Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation
Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation
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DOI：
10.1113/jphysiol.2006.118661
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发表时间：
2006-11-15
影响因子：
5.5
通讯作者：
Romanin, Christoph
中科院分区：
文献类型：
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作者：
Derler, Isabella;Hofbauer, Michael;Romanin, Christoph
钙离子选择性TRPV 6和L型钙离子通道受钙离子结合蛋白钙调素（CaM）的调节。在这里，我们研究了相互作用的钙调素与大鼠（r）TRPV 6细胞内钙的变化，采用钙离子成像和膜片钳技术。此外，利用活细胞上的共聚焦福斯特共振能量转移（FRET）显微镜作为关键方法来可视化对于CaM调节rTRPV 6活性至关重要的体内蛋白质-蛋白质相互作用。为了阐明Ca 2 +/CaM依赖性调控的分子机制并确定生理学上相关的rTRPV 6-CaM相互作用位点，我们研究了过表达CaM或Ca 2+不敏感突变体（CaMMUT）对各种rTRPV 6突变体和片段的影响。一个显着的减少rTRPV 6的活性，以及电流失活的增加，观察到钙调素过表达时，除了内源性钙调素。然而，Ca 2+不敏感的CaMMUT未能影响rTRPV 6衍生的电流。因此，活细胞共聚焦FRET显微镜显示了一个强大的相互作用，钙调素，但不是CaMMUT与rTRPV 6，这表明了严格的Ca 2+依赖性，他们的协会。事实上，rTRPV 6或其C末端与CaM的相互作用随着细胞内Ca 2+水平的升高而增加，如通过动态FRET测量所观察到的。一个rTRPV 6 Delta（695-727）突变体的C-末端删除，产生的钙离子电流与一个显着减少的失活根据缺乏钙调蛋白相互作用的FRET显微镜证实。这些结果与钙调素依赖性L型钙通道失活的结果相反，表明钙调素与rTRPV 6的C-末端（aa 695-727）的动态关联，这使得随着细胞内钙调素浓度的增加，rTRPV 6电流失活的速率加快。
The Ca2+-selective TRPV6 as well as the L-type Ca2+ channel are regulated by the Ca2+-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca2+, employing Ca2+-imaging and patch-clamp techniques. Additionally, confocal Forster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein-protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca2+-insensitive mutant (CaMMUT) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca2+/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6-CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca2+-insensitive CaMMUT, however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaMMUT with rTRPV6, suggesting a strict Ca2+ dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca2+ levels, as observed by dynamic FRET measurements. An rTRPV6 Delta(695-727) mutant with the very C-terminal end deleted, yielded Ca2+ currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca2+ channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695-727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.