Neurodevelopmental disorders are a debilitating category of diseases that initiate in gestation when synaptogenesis is crucial and reliant upon electrochemical ion gradients across the neuronal plasma membrane. For example, the K+/Cl- co-transporter 2 (KCC2) regulates γ-aminobutyric acid (GABA) neurotransmission and has been linked to epilepsy, schizophrenia, and autism spectrum disorders. Importantly, KCC2 is a complex multimeric membrane protein that exhibits reduced cell surface expression and/or activity when disease-associated mutations are present. This observation is indicative of an underlying instability in the KCC2 protein, potentially making it highly susceptible to cellular quality control pathways that encourage proper folding and/or target misfolded proteins for degradation. Despite its critical role in neurodevelopment and disease relevance, little research has been done to define the mechanisms that modulate KCC2 protein stability and cell surface expression. The objective of this study is to elucidate the pathways that regulate KCC2 folding and degradation to better understand its role in the pathogenesis of neurodevelopmental disorders.
To this end, cycloheximide chase assays were conducted in a new yeast expression system for wild-type KCC2 and stability was assessed over time after protein synthesis was arrested. Western blot analysis revealed that KCC2 is highly unstable. To determine which quality control pathway is responsible for the degradation of KCC2, cells were first treated with MG132, an inhibitor of the proteasome. Because MG132 treatment significantly stabilized KCC2, we propose that the transporter is subject to endoplasmic reticulum-associated degradation (ERAD). This hypothesis is supported by an observed increase in KCC2 stability when protein stability was measured in strains lacking two ERAD-requiring E3 ubiquitin ligases. KCC2 stabilization is also observed when yeast expressed a dominant negative version of Otu1, an ERAD deubiquitinase whose levels are altered in synaptosomes isolated from patients with schizophrenia. To validate these findings in a mammalian model, cycloheximide chase assays were conducted in HEK293 cells in the presence or absence of MG132 treatment. Preliminary data suggest that the ER-resident fraction of wild-type KCC2 is unstable and rapidly targeted for proteasome-dependent degradation. Together, these results confirm the underlying instability of KCC2 in two model systems. This study represents the first steps toward elucidating the quality control pathways that regulate KCC2 degradation and suggest that the ERAD of disease-causing mutant forms of KCC2 might contribute to disease onset.