Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
J Neurosci. 2010 Feb 10;30(6):2311-23. doi: 10.1523/JNEUROSCI.5175-09.2010.
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 G(q/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 Ca(2+)/calmodulin (CaM) and PIP(2), we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca(2+)/CaM and PIP(2) 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 M(1) receptors studied under perforated patch-clamp showed cotransfection 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 PIP(2) in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca(2+), to disrupt the interaction of YFP-tagged AKAP79(1-153) with CFP-tagged KCNQ2-5. Tonic depletion of PIP(2) by cotransfection of a PIP(2) phosphatase had no effect, and sudden depletion of PIP(2) did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection 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 PIP(2) depletion.
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(+)转运方面发挥着关键作用，并且受许多通过 G(q/11)介导的信号的受体调节。已经证明，KCNQ2 和毒蕈碱受体的一条途径使用蛋白激酶 C (PKC)，PKC 通过 A-激酶锚定蛋白 (AKAP)79/150 募集到通道。由于 M 型通道可以由 KCNQ1-5 亚基组成，并且已知 M 电流受 Ca(2+)/钙调蛋白 (CaM)和 PIP(2)调节，因此我们探究了 AKAP79/150 在 KCNQ1-5 通道中的作用的普遍性，以及 Ca(2+)/CaM 和 PIP(2)对 AKAP79/150 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 KCNQ1-5 亚基和 AKAP79 的中国仓鼠卵巢 (CHO)细胞中，检查了 AKAP79 靶向的哪些 KCNQ 亚基。CFP 标记的 KCNQ1-5 和 YFP 标记的 AKAP79 之间的供体去猝灭 FRET 揭示了 KCNQ2-5 的关联，但不是 KCNQ1，与 AKAP79 相关联。与这些结果平行，在穿孔膜片钳下研究的稳定表达 M(1)受体的 CHO 细胞表明，AKAP79 的共转染“敏化”了 KCNQ2/3 异源二聚体和 KCNQ2-5，但不是 KCNQ1 同源二聚体对毒蕈碱抑制的反应，表现在对较低浓度的剂量反应关系的转变。PKC 磷酸化位点的 T553A 突变消除了对 KCNQ4 的影响。然后，我们研究了 CaM 和 PIP(2)在这些 AKAP79 作用中的作用。TIRF/FRET 实验表明，野生型 CaM 的共转染，但不是不能结合 Ca(2+)的显性失活 (DN) CaM 的共转染，破坏了 YFP 标记的 AKAP79(1-153)与 CFP 标记的 KCNQ2-5 的相互作用。共转染 PIP(2)磷酸酶以耗竭 tonic PIP(2)对野生型没有影响，并且 PIP(2)的突然耗竭不会使 GFP 标记的 AKAP79 从膜上脱定位。最后，膜片钳实验表明，野生型 CaM 的共转染可防止 KCNQ2/3 异源二聚体对毒蕈碱抑制的 AKAP79 介导的敏化，而 DN CaM 则没有这种作用。因此，AKAP79 作用于 KCNQ2-5，但不是含有 KCNQ1 的通道，其作用受 CaM 钙化破坏，但不受 PIP(2)耗竭的影响。
