Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons
Contribution of Kir3.1, Kir3.2A and Kir3.2C subunits to native G protein-gated inwardly rectifying potassium currents in cultured hippocampal neurons
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DOI：
10.1046/j.1460-9568.2003.02933.x
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发表时间：
2003-10-01
影响因子：
3.4
通讯作者：
Leaney, JL
中科院分区：
文献类型：
--
作者：
Leaney, JL
G蛋白门控内向整流钾通道（GIRK）存在于神经元、心房肌细胞和神经内分泌细胞中。一个典型的特征是它们通过刺激G（i/o）偶联受体而激活。例如，在中枢神经元中，它们被腺苷和GABA激活，因此，它们在神经递质介导的膜兴奋性调节中发挥重要作用。通道是Kir3.x亚基（Kir3.1-3.4加上剪接变体）的四聚体组装体。在这项研究中，我试图确定的通道亚基，有助于本机GIRK电流记录从原代培养的大鼠海马锥体神经元。逆转录-聚合酶链反应显示Kir3.1，3.2A，3.2C和3.3亚基的mRNA表达，共聚焦免疫荧光显微镜用于研究其表达模式。Kir3.1和Kir3.2A在细胞胞体和树突上均呈弥漫性染色，Kir3.2C染色较弱，呈点状。采用全细胞膜片钳技术记录海马锥体神经元的GIRK电流，根据内向整流、反转电位对外部钾离子浓度的依赖性和对特硫平的敏感性鉴定神经元。GIRK电流可被多种G（i/o）偶联受体激活而被百日咳毒素抑制。为了确定哪些Kir3.x亚基负责天然GIRK电流，我将其特性与HEK 293细胞中异源表达的克隆Kir3.1 + 3.2A和Kir3.1 + 3.2C通道的特性进行了比较。
G protein-gated inwardly rectifying potassium (GIRK) channels are found in neurons, atrial myocytes and neuroendocrine cells. A characteristic feature is their activation by stimulation of G(i/o)-coupled receptors. In central neurons, for example, they are activated by adenosine and GABA and, as such, they play an important role in neurotransmitter-mediated regulation of membrane excitability. The channels are tetrameric assemblies of Kir3.x subunits (Kir3.1-3.4 plus splice variants). In this study I have attempted to identify the channel subunits which contribute to the native GIRK current recorded from primary cultured rat hippocampal pyramidal neurons. Reverse transcriptase-polymerase chain reaction revealed the expression of mRNA for Kir3.1, 3.2A, 3.2C and 3.3 subunits and confocal immunofluorescence microscopy was used to investigate their expression patterns. Diffuse staining was observed on both cell somata and dendrites for Kir3.1 and Kir3.2A yet that for Kir3.2C was weaker and punctate. Whole-cell patch clamp recordings were used to record GIRK currents from hippocampal pyramidal neurons which were identified on the basis of inward rectification, dependence of reversal potential on external potassium concentration and sensitivity to tertiapin. The GIRK currents were enhanced by the stimulation of a number of G(i/o)-coupled receptors and were inhibited by pertussis toxin. In order to ascertain which Kir3.x subunits were responsible for the native GIRK current I compared the properties with those of the cloned Kir3.1 + 3.2A and Kir3.1 + 3.2C channels heterologously expressed in HEK293 cells.