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GIRK1/2通道的定量模型表明,其基础活性和诱发活性受Gα和Gβγ不等化学计量的控制。

A Quantitative Model of the GIRK1/2 Channel Reveals That Its Basal and Evoked Activities Are Controlled by Unequal Stoichiometry of Gα and Gβγ.

作者信息

Yakubovich Daniel, Berlin Shai, Kahanovitch Uri, Rubinstein Moran, Farhy-Tselnicker Isabella, Styr Boaz, Keren-Raifman Tal, Dessauer Carmen W, Dascal Nathan

机构信息

Department of Physiology and Pharmacology and Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

Department of Integrative Biology and Pharmacology, University of Texas Health Science Center, Houston, Texas, United States of America.

出版信息

PLoS Comput Biol. 2015 Nov 6;11(11):e1004598. doi: 10.1371/journal.pcbi.1004598. eCollection 2015 Nov.

Abstract

G protein-gated K+ channels (GIRK; Kir3), activated by Gβγ subunits derived from Gi/o proteins, regulate heartbeat and neuronal excitability and plasticity. Both neurotransmitter-evoked (Ievoked) and neurotransmitter-independent basal (Ibasal) GIRK activities are physiologically important, but mechanisms of Ibasal and its relation to Ievoked are unclear. We have previously shown for heterologously expressed neuronal GIRK1/2, and now show for native GIRK in hippocampal neurons, that Ibasal and Ievoked are interrelated: the extent of activation by neurotransmitter (activation index, Ra) is inversely related to Ibasal. To unveil the underlying mechanisms, we have developed a quantitative model of GIRK1/2 function. We characterized single-channel and macroscopic GIRK1/2 currents, and surface densities of GIRK1/2 and Gβγ expressed in Xenopus oocytes. Based on experimental results, we constructed a mathematical model of GIRK1/2 activity under steady-state conditions before and after activation by neurotransmitter. Our model accurately recapitulates Ibasal and Ievoked in Xenopus oocytes, HEK293 cells and hippocampal neurons; correctly predicts the dose-dependent activation of GIRK1/2 by coexpressed Gβγ and fully accounts for the inverse Ibasal-Ra correlation. Modeling indicates that, under all conditions and at different channel expression levels, between 3 and 4 Gβγ dimers are available for each GIRK1/2 channel. In contrast, available Gαi/o decreases from ~2 to less than one Gα per channel as GIRK1/2's density increases. The persistent Gβγ/channel (but not Gα/channel) ratio support a strong association of GIRK1/2 with Gβγ, consistent with recruitment to the cell surface of Gβγ, but not Gα, by GIRK1/2. Our analysis suggests a maximal stoichiometry of 4 Gβγ but only 2 Gαi/o per one GIRK1/2 channel. The unique, unequal association of GIRK1/2 with G protein subunits, and the cooperative nature of GIRK gating by Gβγ, underlie the complex pattern of basal and agonist-evoked activities and allow GIRK1/2 to act as a sensitive bidirectional detector of both Gβγ and Gα.

摘要

G蛋白门控钾通道(GIRK;Kir3)由源自Gi/o蛋白的Gβγ亚基激活,可调节心跳以及神经元的兴奋性和可塑性。神经递质诱发的(I诱发)和非神经递质依赖性基础(I基础)GIRK活性在生理上都很重要,但I基础的机制及其与I诱发的关系尚不清楚。我们之前已证明异源表达的神经元GIRK1/2存在这种情况,现在又在海马神经元的天然GIRK中发现,I基础和I诱发是相互关联的:神经递质的激活程度(激活指数,Ra)与I基础呈负相关。为揭示其潜在机制,我们构建了一个GIRK1/2功能的定量模型。我们对非洲爪蟾卵母细胞中表达的GIRK1/2的单通道电流和宏观电流,以及GIRK1/2和Gβγ的表面密度进行了表征。基于实验结果,我们构建了神经递质激活前后稳态条件下GIRK1/2活性的数学模型。我们的模型准确地概括了非洲爪蟾卵母细胞、HEK293细胞和海马神经元中的I基础和I诱发;正确预测了共表达的Gβγ对GIRK1/2的剂量依赖性激活,并充分解释了I基础与Ra的负相关关系。建模表明,在所有条件下以及不同通道表达水平下,每个GIRK1/2通道有3到4个Gβγ二聚体可用。相比之下,随着GIRK1/2密度的增加,每个通道可用的Gαi/o从约2个减少到少于1个Gα。持续的Gβγ/通道(而非Gα/通道)比例支持GIRK1/2与Gβγ的强关联,这与GIRK1/2将Gβγ而非Gα募集到细胞表面一致。我们的分析表明,每个GIRK1/2通道的最大化学计量比为4个Gβγ,但只有2个Gαi/o。GIRK1/2与G蛋白亚基独特的、不相等的关联,以及Gβγ对GIRK门控的协同性质,构成了基础活性和激动剂诱发活性的复杂模式,并使GIRK1/2能够作为Gβγ和Gα的灵敏双向检测器。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/81c5/4636287/e6fede13b85c/pcbi.1004598.g001.jpg

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