Sienaert I, Missiaen L, De Smedt H, Parys J B, Sipma H, Casteels R
Laboratorium voor Fysiologie, K. U. Leuven Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium.
J Biol Chem. 1997 Oct 10;272(41):25899-906. doi: 10.1074/jbc.272.41.25899.
Structural and functional analyses were used to investigate the regulation of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) by Ca2+. To define the structural determinants for Ca2+ binding, cDNAs encoding GST fusion proteins that covered the complete linear cytosolic sequence of the InsP3R-1 were expressed in bacteria. The fusion proteins were screened for Ca2+ and ruthenium red binding through the use of 45Ca2+ and ruthenium red overlay procedures. Six new cytosolic Ca2+-binding regions were detected on the InsP3R in addition to the one described earlier (Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005-27012). Strong 45Ca2+ and ruthenium red binding domains were localized in the N-terminal region of the InsP3R as follows: two Ca2+-binding domains were located within the InsP3-binding domain, and three Ca2+ binding stretches were localized in a 500-amino acid region just downstream of the InsP3-binding domain. A sixth Ca2+-binding stretch was detected in the proximity of the calmodulin-binding domain. Evidence for the involvement of multiple Ca2+-binding sites in the regulation of the InsP3R was obtained from functional studies on permeabilized A7r5 cells, in which we characterized the effects of Ca2+ and Sr2+ on the EC50 and cooperativity of the InsP3-induced Ca2+ release. The activation by cytosolic Ca2+ was due to a shift in EC50 toward lower InsP3 concentrations, and this effect was mimicked by Sr2+. The inhibition by cytosolic Ca2+ was caused by a decrease in cooperativity and by a shift in EC50 toward higher InsP3 concentrations. The effect on the cooperativity occurred at lower Ca2+ concentrations than the inhibitory effect on the EC50. In addition, Sr2+ mimicked the effect of Ca2+ on the cooperativity but not the inhibitory effect on the EC50. The different [Ca2+] and [Sr2+] dependencies suggest that three different cytosolic interaction sites were involved. Luminal Ca2+ stimulated the release without affecting the Hill coefficient or the EC50, excluding the involvement of one of the cytosolic Ca2+-binding sites. We conclude that multiple Ca2+-binding sites are localized on the InsP3R-1 and that at least four different Ca2+-interaction sites may be involved in the complex feedback regulation of the release by Ca2+.
采用结构和功能分析方法研究钙离子（Ca2+）对1,4,5-三磷酸肌醇（InsP3）受体（InsP3R）的调控作用。为确定Ca2+结合的结构决定因素，编码覆盖InsP3R-1完整线性胞质序列的GST融合蛋白的cDNA在细菌中表达。通过使用45Ca2+和钌红覆盖法筛选融合蛋白与Ca2+和钌红的结合情况。除先前描述的一个区域外（Sienaert, I., De Smedt, H., Parys, J. B., Missiaen, L., Vanlingen, S., Sipma, H., and Casteels, R. (1996) J. Biol. Chem. 271, 27005 - 27012），在InsP3R上又检测到六个新的胞质Ca2+结合区域。强45Ca2+和钌红结合结构域定位于InsP3R的N端区域，具体如下：两个Ca2+结合结构域位于InsP3结合结构域内，三个Ca2+结合片段定位于InsP3结合结构域下游的一个500个氨基酸区域。在钙调蛋白结合结构域附近检测到第六个Ca2+结合片段。通过对通透的A7r5细胞进行功能研究，获得了多个Ca2+结合位点参与InsP3R调控的证据，在该研究中我们表征了Ca2+和Sr2+对InsP3诱导的Ca2+释放的半数有效浓度（EC50）和协同性的影响。胞质Ca2+的激活是由于EC50向较低InsP3浓度的偏移，Sr2+可模拟此效应。胞质Ca2+的抑制作用是由于协同性降低以及EC50向较高InsP3浓度的偏移。对协同性的影响发生在比EC50抑制作用更低的Ca2+浓度下。此外，Sr2+模拟了Ca2+对协同性的影响，但未模拟对EC50的抑制作用。不同的[Ca2+]和[Sr2+]依赖性表明涉及三个不同的胞质相互作用位点。内质网腔Ca2+刺激释放但不影响希尔系数或EC50，排除了其中一个胞质Ca2+结合位点的参与。我们得出结论，多个Ca2+结合位点定位于InsP3R-1上，并且至少四个不同的Ca2+相互作用位点可能参与Ca2+对释放的复杂反馈调节。
