Post-translational regulation of KCC2 in the rat hippocampus

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http://urn.fi/URN:ISBN:978-952-10-8037-1
Title: Post-translational regulation of KCC2 in the rat hippocampus
Author: Ahmad, Faraz
Contributor: University of Helsinki, Faculty of Biological and Environmental Sciences, Department of Biosciences, division of physiology and neuroscience
finnish graduate school of neuroscience
Thesis level: Doctoral dissertation (article-based)
Abstract: KCC2 is an important K+-Cl- co-transporter that along with Na+-K+-2Cl- co-transporter, NKCC1 is largely responsible for the regulation of intracellular chloride concentration in neurons which determines whether the ionotropic GABAergic/glycinergic responses are depolarizing or hyperpolarizing. There are spatiotemporal differences in the intracellular chloride concentration in individual neurons which are attributable to a differential temporal and spatial activation of cation chloride co-transport mediated by KCC2 and NKCC1. Post-translational modulation of proteins is a fundamental cellular mechanism for such spatiotemporal regulation of protein activity. This thesis deals with the work that has been ongoing in our laboratory to understand the mechanisms of post-translational regulation of KCC2 function. In Study I, we have demonstrated a fast post-translational increase in KCC2 co-transport function in neonatal rat hippocampus after a single seizure episode. This TrkB-dependent effect was caused by an increased surface expression of KCC2. Study II deals with the establishment of a modified protease cleavage method for quantitative analysis of surface expression of proteins using a cold-adapted trypsin. This can serve as a fast and reliable procedure and can be easily applied to brain slice preparations as well as cell culture systems. In study II, we have also shown that KCC2 has a low surface expression in the rat hippocampus but a very fast turn-over rate of the plasmalemmal pool. Not surprisingly, modifications in the turn-over rate of the surface pool can be employed as a mechanism to regulate the surface expression of KCC2 and consequently its function. Study III deals with another post-translational cellular strategy to regulate KCC2 function in the rat hippocampus under patho-physiological conditions. While the KCC2 protein is quite stable in the rat hippocampus and has a slow turn-over rate under basal conditions, epileptiform activity and excitotoxicity can induce a rapid calpain-mediated cleavage of KCC2 with a consequent loss of its co-transport function.KCC2 is an important K+-Cl- co-transporter that along with Na+-K+-2Cl- co-transporter, NKCC1 is largely responsible for the regulation of intracellular chloride concentration in neurons which determines whether the ionotropic GABAergic/glycinergic responses are depolarizing or hyperpolarizing. There are spatiotemporal differences in the intracellular chloride concentration in individual neurons which are attributable to a differential temporal and spatial activation of cation chloride co-transport mediated by KCC2 and NKCC1. This thesis deals with the work that has been ongoing in our laboratory to understand the mechanisms of post-translational regulation of KCC2 function. Specifically, the effects of changes in the surface expression and protein degradation on the co-transport function of KCC2 have been outlined. In parallel, an improved method for quantitative analysis of surface expression of membrane proteins has also been devised. KCC2 on solukalvolla sijaitseva kalium-kloridi-kuljettaja, joka yhdessä natrium-kalium-kloridi-kuljettajan (NKCC1) kanssa säätelee neuronien solunsisäistä kloridipitoisuutta. Kloridipitoisuuden muutokset puolestaan paljolti määrittävät ovatko hermosolun GABA- tai glysiinireseptorien vasteet solukalvoa depolarisoivia vai hyperpolarisoivia. Yksittäisten hermosolujen välillä ja myös yksittäisten hermosolujen sisällä on eroja solunsisäisessä kloridipitoisuudessa, jotka perustuvat eroihin KCC2- ja NKCC1-molekyylien mikroanatomisessa sijainnissa sekä näiden kuljettajaproteiinien aktiivisuuden tasoissa. Tässä väitöskirjassa tutkitaan mekanismeja jotka säätelevät KCC2-kuljettajan toimintaa post-translationaalisella tasolla. Erityisen huomion kohteena ovat solukalvolla sijaitsevan KCC2-proteiinin tasojen muutokset ja KCC2-proteiiniin kohdistuva entsymaattinen hajoitus, sekä näiden kahden tekijän merkitys KCC2:n kokonaisaktiivisuudessa. Tämän tutkimuksen osana on kehitetty myös uusi menetelmä solukalvoproteiinien kvantitatiiviseen määritykseen.
URI: URN:ISBN:978-952-10-8037-1
http://hdl.handle.net/10138/33252
Date: 2012-06-04
Subject: neuroscience
Rights: This publication is copyrighted. You may download, display and print it for Your own personal use. Commercial use is prohibited.


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