Odorant–Binding Protein–1a Regulates Aquaporin–5 Gating in Mouse

Abstract

Abstract: : Purpose: Aquaporin5 (AQP5) is a water–selective channel protein and located at apical membranes of the acinar cells of the lacrimal glands. AOP5 plays an important role in tear secretion. The tear secretion is regulated exactly by on/off mechanism, suggesting that AQP5 is regulated by gating. However, the gating mechanisms are still unknown. Our purpose is, therefore, to clarify the gating mechanisms of AQP5 in the lacrimal glands. Methods: Poly(A) RNAs from the lacrimal glands were prepared from 5–6 week–old BALB/c mouse. The poly(A) RNAs and AQP5 cRNA were microinjected into Xenopus laevis oocytes to examine if AQP5 inhibitory factor exists in the lacrimal glands. Antisense DNA or dibutyryl cAMP was added to the extra–oocytes solution to test the water permeability of AQP5 is regulated. For cross–linking analysis, the oocytes microinjected with the poly(A)RNAs and AQP5 cRNA were homogenized and incubated with imidoester–type crosslinker. Immunoblot analysis was performed with anti–mouse AQP5 antibodies. The inhibitory factors were purified from the mouse lacrimal glands by affinity chromatography coupled to the C–terminal peptides of mouse AQP5. Results: The osmotic water permeability of AQP5 was inhibited by the co–injection of poly(A)RNAs from the lacrimal glands into oocytes, and dibutyryl–cAMP restored the suppressed water permeability of AQP5 oocytes. The inhibitory factors that bind to AQP5 were shown by the administration of imidoester–type cross–linker. Using affinity chromatography, odorant–binding protein–1a (OBP–1a) is isolated from mouse lacrimal glands as one of the inhibitory factors for AQP5. Antisense DNA to OBP–1a also restored the suppressed water permeability of AQP5 oocytes. Conclusions: AQP5 is gated by binding of OBP–1a to the C–terminus of the molecule, which may be regulated by phosphorylation.

Keywords: cornea: tears/tear film/dry eye • lacrimal gland • phosphorylation