The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.T he neuronal K-Cl cotransporter KCC2 transports chloride using the electrochemical gradient of K + ions (1). In mature neurons, this action maintains a low intraneuronal chloride concentration that ensures a hyperpolarizing effect of GABA at chloride-permeable GABA A receptors. KCC2 expression, activity, and membrane traffic are tightly regulated by neuronal activity, particularly through the phosphorylation of its carboxylterminal domain (CTD) (2-4). Activation of postsynaptic glutamate receptors, for instance, reduces KCC2 activity through dephosphorylation and endocytosis within minutes (3, 5). KCC2 expression is also suppressed in pathological conditions associated with enhanced neuronal activity (6), leading to a rise in intraneuronal chloride and an alteration of GABA function (7-9). KCC2 therefore appears to mediate a functional cross-talk between synaptic excitation and inhibition in neurons.Although KCC2 function primarily influences the efficacy of GABAergic signaling, its presence in dendritic spines (10) raises the question of its role in spine morphogenesis and function. Genetic ablation of KCC2 in mice compromises spine maturation and excitatory synapse formation in immature hippocampal neurons (11). This effect appears to be independent of KCC2 function but, instead, involves KCC2 interaction with the neuronal FERM-domain protein 4.1N (12). However, KCC2 expression is up-regulated during postnatal development and is maximal in mature neurons (13), after spine formation, where its role in the maintenance and function of dendritic spines remains unknown. Here, we show that suppression of KCC2 after spine morphogenesis reduces postsynaptic glutamate recept...

Focal adhesion kinase (FAK) is activated following integrin engagement or stimulation of transmembrane receptors. Autophosphorylation of FAK on Tyr-397 is a critical event, allowing binding of Src family kinases and activation of signal transduction pathways. Tissue-specific alternative splicing generates several isoforms of FAK with different autophosphorylation rates. Despite its importance, the mechanisms of FAK autophosphorylation and the basis for differences between isoforms are not known. We addressed these questions using isoforms of FAK expressed in brain. Autophosphorylation of FAK ؉ , which is identical to that of "standard" FAK, was intermolecular in transfected cells, although it did not involve the formation of stable multimeric complexes. Coumermycin-induced dimerization of gyrase B-FAK ؉ chimeras triggered autophosphorylation of Tyr-397. This was independent of cell adhesion but required the C terminus of the protein. In contrast, the elevated autophosphorylation of FAK ؉6,7 , the major neuronal splice isoform, was not accounted for by transphosphorylation. Specifically designed immune precipitate kinase assays confirmed that autophosphorylation of FAK ؉ was intermolecular, whereas autophosphorylation of FAK ؉6,7 or FAK ؉7 was predominantly intramolecular and insensitive to the inhibitory effects of the N-terminal domain. Our results clarify the mechanisms of FAK activation and show how alternative splicing can dramatically alter the mechanism of autophosphorylation of a protein kinase.