SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro

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• 作者：R.A. Saadi^a ; K. He^a ; K.A. Hartnett^a ; K. Kandler^a ; ^b ; M. Hershfinkel^c ; E. Aizenman^a ; ^c ; ^{redox@pitt.edu}
• 关键词：zinc ; metabotropic zinc receptor ; GPR39 ; KCC2 ; botulinum toxin ; SNARE
• 刊名：Neuroscience
• 出版年：2012
• 期刊代码：50_03064522
• 类别：neu
• 出版时间：17 May, 2012
• 卷：210
• 期：Complete
• 页码：38-46
• 文件大小：644 K

摘要

The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABA_A receptor channels. In a recent study, we showed that Zn²⁺ regulates GABA_A reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn²⁺ initiates this process by activating the Gq-coupled metabotropic Zn²⁺ receptor/G protein-linked receptor 39 (mZnR/GPR39). Here, we first demonstrated that mZnR/GPR39 is functional in cortical neurons in culture, and then tested the hypothesis that the increase in KCC2 activity is mediated through a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent process. We established the presence of functional mZnR in rat cultured cortical neurons by loading cells with a Ca²⁺ indicator and exposing cells to Zn²⁺, which triggered consistent Ca²⁺ responses that were blocked by the Gq antagonist YM-254890, but not by the metabotropic glutamate receptor antagonist (RS)-伪-methyl-4-carboxyphenylglycine (MCPG). Importantly, Zn²⁺ treatment under these conditions did not increase the intracellular concentrations of Zn²⁺ itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH₄⁺ influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn²⁺ pretreatment induced a Ca²⁺-dependent increase in KCC2 activity. The effects of Zn²⁺ on KCC2 activity were also observed in wild-type mouse cortical neurons in culture, but not in neurons obtained from mZnR/GPR39^{鈭?鈭?/sup> mice, suggesting that Zn2+ acts through mZnR/GPR39 activation to upregulate KCC2 activity. We next transfected rat cortical neurons with a plasmid encoding botulinum toxin C1 (Botox C1), which cleaves the SNARE proteins syntaxin 1 and synaptosomal-associated protein 25 (SNAP-25). Basal KCC2 activity was similar in both transfected and non-transfected neurons. Non-transfected cells, or cells transfected with marker vector alone, showed a Zn2+-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn2+. These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn2+ stimulation of mZnR/GPR39.}