A.S.Sengar and W.Wang contributed equally to this workClathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Chronic pain hypersensitivity depends upon N-methyl-D-aspartate receptors (NMDARs). However, clinical use of NMDAR blockers is limited by side effects from suppressing physiological functions of these receptors. Here we report a means to suppress pain hypersensitivity without blocking NMDARs but rather by inhibiting the binding of a key enhancer of NMDAR function, the protein tyrosine kinase Src. We show that a peptide consisting of amino acids 40-49 of Src fused to the protein transduction domain of the HIV Tat protein (Src40-49Tat) prevented pain behaviors induced by intraplantar formalin and reversed pain hypersensitivity produced by intraplantar injection of complete Freund's adjuvant or by peripheral nerve injury. Src40-49Tat had no effect on basal sensory thresholds, acute nociceptive responses, or cardiovascular, respiratory, locomotor or cognitive functions. Thus, by targeting Src-mediated enhancement of NMDARs, inflammatory and neuropathic pain are suppressed without deleterious consequences of directly blocking NMDARs, an approach that may be of broad relevance to managing chronic pain.Chronic pain is categorized as inflammatory or neuropathic, each involving neuroplastic changes leading to hypersensitivity in peripheral and central nociceptive systems 1,2 . Multiple mechanisms including increased primary afferent excitability 3 , enhanced transmission in the dorsal horn 1 , changes in gene expression 4 , aberrant neuron-glia interactions 5,6 and neuronal apoptosis 7 are implicated in hypersensitivity in chronic pain models. Abundant pre-clinical evidence indicates that N-methyl-D-aspartate receptor (NMDARs) 8 are critically involved in pain hypersensitivity 9-11 . However, pharmacological blockade of these receptors in humans is deleterious because the activity of NMDARs is essential for many important physiological functions including breathing and locomotion 9,12,13 . A crucial signaling event for NMDAR-dependent neuroplasticity, including pain hypersensitivity 1,14 , is upregulation of NMDAR currents by mechanisms including relieving Mg 2+ blockade and receptor phosphorylation 15,16 . Thus, preferentially inhibiting mechanisms which upregulate NMDARs without affecting basal channel activity represents a strategy that may suppress pain hypersensitivity without impairing key physiological functions.

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl-dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.