Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain.
Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain.
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DOI：
10.1523/jneurosci.6089-11.2012
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发表时间：
2012-06-20
期刊：
影响因子：
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通讯作者：
Woodin MA
中科院分区：
文献类型：
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作者：
Acton BA;Mahadevan V;Mercado A;Uvarov P;Ding Y;Pressey J;Airaksinen MS;Mount DB;Woodin MA
KCC2 是 K+-Cl− 协同转运蛋白基因家族的神经元特异性成员。它也是其家族中唯一在生理正常条件下、在没有渗透压的情况下保持活跃的成员。通过在等渗条件下从神经元中挤出 Cl−，该转运蛋白可维持神经元 Cl− 的低浓度，这对于成熟神经系统中 GABA 和甘氨酸的快速抑制性突触传递至关重要。该 K+-Cl− 协同转运蛋白基因家族的其他成员完全是肿胀激活的。在这里，我们证明了 KCC2 特有的靠近 C 末端的 15 个氨基酸区域（称为 ISO 结构域）是 KCC2 在等渗条件下将 K+ 和 Cl− 共同转运出神经元所必需的。我们通过在从 Sprague Dawley 大鼠胚胎制备的培养海马神经元中过度表达嵌合 KCC2-KCC4 cDNA 构建体并通过短杆菌肽穿孔膜片钳记录测定神经元 Cl- 来实现这一发现。我们发现，当神经元被缺乏独特 ISO 结构域的嵌合 KCC2 转染时，对 GABA 的超极化反应被消除。这一发现表明 ISO 结构域是神经元 Cl− 调节所必需的。此外，我们发现，当 KCC2 缺乏 ISO 结构域时，它仍然保留肿胀激活的转运，这表明神经元中等渗和肿胀诱导的 K+-Cl− 共转运存在独特的分子决定因素。
KCC2 is the neuron-specific member of the of K+-Cl− cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl− from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl−, which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K+-Cl− cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 amino acid region near the end of the C-terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K+ and Cl− out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl− through gramicidin perforated patch clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl− regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K+-Cl− cotransport in neurons.