Bal Manjot, Zaika Oleg, Martin Pamela, Shapiro Mark S
University of Texas Health Science Center at San Antonio, Department of Physiology, MS 7756, San Antonio, TX 78229, USA.
J Physiol. 2008 May 1;586(9):2307-20. doi: 10.1113/jphysiol.2008.152777. Epub 2008 Mar 13.
Calmodulin (CaM) binds to KCNQ2-4 channels within their carboxy termini, where it regulates channel function. The existing data have not resolved the Ca2+ dependence of the interaction between the channels and CaM. We performed glutathione S-transferase (GST)-pull-down assays between purified KCNQ2-4 carboxy termini and CaM proteins to determine the Ca2+ dependence of the interaction in vitro. The assays showed substantial Ca2+ dependence of the interaction of the channels with wild-type (WT) CaM, but not with dominant-negative (DN) CaM. To demonstrate CaM-channel interactions in individual living cells, we performed fluorescence resonance energy transfer (FRET) between ECFP-tagged KCNQ2-4 channels and EYFP-tagged CaM expressed in CHO cells, performed under total internal reflection fluorescence (TIRF) microscopy, in which excitation light only penetrates several hundred nanometres into the cell, thus isolating membrane events. FRET was assayed between the channels and either WT or DN CaM, performed under conditions of normal [Ca2+]i, low [Ca2+]i or high [Ca2+]i induced by empirically optimized bathing solutions. The FRET data suggest a strong Ca2+ dependence for the interaction between WT CaM and KCNQ2, but less so for KCNQ3 and KCNQ4. FRET between all KCNQ2-4 channels and DN CaM was robust, and not significantly Ca2+ dependent. These data show interactions between CaM and KCNQ channels in living cells, and suggest that the interactions between KCNQ2-4 channels and CaM are likely to have Ca2+-dependent and Ca2+-independent components.
钙调蛋白（CaM）与其羧基末端的KCNQ2 - 4通道结合，在该处调节通道功能。现有数据尚未明确通道与CaM之间相互作用的钙离子依赖性。我们进行了纯化的KCNQ2 - 4羧基末端与CaM蛋白之间的谷胱甘肽S - 转移酶（GST）下拉实验，以确定体外相互作用的钙离子依赖性。实验表明通道与野生型（WT）CaM的相互作用存在显著的钙离子依赖性，但与显性负性（DN）CaM的相互作用则不然。为了证明单个活细胞中CaM与通道的相互作用，我们在全内反射荧光（TIRF）显微镜下，对CHO细胞中表达的ECFP标记的KCNQ2 - 4通道和EYFP标记的CaM进行了荧光共振能量转移（FRET）实验，其中激发光仅穿透细胞几百纳米，从而分离出膜相关事件。在正常[Ca2 +]i、低[Ca2 +]i或由经验优化的浴液诱导的高[Ca2 +]i条件下，对通道与WT或DN CaM之间进行FRET检测。FRET数据表明WT CaM与KCNQ2之间的相互作用对钙离子有很强的依赖性，但对KCNQ3和KCNQ4的依赖性较小。所有KCNQ2 - 4通道与DN CaM之间的FRET很强，且对钙离子的依赖性不显著。这些数据显示了活细胞中CaM与KCNQ通道之间的相互作用，并表明KCNQ2 - 4通道与CaM之间的相互作用可能具有钙离子依赖性和非钙离子依赖性成分。
