Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca(2+) release and entry in DT40 B lymphocytes.
Functional analysis of the green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3 in Ca(2+) release and entry in DT40 B lymphocytes.
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绿色荧光蛋白标记的肌醇 1,4,5-三磷酸受体 3 型在 DT40 B 淋巴细胞 Ca(2) 释放和进入中的功能分析。
作者：
T. Morita;A. Tanimura;A. Nezu;T. Kurosaki;Y. Tojyo
我们利用突变的DT40细胞株(IP(3)R-KO)检测了绿色荧光蛋白标记的肌醇1，4，5-三磷酸受体3(GFP-IP(3)R3)在Ca(2+)释放和进入中的作用。GFP-IP(3)R3的荧光主要与内质网的分布重叠，而GFP-IP(3)R3的部分荧光明显与质膜共定位。将IP(3)应用于通透性野生型DT40细胞，可诱导细胞内钙离子释放。虽然在IP(3)R-KO细胞中没有发生这种情况，但通过表达GFP-IP(3)R3可以恢复这种情况。在完整细胞中，BCR(B细胞受体)激活剂抗IgM或蛋白酶激活受体2激动剂胰酶不引起IP(3)R-KO细胞的任何钙反应，而这些处理在表达GFP-IP(3)R3的IP(3)R-KO细胞和WT细胞中诱导振荡或瞬时的Ca(2+)反应。此外，在表达IP(3)R-KO的WT和GFP-IP(3)R-KO细胞中，BCR激活可引起Ca(2+)内流，而在IP(3)R-KO细胞中则不能。这种BCR介导的Ca(2+)内流是在La(3+)存在的情况下观察到的，La(3+)可阻断容积性Ca(2+)内流。与GFP-IP(3)R3表达无关，thapsigargin可耗尽IP(3)R-KO细胞内的Ca(2+)储存，并导致Ca(2+)进入细胞内。与BCR刺激相比，thapsigargin诱导的Ca(2+)内流可被La(3+)完全阻断，提示BCR介导的Ca(2+)内流途径有别于容纳性的Ca(2+)内流途径。本研究表明，GFP-IP(3)R3在IP(3)诱导的Ca(2+)释放和BCR介导的Ca(2+)内流中均能补偿天然IP(3)R。
We examined the function of GFP-IP(3)R3 (green fluorescent protein-tagged inositol 1,4,5-trisphosphate receptor type 3) in Ca(2+) release and entry using a mutant DT40 cell line (IP(3)R-KO) in which all three IP(3)R genes had been disrupted. GFP-IP(3)R3 fluorescence largely overlapped with the distribution of endoplasmic reticulum, whereas a portion of GFP-IP(3)R3 apparently co-localized with the plasma membrane. The application of IP(3) to permeabilized WT (wild-type) DT40 cells induced Ca(2+) release from internal stores. Although this did not occur in IP(3)R-KO cells it was restored by expression of GFP-IP(3)R3. In intact cells, application of anti-IgM, an activator of the BCR (B-cell receptor), or trypsin, a protease-activated receptor 2 agonist, did not cause any Ca(2+) response in IP(3)R-KO cells, whereas these treatments induced oscillatory or transient Ca(2+) responses in GFP-IP(3)R3-expressing IP(3)R-KO cells, as well as in WT cells. In addition, BCR activation elicited Ca(2+) entry in WT and GFP-IP(3)R3-expressing IP(3)R-KO cells but not in IP(3)R-KO cells. This BCR-mediated Ca(2+) entry was observed in the presence of La(3+), which blocks capacitative Ca(2+) entry. Thapsigargin depleted Ca(2+) stores and led to Ca(2+) entry in IP(3)R-KO cells irrespective of GFP-IP(3)R3 expression. In contrast with BCR stimulation, thapsigargin-induced Ca(2+) entry was completely blocked by La(3+), suggesting that the BCR-mediated Ca(2+) entry pathway is distinct from the capacitative Ca(2+) entry pathway. The present study demonstrates that GFP-IP(3)R3 could compensate for native IP(3)R in both IP(3)-induced Ca(2+) release and BCR-mediated Ca(2+) entry.
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--
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