Evidence for an InsP₃-gated channel protein in isolated rat olfactory cilia

Stimulation of rat olfactory cilia (ROC) with odorants leads to a transient elevation in the levels of either cAMP or inositol trisphosphate (InsP₃). We have characterized the binding of [³H]InsP₃ to isolated ROC. Unlabeled InsP₃ displaced [³H]InsP₃ binding in a dose-dependent manner (dissociation constant = 3.9 ± 0.65 μM). Binding was stereospecific and dependent on the number of phosphates in the inositol ring. A ciliary protein of 120 kDa molecular mass was labeled specifically upon exposure of cilia membranes to ultraviolet light in the presence of the ¹²⁵I-labeled InsP₃ analogue 1-O-[N-(4-azidosaliciloxy)-3-aminopropyl-1-phopspho]-myo-inositol 4,5-bisphosphate. Labeling of this protein displayed the same stereospecificity as binding of [³H]InsP₃ to ROC. In addition, ROC membranes incorporated into a phospholipid bilayer at the tip of a patch pipette displayed an increase in conductance upon exposure to micromolar D- myo-inositol 1,4,5-trisphosphate in 45% of the trials (n = 88). The InsP₃- gated conductance is relatively nonspecific for cations and is distinct from the cAMP-gated conductance. The conductance displayed stereospecificity consistent with the InsP₃ binding experiments. The results suggest that the site of action for odorant-stimulated elevations in InsP₃ concentration in rat olfactory cilia is at a ciliary InsP₃-gated channel.