We have identified the first putative integral membrane pentraxin and named it neuronal pentraxin receptor (NPR). NPR is enriched by affinity chromatography on columns of a snake venom toxin, taipoxin, and columns of the taipoxin-binding proteins neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and taipoxinassociated calcium-binding protein 49 (TCBP49). The predominant form of NPR contains an putative NH 2 -terminal transmembrane domain and all forms of NPR are glycosylated. NPR has 49 and 48% amino acid identity to NP1 and NP2, respectively, and NPR message is expressed in neuronal regions that express NP1 and NP2. We suggest that NPR, NP1, NP2, and TCBP49 are involved in a pathway responsible for the transport of taipoxin into synapses and that this may represent a novel neuronal uptake pathway involved in the clearance of synaptic debris.We identified two taipoxin binding proteins for a presynaptic-acting snake venom neurotoxin, taipoxin, that blocks recycling of synaptic vesicles (1, 2). Affinity chromatography of solubilized rat brain membranes on columns of immobilized taipoxin enriches two major proteins: (i) neuronal pentraxin 1 (NP1), 1 a neuronally secreted protein with homology to serum pentraxins (2), and (ii) taipoxin-associated calcium-binding protein 49 (TCBP49), a reticular calcium-binding protein (3). NP1 has homology to previously identified pentraxins, such as serum amyloid P protein and C-reactive protein, which are elevated in the serum during acute phase response. Although the exact functions of these previously identified pentraxins are not known, they have been shown to bind, in a calcium-dependent manner, a wide variety of ligands and have been proposed to mediate the uptake of bacteria, toxins, and extracellular debris (4, 5). Homology to serum pentraxins, as well as the presence of a cleaved signal peptide and N-linked glycosylation sites, suggests that NP1 is secreted. The abundance of NP1 mRNA and rarity of NP1 protein suggest that NP1 protein has a rapid turnover. We have proposed that NP1 has a role in uptake at the synapse and that NP1 mediates the uptake of taipoxin into neurons. By low stringency screening, we identified an additional neuronal pentraxin (NP2) in human that has 54% amino acid identity with NP1 and is expressed in brain and multiple other tissues (6). Potential homologs of NP2 have been identified in guinea pig as a sperm acrosomal protein, apexin/p50 (7,8), and in rat as a neural activity-regulated pentraxin, narp (9). The second taipoxin-binding protein, TCBP49, binds calcium via six EF-hand calcium binding motifs and is localized to the lumen of reticular membranes in neurons and glia (3). It contains the carboxyl-terminal sequence HDEL which has been shown to occasionally mediate endoplasmic reticulum retention in mammalian cells (10 -12). We have suggested that NP1 binds to synaptic material and is taken up into a compartment containing TCBP49 (2, 3). We have also suggested that NP1 allows the internalization of taipoxin or a taipoxin⅐NP1 complex and t...

The buccal ganglia of Aplysia contain a central pattern generator (CPG) that mediates rhythmic movements of the buccal apparatus during feeding. Activity in this CPG is believed to be regulated, in part, by extrinsic serotonergic inputs and by an intrinsic and extrinsic system of putative dopaminergic cells. The present study investigated the roles of dopamine (DA) and serotonin (5-HT) in regulating feeding movements of the buccal apparatus and properties of the underlying neural circuitry. Perfusing a semi-intact head preparation with DA (50 microM) or the metabolic precursor of catecholamines (L-3-4-dihydroxyphenylalanine, DOPA, 250 microM) induced feeding-like movements of the jaws and radula/odontophore. These DA-induced movements were similar to bites in intact animals. Perfusing with 5-HT (5 microM) also induced feeding-like movements, but the 5-HT-induced movements were similar to swallows. In preparations of isolated buccal ganglia, buccal motor programs (BMPs) that represented at least two different aspects of fictive feeding (i.e., ingestion and rejection) could be recorded. Bath application of DA (50 microM) increased the frequency of BMPs, in part, by increasing the number of ingestion-like BMPs. Bath application of 5-HT (5 microM) did not significantly increase the frequency of BMPs nor did it significantly increase the proportion of ingestion-like BMPs being expressed. Many of the cells and synaptic connections within the CPG appeared to be modulated by DA or 5-HT. For example, bath application of DA decreased the excitability of cells B4/5 and B34, which in turn may have contributed to the DA-induced increase in ingestion-like BMPs. In summary, bite-like movements were induced by DA in the semi-intact preparation, and neural correlates of these DA-induced effects were manifest as an increase in ingestion-like BMPs in the isolated ganglia. Swallow-like movements were induced by 5-HT in the semi-intact preparation. Neural correlates of these 5-HT-induced effects were not evident in isolated buccal ganglia, however.

To interpret the recent atomic structures of the Kv (voltage-dependent potassium) channel T1 domain in a functional context, we must understand both how the T1 domain is integrated into the full-length functional channel protein and what functional roles the T1 domain governs. The T1 domain clearly plays a role in restricting Kv channel subunit heteromultimerization. However, the importance of T1 tetramerization for the assembly and retention of quarternary structure within full-length channels has remained controversial. Here we describe a set of mutations that disrupt both T1 assembly and the formation of functional channels and show that these mutations produce elevated levels of the subunit monomer that becomes subject to degradation within the cell. In addition, our experiments reveal that the T1 domain lends stability to the full-length channel structure, because channels lacking the T1 containing N terminus are more easily denatured to monomers. The integration of the T1 domain ultrastructure into the full-length channel was probed by proteolytic mapping with immobilized trypsin. Trypsin cleavage yields an N-terminal fragment that is further digested to a tetrameric domain, which remains reactive with antisera to T1, and that is similar in size to the T1 domain used for crystallographic studies. The trypsin-sensitive linkages retaining the T1 domain are cleaved somewhat slowly over hours. Therefore, they seem to be intermediate in trypsin resistance between the rapidly cleaved extracellular linker between the first and second transmembrane domains, and the highly resistant T1 core, and are likely to be partially structured or contain dynamic structure. Our experiments suggest that tetrameric atomic models obtained for the T1 domain do reflect a structure that the T1 domain sequence forms early in channel assembly to drive subunit protein tetramerization and that this structure is retained as an integrated stabilizing structural element within the fulllength functional channel.The structural elements of potassium channels have begun to be characterized in atomic detail, allowing much increased sophistication in our understanding of their mechanism of action and biological function. For voltage-dependent potassium (Kv) 1 channels, the structure of the highly conserved cytoplasmic N-terminal T1 domain has been determined as a rotationally symmetric tetramer from three different Kv channels and in complex with an auxiliary ␤-subunit protein (1-4). Because the T1 domain structures are determined from isolated soluble protein domains, questions arise as to the relevance of the determined structures to the ultrastructure of the full-length Kv channel and in how the tetrameric domain is integrated into the remainder of the channel. Recent published studies have suggested that the T1 structure within the channel is likely to be very similar to the tetrameric structure of the isolated domain (3, 5-7). However, crystallography also has shown that the T1 domain can adopt several different conformations within the cha...

The T1 domain, a highly conserved cytoplasmic portion at the N-terminus of the voltage-dependent K+ channel (Kv) alpha-subunit, is responsible for driving and regulating the tetramerization of the alpha-subunits. Here we report the identification of a set of mutations in the T1 domain that alter the gating properties of the Kv channel. Two mutants produce a leftward shift in the activation curve and slow the channel closing rate while a third mutation produces a rightward shift in the activation curve and speeds the channel closing rate. We have determined the crystal structures of T1 domains containing these mutations. Both of the leftward shifting mutants produce similar conformational changes in the putative membrane facing surface of the T1 domain. These results suggest that the structure of the T1 domain in this region is tightly coupled to the channel's gating states.

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