane domain. DAPI (blue) stains cell nuclei. Bar = 10 µm.</p

V-ATPase localization in the mouse olfactory epithelium.

<p>(A) A schematic diagram showing the subunit composition of the V-ATPase. The cytosolic V₁ domain is composed of subunits A through H (shown in white or light gray, marked with blue letters). The transmembrane V₀ domain is composed of subunits a, c, c” (or b), d, e, and Ac45 (shown in blue, marked with white letters). Some of the subunit interactions are putative. (B) Section from a 3-D image reconstruction showing that B1 V-ATPase (red) localizes to the microvilli of olfactory sustentacular cells in a 2-week old female mouse pup. Apical cilia of olfactory sensory neurons are immunostained for CNGA2 (green). DAPI (blue) stains cell nuclei. Bar = 30 µm.</p