Phospho-dependent modulation of potassium chloride co-transporter KCC2

• Main Author: Lee, H. C.
• Published: University College London (University of London) 2009
• Subjects: 612.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.564659

The neuronal-specific potassium chloride co-transporter 2, KCC2, is a major chloride extruder in brain. The expression of KCC2 during neuronal development is fundamental to the switch of GABAergic response from excitatory to inhibitory. Malfunction of KCC2 can cause impairment of chloride homeostasis in neurons and is implicated in neurological disorders such as epilepsy. To date the role of protein phosphorylation in the regulation of KCC2 remains elusive. In this thesis, direct phosphorylation of KCC2 by PKC and Src tyrosine kinase was shown in vitro and in cultured neurons using the radioactive isotope 32P. Single mutation of serine residue at position 940 in the intracellular domain of KCC2 (Ser940) to alanine (S940A) blocked the phosphorylation of KCC2 under PKC activation. However, tyrosine phosphorylation of KCC2 was shown to not affect Tyr1087, the putative tyrosine kinase phosphorylation site. To better understand phosphorylation of KCC2 at Ser940, a phospho-specific antibody against this residue - namely p-S940 - was developed. Interestingly, agents inhibiting PKC and phosphatases altered signal of p-S940, indicating involvement of PKC, phosphatase-1 (PP1) and phosphatase-2A (PP2A) in the regulation of Ser940 phosphorylation. In an in vitro method using p-S940, it was shown that PP1 and PP2A dephosphorylated KCC2.