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 microM). 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-phospho]-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-myoinositol 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.
Restrepo
D
D
Monell Chemical Senses Center, Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia.
Teeter
J H
JH
Honda
E
E
Boyle
A G
AG
Marecek
J F
JF
Prestwich
G D
GD
Kalinoski
D L
DL
D000818:Animals; D001667:Binding, Competitive; D002923:Cilia / Q000378:metabolism; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007295:Inositol Phosphates / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D009831:Olfactory Mucosa / Q000378:metabolism*; D009994:Osmolar Concentration; D051381:Rats
D007295:Inositol Phosphates; D007473:Ion Channels; D015544:Inositol 1,4,5-Trisphosphate