CHARACTERIZATION OF A NOVEL INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IN ISOLATED OLFACTORY CILIA

Inositol 1,4,5-trisphosphate (InsP(a)), 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 [H-3]InsP3 to isolated olfactory cilia, microsomes and brain membranes from the channel catfish (Ictalurus punctatus) and, by photoaffinity labelling with an InsP3 analogue {I-125-labelled 1-[3-(4-azidosalicyloxy)-aminopropyl]inositol 1,4,5-trisphosphate (I-125-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-mu-M and a maximum number of binding sites (B(max)) of 17.6 +/- 5.8 pmol/mg. The rank order for potency of inhibition of [H-3]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. light in the presence of I-125-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 [H-3]InsP3 binding and photoaffinity labelling of the M(r)-107000 protein were displaced by heparin. The K(d) and the inhibition of [H-3]InsP3 binding and of photoaffinity labelling by inositol phosphates and heparin are consistent with the ability of micromolar concentrations of Ins(1,4,5)P2 [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.