Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high affinity interactions with odorant organic anions

Abstract

We have characterized the expression of organic anion transporter 6, Oat6 (slc22a20), in olfactory mucosa, as well as its interaction with several odorant organic anions. In situ hybridization reveals diffuse Oat6 expression throughout olfactory epithelium, yet olfactory neurons laser-capture microdissected from either the main olfactory epithelium (MOE) or the vomeronasal organ (VNO) did not express Oat6 mRNA. These data suggest that Oat6 is expressed in non-neuronal cells of olfactory tissue, such as epithelial and/or other supporting cells. We next investigated interaction of Oat6 with several small organic anions that have previously been identified as odortype components in mouse urine. We find that each of these compounds, propionate, 2- and 3-methylbutyrate, benzoate, heptanoate, and 2-ethylhexanoate, inhibits Oat6-mediated uptake of a labeled tracer, estrone sulfate, consistent with their being Oat6 substrates. Previously, we noted defects in the renal elimination of odortype and odortype-like molecules in Oat1 knockout mice. The finding that such molecules interact with Oat6 raises the possibility that odorants secreted into the urine through one OAT-mediated mechanism (Eraly et al., JBC 2006) are transported through the olfactory mucosa through another OAT-mediated mechanism. Oat6 might play a direct or indirect role in olfaction, such as modulation of the availability of odorant organic anions at the mucosal surface for presentation to olfactory neurons or facilitation of delivery to a distal site of chemosensation, among other possibilities that we discuss.

Figure 1

Expression of Oat6 and OMP in olfactory epithelium. Coronal sections through the nasal mucosa were hybridized to antisense (A) or sense (B) Oat6 probes, or to an antisense OMP probe (C). A diffuse Oat6-specific signal was observed throughout the olfactory epithelium. A more intense signal with a similar distribution was observed for OMP. Scale bar =100μm.

Figure 2

Amplification from comparable quantities of cDNA prepared from laser captured MOE sensory neurons (1-3), VNO sensory neurons (4-6), whole MOE epithelium (7), and whole VNO epithelium (8), with no template control (9). (A) Oat6 amplification (100bp ladder illustrates 492bp amplicon). (B) β3-Tubulin amplification (illustrates cDNA integrity). (C) Olfactory Marker Protein (OMP) amplification (illustrates neuronal cell identity).

Figure 3

(A) Time course of the accumulation of [3H]-ES in Oat6-injected oocytes, of [3H]-PAH in Oat1-injected oocytes, and of both [3H]-labeled tracers in uninjected oocytes. Each data point represents Mean ± SE of triplicate samples. (B) Concentration-dependent inhibition of the clearance of tracer substrate ([3H]-ES in Oat6-injected oocytes or [3H]-PAH in Oat1-injected oocytes) by ‘cold’ (unlabeled) ES and PAH. Each data point represents Mean ± SE of quadruplicate samples. Best-fit curves and IC₅₀ values were calculated using GraphPad Prism software.

Figure 4

Concentration-dependent inhibition of [3H]-labeled tracer clearance in Oat6- and Oat1-injected oocytes by odorant organic anions.