Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels.
Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels.
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对磷脂酰肌醇 4,5-二磷酸的亲和力决定了 Kv7 K+ 通道的毒蕈碱激动剂敏感性。
DOI：
10.1085/jgp.200910313
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发表时间：
2009-11
期刊：
影响因子：
--
通讯作者：
Shapiro MS
中科院分区：
文献类型：
--
作者：
Hernandez CC;Falkenburger B;Shapiro MS
Kv7 K+通道亚基与 PIP2 的表观亲和力不同，并且根据它们在这些组织中的生理作用而在神经、肌肉和上皮细胞中差异表达。为了研究 PIP2 亲和力如何影响对生理刺激（例如受体刺激）的反应，我们将同聚和异聚 Kv7.2、7.3 和 7.4 通道暴露于异源表达系统中一系列浓度的毒蕈碱受体激动剂氧化震颤素-M (oxo-M)。 oxo-M 激活 M1 受体会通过 Gq 和磷脂酶 C (PLC) 导致 PIP2 耗尽。用 Kv7 亚基和 M1 受体瞬时转染中国仓鼠卵巢细胞，并在穿孔贴片电压钳下进行研究。对于 Kv7.2/7.3 异聚体，电流抑制的 EC50 为 0.44 ± 0.08 µM，最大抑制 (Inhibmax) 为 74 ± 3% (n = 5–7)。当 PIP 5 激酶过表达增加补品 PIP2 丰度时，EC50 向右移动三倍 (1.2 ± 0.1 µM)，但 Inhibmax 没有显着变化 (73 ± 4%，n = 5)。为了研究 Kv7.3 同聚体的毒蕈碱敏感性，我们使用了 A315T 孔突变体 (Kv7.3T)，该突变体将全细胞电流增加了 30 倍，而表观 PIP2 亲和力没有任何变化。与 Kv7.2/7.3 异聚体 (1.0 ± 0.8 µM) 相比，Kv7.3T 电流的 EC50 略有右移，并且 Inhibmax 大幅降低 (39 ± 3%)。相反，同聚 Kv7.4 通道的剂量反应曲线显着向左移动 (66 ± 8 nM)，Inhibmax 略有增加 (81 ± 6%，n = 3-4)。然后，我们通过将 Kv7.2 突变体与 Kv7.3T 亚基共表达来提高电流振幅，研究了几种对 PIP2 具有明显亲和力改变的 Kv7.2 突变体。对于较低亲和力 (Kv7.2 (R463Q)/Kv7.3T) 或较高亲和力 (Kv7.2 (R463E)/Kv7.3T) 通道，EC50 和 Inhibmax 与 Kv7.4 或 Kv7.3T 同聚物相似（0.12 ± 0.08 µM 和 79 ± 6% [n = 3–4] 和 0.58 ±分别为 0.07 µM 和 27 ± 3% [n = 3–4]）。亲和力极低的 Kv7.2（R452E、R459E 和 R461E）三重突变体也与 Kv7.3T 共表达。所得异聚体显示出非常低的抑制 EC50 (32 ± 8 nM) 和略微增加的 Inhibmax (83 ± 3%, n = 3–4)。然后，我们构建了一个细胞模型，其中包含 oxo-M 的 PLC 激活、PIP2 水解、PIP2 与 Kv7 通道亚基的结合以及通过 Kv7 四聚体的 K+ 电流。我们能够完全重现我们的数据并提取一组一致的 PIP2 亲和力。
Kv7 K+-channel subunits differ in their apparent affinity for PIP2 and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP2 affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M1 receptors by oxo-M leads to PIP2 depletion through Gq and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M1 receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC50 for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhibmax) was 74 ± 3% (n = 5–7). When tonic PIP2 abundance was increased by overexpression of PIP 5-kinase, the EC50 was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhibmax (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3T) that increases whole-cell currents by 30-fold without any change in apparent PIP2 affinity. Kv7.3T currents had a slightly right-shifted EC50 as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhibmax (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhibmax was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP2 by coexpressing them with Kv7.3T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3T) or higher affinity (Kv7.2 (R463E)/Kv7.3T) channels, the EC50 and Inhibmax were similar to Kv7.4 or Kv7.3T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3T. The resulting heteromer displayed a very low EC50 for inhibition (32 ± 8 nM) and a slightly increased Inhibmax (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP2 hydrolysis, PIP2 binding to Kv7-channel subunits, and K+ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP2 affinities.
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影响因子：
5.3
作者：
Li, Y;Gamper, N;Shapiro, MS
通讯作者：
Shapiro, MS
DOI：
10.1073/pnas.95.12.7151
发表时间：
1998-06-09
影响因子：
11.1
作者：
Cruzblanca, H;Koh, DS;Hille, B
通讯作者：
Hille, B
影响因子：
5.3
作者：
Gamper, N;Reznikov, V;Shapiro, MS
通讯作者：
Shapiro, MS
影响因子：
5.3
作者：
Ford, CP;Stemkowski, PL;Smith, PA
通讯作者：
Smith, PA
影响因子：
64.5
作者：
Kubisch, C;Schroeder, BC;Jentsch, TJ
通讯作者：
Jentsch, TJ