Restrepo D, Teeter J H, Honda E, Boyle A G, Marecek J F, Prestwich G D, Kalinoski D L
Monell Chemical Senses Center, Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia.
Am J Physiol. 1992 Sep;263(3 Pt 1):C667-73. doi: 10.1152/ajpcell.1992.263.3.C667.
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.
用气味剂刺激大鼠嗅觉纤毛（ROC）会导致环磷酸腺苷（cAMP）或肌醇三磷酸（InsP3）水平短暂升高。我们已对[3H]InsP3与分离的ROC的结合特性进行了表征。未标记的InsP3以剂量依赖性方式取代[3H]InsP3的结合（解离常数 = 3.9 +/- 0.65 microM）。结合具有立体特异性，且取决于肌醇环中的磷酸数量。在125I标记的InsP3类似物1-O-[N-(4-叠氮水杨氧基)-3-氨基丙基-1-磷酸]-肌醇4,5-二磷酸存在的情况下，将纤毛膜暴露于紫外线下时，一种分子量为120 kDa的纤毛蛋白会被特异性标记。该蛋白的标记显示出与[3H]InsP3与ROC结合相同的立体特异性。此外，在88次试验中的45%（n = 88）中，当将ROC膜整合到膜片吸管尖端的磷脂双分子层中并暴露于微摩尔浓度的D-肌醇1,4,5-三磷酸时，电导会增加。InsP3门控电导对阳离子相对非特异性，且与cAMP门控电导不同。该电导显示出与InsP3结合实验一致的立体特异性。结果表明，气味剂刺激大鼠嗅觉纤毛中InsP3浓度升高的作用位点是在纤毛InsP3门控通道处。
