title: SNARE-dependent upregulation of potassium chloride co-transporter 2 activity after metabotropic zinc receptor activation in rat cortical neurons in vitro.
abstract: 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 Zn(2+) regulates GABA(A) reversal potentials in the hippocampus by enhancing the activity of KCC2 through an increase in its surface expression. Zn(2+) initiates this process by activating the Gq-coupled metabotropic Zn(2+) 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 Ca(2+) indicator and exposing cells to Zn(2+), which triggered consistent Ca(2+) 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, Zn(2+) treatment under these conditions did not increase the intracellular concentrations of Zn(2+) itself. We then measured KCC2 activity by monitoring both the rate and relative amount of furosemide-sensitive NH(4)(+) influx through the co-transporter using an intracellular pH-sensitive fluorescent indicator. We observed that Zn(2+) pretreatment induced a Ca(2+)-dependent increase in KCC2 activity. The effects of Zn(2+) 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 Zn(2+) 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 Zn(2+)-dependent increase in KCC2 activity. In contrast, KCC2 activity in neurons expressing Botox C1 was unchanged by Zn(2+). These results suggest that SNARE proteins are necessary for the increased activity of KCC2 after Zn(2+) stimulation of mZnR/GPR39.
authors.affiliation: Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: Saadi
authors.forename: K
authors.initials: K
authors.lastname: He
authors.forename: K A
authors.initials: KA
authors.lastname: Hartnett
authors.forename: K
authors.initials: K
authors.lastname: Kandler
authors.forename: M
authors.initials: M
authors.lastname: Hershfinkel
authors.forename: E
authors.initials: E
authors.lastname: Aizenman
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000378:metabolism*; D015151:Immunoblotting; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D027981:Symporters / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation; D015032:Zinc / Q000378:metabolism; D000096922:K Cl- Cotransporters
chemical_list: D043562:Receptors, G-Protein-Coupled; D050600:SNARE Proteins; D027981:Symporters; D015032:Zinc; D000096922:K Cl- Cotransporters; C535178:GPR39 protein, rat