Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein with a domain structure similar to the neuron-specific assembly protein AP180. We have previously found that CALM is expressed in neurons and present in synapses. We now report that CALM has a neuron-related function: it facilitates the endocytosis of the synaptic vesicle protein VAMP2 from the plasma membrane. Overexpression of CALM leads to the reduction of cell surface VAMP2, whereas knockdown of CALM by RNA interference results in the accumulation of surface VAMP2. The AP180 N-terminal homology (ANTH) domain of CALM is required for its effect on VAMP2 trafficking, and the ANTH domain itself acts as a dominant-negative mutant. Thus, our results reveal a role for CALM in directing VAMP2 trafficking during endocytosis. Neurotransmitter release is crucial to neuron function. In the presynaptic terminal, neurotransmitter release begins with the fusion of synaptic vesicles (SV) to the presynaptic plasma membrane (1). SV fusion is mediated by the SNARE proteins, which include the VAMP2 (also known as synap-tobrevin 2) on the vesicle and syntaxin 1 and SNAP25 on the plasma membrane (2,3). A recent quantitative analysis of SV constituents reveals a startling abundance of VAMP2 on SV, twice that of the next most abundant SV protein-synaptophysin (4). This finding emphasizes the importance of VAMP2 for SV. A fundamental question concerning the function of SV is how the neuron precisely constitutes and effectively preserves the vesicle components. Following SV fusion and subsequent exocytosis to release neurotransmitters, the retrieval of SV components that have dispersed to the presynaptic plasma membrane is thought to be accomplished primarily by clathrin-mediated endocytosis (5-7). Live cell image studies of hippocampal neurons show that surface VAMP2, most of which originates from SV, does not confine itself to the fusion site; instead, the protein diffuses along the axonal membrane even beyond the synapse (8,9). Although there is no doubt that endocytosis plays an indispensable role in the retrieval of SV components, it is unclear exactly how the SV acquires and maintains the individual SV-associated proteins-including VAMP2-through repeated cycles of exocytosis and endocytosis. Studies of Caenorhabditis elegans (10,11) and Drosophila (12) mutants have associated the loss of functional assembly protein (AP)180 with the misplacement of VAMP2 from its usual location on SV to the plasma membrane. AP180, a clathrin assembly protein, has been well characterized for its function in promoting the assembly of clathrin-coated vesicles at the plasma membrane (13,14). The membrane accumulation of VAMP2 in the AP180 mutants suggests that the assembly protein AP180, at least in Caenorhabditis elegans and Drosophila, has a specialized role in recruiting and directing VAMP2 from the plasma membrane to SV during endocytosis. Clathrin assembly lymphoid myeloid leukemia protein (CALM) is a clathrin assembly protein that structurally resembles A...

Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The clathrin assembly proteins AP180 and CALM (clathrin assembly lymphoid myeloid protein) are known to be involved in clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALMdeficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

The clathrin assembly protein, CALM, promotes the assembly of clathrin-coated vesicles. In a previous study, we showed that CALM controls the level of the synaptic vesicle protein VAMP2 at the plasma membrane by regulating VAMP2 endocytosis. Here, we provide evidence that CALM also influences the cell surface level of the AMPA receptor subunit GluR2. Although mechanistic details as well as the physiological relevance of CALM and GluR2 in the neuron have yet to be established, CALM-mediated trafficking could function as a component of a dedicated system for controlling postsynaptic abundance of GluR2.

Transient receptor potential vanilloid 1 (TRPV1) is a pronociceptive cation channel involved in persistent inflammatory and neuropathic pain. Herpes simplex virus (HSV) vector expression of TRPV1 causes cell death in the presence of capsaicin, thereby completely blocking virus replication. Here we describe a selection system for negative regulators of TRPV1 based on rescue of virus replication. HSV-based coexpression of TRPV1 and a PC12 cell-derived cDNA library identified protein phosphatase 1α (PP1α) as a negative regulator of TRPV1, mimicking the activity of “poreless” (PL), a dominant-negative mutant of TRPV1. Vectors expressing PP1α or PL reduced thermal sensitivity following virus injection into rat footpads, but failed to reduce the nocifensive responses to menthol/icilin-activated cold pain or formalin, demonstrating that the activity identified in vitro is functional in vivo with a degree of specificity. This system should prove powerful for identifying other cellular factors that can inhibit ion channel activity.

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