Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels.
Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels.
复制标题
DOI：
10.1523/jneurosci.5175-09.2010
复制
发表时间：
2010-02-10
期刊：
影响因子：
--
通讯作者：
Shapiro MS
中科院分区：
文献类型：
--
作者：
Bal M;Zhang J;Hernandez CC;Zaika O;Shapiro MS
M型通道定位于神经元、心血管和上皮组织，在控制兴奋性和K+转运中起关键作用，并通过Gq/11介导的信号由许多受体调节。KCNQ 2和毒蕈碱受体显示的一种途径使用PKC，通过A-激酶锚定蛋白（AKAP）79/150募集到通道中。由于KCNQ 1 -5亚基组成的M型通道多种多样，且M电流受Ca ~（2+）/钙调素（CaM）和PIP 2的调节，我们探讨了AKAP 79/150在KCNQ 1 -5通道中作用的普遍性，以及Ca ~（2+）/CaM和PIP 2对AKAP 79/150作用的影响。我们首先使用全内反射荧光（TIRF）显微镜下的荧光共振能量转移（FRET）和膜片钳分析，在异源表达KCNQ 1 -5亚基和AKAP 79的中国仓鼠卵巢（CHO）细胞中检查AKAP 79靶向哪些KCNQ亚基。CFP标记的KCNQ 1 -5和YFP标记的AKAP 79之间的供体去猝灭FRET揭示了KCNQ 2 -5与AKAP 79的关联，而不是KCNQ 1。与这些结果平行的是，在穿孔膜片钳下研究的稳定表达M1受体的CHO细胞显示AKAP 79共转染使KCNQ 2/3异聚体和KCNQ 2 -5（但不是KCNQ 1）同聚体对毒蕈碱抑制“敏感”，表现为剂量-反应关系向较低浓度的转变。对KCNQ 4的影响被假定的PKC磷酸化位点的T553 A突变所消除。然后，我们探讨了CaM和PIP 2在这些AKAP 79作用中的作用。TIRF/FRET实验揭示了野生型CaM的共转染，而不是不能结合Ca 2+的显性阴性（DN）CaM，以破坏YFP标记的AKAP 791 -153与CFP标记的KCNQ 2 -5的相互作用。通过共转染PIP 2磷酸酶对PIP 2进行紧张性消耗没有影响，并且PIP 2的突然消耗不会使GFP标记的AKAP 79从膜上去定位。最后，膜片钳实验表明，野生型，但不是DN，CaM的共转染，以防止AKAP 79介导的KCNQ 2/3异聚体的致敏毒蕈碱抑制。因此，AKAP 79作用于KCNQ 2 -5，但不作用于含KCNQ 1的通道，其作用被钙化的CaM破坏，但不被PIP 2耗尽破坏。
M-type channels are localized to neuronal, cardiovascular and epithelial tissues, where they play critical roles in control of excitability and K+ transport, and are regulated by numerous receptors via Gq/11-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1–5 subunits, and M current is known to be regulated by Ca2+/calmodulin (CaM) and PIP2, we probed the generality of AKAP79/150 actions among KCNQ1–5 channels, and the influence of Ca2+/CaM and PIP2 on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1–5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1–5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M1 receptors studied under perforated-patch clamp showed co-transfection of AKAP79 to “sensitize” KCNQ2/3 heteromers and KCNQ2–5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose-response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP2 in these AKAP79 actions. TIRF/FRET experiments revealed co-transfection of wild-type, but not dominant-negative (DN) CaM that cannot bind Ca2+, to disrupt the interaction of YFP-tagged AKAP791–153 with CFP-tagged KCNQ2–5. Tonic depletion of PIP2 by co-transfection of a PIP2 phosphatase had no effect, and sudden depletion of PIP2 did not de-localize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed co-transfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2–5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP2 depletion.