Molekulare Pflanzenphysiologie and Erlangen Center of Plant Science, Department Biology, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstraße 5, D-91058 Erlangen, Germany.
Plant Cell Physiol. 2013 Apr;54(4):573-84. doi: 10.1093/pcp/pct021. Epub 2013 Feb 5.
Cyclic nucleotide-gated channels (CNGCs) form non-selective cation entry pathways regulated by calmodulin (CaM), a universal Ca(2+) sensor in eukaryotes. Although CaM binding has been shown to be important for Ca(2+)-dependent feedback regulation of CNGC activity, the CaM-binding properties of these channels have been investigated in a few cases only. We show that CNGC20 from Arabidopsis thaliana binds CaM in a Ca(2+)-dependent manner and interacts with all AtCaM isoforms but not with the CaM-like proteins CML8 and CML9. CaM interaction with the full-length channel was demonstrated in planta, using bimolecular fluorescence complementation. This interaction occurred at the plasma membrane, in accordance with our localization data of green fluorescent protein (GFP)-fused CNGC20 proteins. The CaM-binding site was mapped to an isoleucine glutamine (IQ) motif, which has not been characterized in plant CNGCs so far. Our results show that compared with the overlapping binding sites for cyclic nucleotides and CaM in CNGCs studied so far, they are sequentially organized in CNGC20. The presence of two alternative CaM-binding modes indicates that ligand regulation of plant CNGCs is more complex than previously expected. Since the IQ domain is conserved among plant CNGCs, this domain adds to the variability of Ca(2+)-dependent channel control mechanisms underlining the functional diversity within this multigene family.
环核苷酸门控通道（CNGCs）形成非选择性阳离子进入途径，受钙调蛋白（CaM）调节，CaM 是真核生物中普遍存在的 Ca(2+) 传感器。尽管已经表明 CaM 结合对于 CNGC 活性的 Ca(2+)-依赖性反馈调节很重要，但这些通道的 CaM 结合特性仅在少数情况下进行了研究。我们表明，拟南芥的 CNGC20 以 Ca(2+) 依赖性方式结合 CaM，并与所有 AtCaM 同工型相互作用，但不与 CaM 样蛋白 CML8 和 CML9 相互作用。通过双分子荧光互补，在植物体内证明了 CaM 与全长通道的相互作用。这种相互作用发生在质膜上，与我们 GFP 融合的 CNGC20 蛋白的定位数据一致。CaM 结合位点被映射到一个异亮氨酸谷氨酰胺（IQ）基序，迄今为止尚未在植物 CNGC 中进行表征。我们的结果表明，与迄今为止研究的 CNGCs 中循环核苷酸和 CaM 的重叠结合位点相比，它们在 CNGC20 中是顺序排列的。两种替代的 CaM 结合模式的存在表明，植物 CNGCs 的配体调节比预期的更为复杂。由于 IQ 结构域在植物 CNGC 中保守，该结构域增加了 Ca(2+) 依赖性通道控制机制的可变性，突出了该多基因家族内的功能多样性。
