Sienaert I, De Smedt H, Parys J B, Missiaen L, Vanlingen S, Sipma H, Casteels R
Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
J Biol Chem. 1996 Oct 25;271(43):27005-12. doi: 10.1074/jbc.271.43.27005.
To study the Ca2+ regulation of the inositol 1,4,5-trisphosphate receptor (InsP3R) at the molecular level, we expressed various cytosolic and luminal regions of the mouse type I InsP3R as glutathione S-transferase fusion proteins. 45Ca2+ and ruthenium red overlay studies and Stains-all spectra and staining revealed both a cytosolic and a luminal Ca2+ binding site. The luminal Ca2+ binding site was mapped to the nonconserved acidic subregion of the luminal loop between amino acids 2463 and 2528. A K0.5 of 0.3 microM and a Hill coefficient of 1.1 were determined by 45Ca2+ overlay by quantification of 45Ca2+ binding on blots. The cytosolic Ca2+ binding site was localized in a region just preceding the transmembrane domain M1. The Ca2+ binding was mapped to a 23-amino acid stretch between amino acids 2124 and 2146. This cytosolic region showed a single high affinity site for Ca2+, with a K0.5 of 0. 8 microM and a Hill coefficient of 1.0. Neither of the identified Ca2+ binding regions contained an EF-hand motif. We conclude that the type I InsP3R has at least two quite distinct types of Ca2+ binding sites, which are localized in different structural regions of the protein.
为了在分子水平上研究钙离子（Ca2+）对1,4,5-三磷酸肌醇受体（InsP3R）的调节作用，我们将小鼠I型InsP3R的各种胞质区和腔区表达为谷胱甘肽S-转移酶融合蛋白。45Ca2+和钌红覆盖研究以及全染光谱和染色显示存在一个胞质Ca2+结合位点和一个腔Ca2+结合位点。腔Ca2+结合位点被定位到氨基酸2463和2528之间腔环的非保守酸性亚区域。通过对印迹上45Ca2+结合进行定量的45Ca2+覆盖法测定出K0.5为0.3微摩尔，希尔系数为1.1。胞质Ca2+结合位点位于跨膜结构域M1之前的一个区域。Ca2+结合被定位到氨基酸2124和2146之间的一段23个氨基酸的序列。该胞质区域显示出一个对Ca2+的单一高亲和力位点，K0.5为0.8微摩尔，希尔系数为1.0。所鉴定的两个Ca2+结合区域均不包含EF手基序。我们得出结论，I型InsP3R至少有两种截然不同类型的Ca2+结合位点，它们位于该蛋白的不同结构区域。
