The fine-structural distribution of G-protein receptor kinase 3, β-arrestin-2, Ca2+/calmodulin-dependent protein kinase II and phosphodiesterase PDE1C2, and a Cl−-cotransporter in rodent olfactory epithelia

Abstract

The sequentially activated molecules of olfactory signal-onset are mostly concentrated in the long, thin distal parts of olfactory epithelial receptor cell cilia. Is this also true for molecules of olfactory signal-termination and -regulation? G-protein receptor kinase 3 (GRK3) supposedly aids in signal desensitization at the level of odor receptors, whereas beta-arrestin-2, Ca2+/calmodulin-dependent protein kinase II (CaMKII) and phosphodiesterase (PDE) PDE1C2 are thought to do so at the level of the adenylyl cyclase, ACIII. The Na+, K(+)-2Cl(-)-cotransporter NKCC1 regulates Cl(-)-channel activity. In an attempt to localize the subcellular sites olfactory signal-termination and -regulation we used four antibodies to GRK3, two to beta-arrestin-2, five to CaMKII (one to both the alpha and beta form, and two each specific to CaMKII alpha and beta), two to PDE1C2, and three to Cl(-)-cotransporters. Only antibodies to Cl(-)-cotransporters labeled cytoplasmic compartments of, especially, supporting cells but also those of receptor cells. For all other antibodies, immunoreactivity was mostly restricted to the olfactory epithelial luminal border, confirming light microscopic studies that had shown that antibodies to GRK3, beta- arrestin-2, CaMKII, and PDE1C2 labeled this region. Labeling did indeed include receptor cell cilia but occurred in microvilli of neighboring supporting cells as well. Apical parts of microvillous cells that are distinct from supporting cells, and also of ciliated respiratory cells, immunoreacted slightly with most antibodies. When peptides were available, antibody preabsorption with an excess of peptide reduced labeling intensities. Though some of the antibodies did label apices and microvilli of vomeronasal (VNO) supporting cells, none immunoreacted with VNO sensory structures.

Ca2+–Calmodulin Feedback Mediates Sensory Adaptation and Inhibits Pheromone-Sensitive Ion Channels in the Vomeronasal Organ

“…5H ). This finding is consistent with the absence of CaMKII immunoreactivity in rodent VSNs (Menco, 2005).…”

Molecular components of signal amplification in olfactory sensory cilia

“…In an electron microscopic study in rat olfactory epithelium, the T4 antibody reported NKCC1 expression in supporting cells as well as in ORN cilia (34). Because the Nkcc1 gene is not expressed in supporting cells (3), and because the T4 antibody shows nonspecific staining in retina (35), these data must be interpreted as a cross-reaction of T4.…”

β-Arrestin2-Mediated Internalization of Mammalian Odorant Receptors

“…Our Ca 2ϩ -imaging results provide a clear indication that ORs are internalized via clathrin coated-pits both in the heterologous expression system and in vivo in ORNs. Both, ␤-arrestin2 (Menco, 2005) and ORs (Barnea et al, 2004;Strotmann et al, 2004) are localized in the olfactory cilia and dendritic knobs of ORNs. We found that ␤-arrestin2 seems to be involved in OR signaling in vivo, because it accumulated into dendritic knobs of ORNs during treatment of mice with a complex odorant mixture.…”