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

• Published in: Neuroscience 2012-05, Vol.210, p.38-46
• Main Authors: Saadi, R.A, He, K, Hartnett, K.A, Kandler, K, Hershfinkel, M, Aizenman, E
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; botulinum toxin; Cerebral Cortex - metabolism; GPR39; Immunoblotting; In Vitro Techniques; K Cl- Cotransporters; KCC2; metabotropic zinc receptor; Mice; Mice, Knockout; Neurology; Neurons - metabolism; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled - metabolism; Signal Transduction - physiology; SNARE; SNARE Proteins - metabolism; Symporters - metabolism; Transfection; Up-Regulation; zinc; Zinc - metabolism
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2012.03.001

Abstract The major outward chloride transporter in neurons is the potassium chloride co-transporter 2 (KCC2), critical for maintaining an inhibitory reversal potential for GABAA receptor channels. In a recent study, we showed that Zn2+ regulates GABAA 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)-α-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−/− 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.