Characterization of a novel inositol 1,4,5-trisphosphate receptor in isolated olfactory cilia

Inositol 1,4,5-trisphosphate (InsP₃), a product of G-protein-mediated receptor activation of phosphoinositide turnover, plays the role of a second messenger when olfactory neurons are stimulated with certain olfactory stimuli. In this paper we examine the specific binding of [³H]InsP₃ to isolated olfactory cilia, microsomes and brain membranes from the channel catfish (Ictalurus punctatus) and, by photoaffinity labelling with an InsP₃ analogue {¹²⁵I-labelled 1-[3-(4-azidosalicyloxy)- aminopropyl]inositol 1,4,5-trisphosphate (¹²⁵I-ASA-InsP₃)}, we tentatively identify the major InsP3-binding protein in catfish olfactory cilia. InsP3 binding to ciliary membranes is specific and saturable, with a K(d) of 1.10±0.31 μM and a maximum number of binding sites (B(max) of 17.6±5.8 pmol/mg. The rank order for potency of inhibition of [³H]InsP₃ binding is Ins(1,4)P₂ < Ins(1,3,4)P₃ < Ins(1,3,4,5)P₄ = Ins(1,4,5)P₃ < Ins(2,4,5)P₃. Exposure of cilia membranes to u.v. Iight in the presence of ¹²⁵I-ASA-InsP₃ results in the labelling of a protein with apparent M(r) 107000. Labelling is specifically prevented by Ins(1,4,5)P₃, Ins(2,4,5)P₃ and Ins(1,3,4,5)P₄, but not by Ins(1,4)P₂ or Ins(1,3,4)P₃. Both specific [³H]InsP₃ binding and photoaffinity labelling of the M(r)-107000 protein were displaced by heparin. Thc K(d) and the inhibition of [³H]InsP₃ binding and of photoaffinity labelling by inositol phosphates and heparin are consistent with the ability of micromolar concentrations of Ins(1,4,5)P₃ [but not Ins(1,3,4)P₃] to activate the InsP₃-gated currents in patch-clamp experiments with olfactory neurons. These results suggest that InsP₃ binding to a M(r)-107000 cilia membrane protein may represent binding to the olfactory InsP₃-gated cation channel.