Both KCC2-FL and KCC2-ΔNTD can interact with the actin cytoskeleton by direct structural interaction of the intracellular C-terminus with the actin-binding protein 4.1N (Li et al., 2007). We found aberrant actin and 4.1N

patterns in the neural tube of transgenic embryos. The cells of the neural tube had diffuse cytoplasmic levels of actin and 4.1N. Similar results were obtained in the neural cell line C17.2. The cytoplasmic staining in KCC2-overexpressing cells points to a redistribution of the 4.1N protein within the cell, perhaps leading to a defective formation of F-actin. KCC2-C568A did not produce similar effects on the actin cytoskeleton, indicating that the point mutation rendered KCC2 less effective in binding to CAL-101 molecular weight click here 4.1N. Indeed, immunoprecipitation of the three variants of the KCC2 protein demonstrated a significantly lower binding of KCC2-C568A to 4.1N (Fig. 8). Previous studies have employed KCC2-C568A as a control for KCC2-FL overexpression (Cancedda et al., 2007; Reynolds et al., 2008). The lack of effects of KCC2-C568A was

suggested to be due to inactivation of the ion transport function. However, this interpretation does not exclude a structural effect of KCC2, as our data suggest. It is not clear whether the C568A mutation interferes with the folding or intracellular trafficking not of the protein or resides in an important 4.1N-binding structure. However, the mutation lies within a central domain of the KCC2 protein, and the 4.1N-binding domain has been localized to the C-terminus (Li et al., 2007). As we have detected expression of KCC2-C568A at the protein level, we propose that the mutation has a major influence on the tertiary structure of KCC2, yielding a protein inactive both as an ion transporter and as an interacting partner of 4.1N. Taken together,

our results indicate that KCC2 regulates early neuronal differentiation and migration by effects mediated through direct structural interaction with 4.1N and the actin cytoskeleton. This interaction may be essential for neural tube development. We wish to thank Ruth Detlofsson, Panagiotis Papachristou, Maria Lindqvist and the Karolinska Center for Transgene Technologies for technical support, and Evan Y. Snyder for the C17.2 cells. This study was supported by grants from the Swedish Research Council, Stockholm County Council, M&M Wallenberg, Sällskapet Barnavård, Swedish Heart and Lung Foundations (E.H.), the Academy of Finland and the Sigrid Jusélius Foundation (K.K.). Z.H. is supported by the League of European Research Universities (LERU). K.K. is a member of the Finnish Center of Excellence in Molecular and Integrative Neuroscience Research.