The mechanisms that regulate the activity of the neuron specific K+Cl- cotransporter (KCC2) remain poorly understood, despite the critical importance of this transporter in inhibitory synaptic transmission and plasticity. In this thesis I made three novel discoveries which reveal the cellular and molecular mechanisms of KCC2 regulation. First, I assayed the K+Cl- cotransport function of KCC2 under isotonic conditions and determined the molecular domain of the cotransporter required for constitutive Cl- transport in hippocampal neurons (Acton et al 2012). Specifically, I identified the 15 amino acid domain of the C-terminus in neurons that is responsible for the ability of KCC2 to cotransport K+Cl- under basal isotonic conditions, allowing it to remain constitutively active to create the steep Cl- gradient across the neuronal membrane required for synaptic inhibition. Secondly, I investigated a novel KCC2-interacting protein named Neto2 and determined its effect on the postsynaptic action of GABA (Ivakine et al 2013). I have found that Neto2, which is also an auxiliary protein of kainate-type ionotropic receptors, can also regulate the activity of the KCC2. Neto2 is required for neurons to maintain low [Cl-]i and strong synaptic inhibition. Third, I examined the functional relevance of the KCC2:Neto2:KAR multiprotein complex and found that this complex regulates the surface level
membrane expression pattern of KCC2 and the stability of the cotransporter in the membrane. Moreover, I have provided the first evidence that the interactions of KCC2:Neto2:GluK2 regulate KCC2 via a PKC-mediated phosphorylation of the cotransporter. Taken together, these results resolve three novel mechanisms of KCC2 regulation: the identity of the key C-terminal domain of KCC2 required for isotonic transport, the functional significance of the KCC2:Neto2 interaction, and the mechanism by which the KCC2:Neto2:KAR complex regulates KCC2 expression and mobility in the neuronal membrane.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/43522 |
| Date | 08 January 2014 |
| Creators | Acton, Brooke Ashley |
| Contributors | Woodin, Melanie A. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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