Here we investigate differences in the temporal and spatial patterning, and subunit interactions of two of the major Na+ channel α‐subunit isoforms in mammalian brain, the type I and type II Na+ channels. By using subtype‐specific antibodies, we find that both isoforms are abundant in adult rat brain, where both interact with the covalently bound β2 auxiliary subunit. Immunoblot analysis reveals complementary levels of type I and type II in different brain regions, with the highest levels of type I in brainstem, cortex, substantia nigra, and caudate, where it is found predominantly on the soma of neurons, and the highest levels of type II in globus pallidus, hippocampus and thalamus, where it is preferentially localized to axons. Developmentally, type I Na+ channel polypeptide expression in brain increases dramatically during the third postnatal week, peaks at the end of the first postnatal month, and then decreases such that adult levels are ≈ 50% of those at peak. Type II Na+ channel polypeptide expression in brain also undergoes large increases in the third postnatal week, but levels continue to increase such that peak expression levels are maintained in adult animals. Type I Na+ channels are found associated with the auxiliary β2 subunit at all ages, whereas free type II Na+ channels exist during the first two postnatal weeks. Thus, although expression of these two Na+ channel α subunits in heterologous systems yields currents with very similar electrophysiological and pharmacological properties, their distinct spatial and temporal patterning, and association with auxiliary subunits in brain, suggest that they perform distinct, nonoverlapping functions in situ. J. Comp. Neurol. 412:342–352, 1999. © 1999 Wiley‐Liss, Inc.

Voltage-gated Kv1 potassium channels consist of poreforming ␣ subunits and cytoplasmic Kv␤ subunits. The latter play diverse roles in modulating the gating, stability, and trafficking of Kv1 channels. The crystallographic structure of the Kv␤2 subunit revealed surprising structural homology with aldo-keto reductases, including a triosephosphate isomerase barrel structure, conservation of key catalytic residues, and a bound NADP ؉ cofactor (Gulbis, J. M., Mann, S., and MacKinnon, R. (1999) Cell 90, 943-952). Each Kv1-associated Kv␤ subunit (Kv␤1.1, Kv␤1.2, Kv␤2, and Kv␤3) shares striking amino acid conservation in key catalytic and cofactor binding residues. Here, by a combination of structural modeling and biochemical and cell biological analyses of structure-based mutations, we investigate the potential role for putative Kv␤ subunit enzymatic activity in the trafficking of Kv1 channels. We found that all Kv␤ subunits promote cell surface expression of coexpressed Kv1.2 ␣ subunits in transfected COS-1 cells. Kv␤1.1 and Kv␤2 point mutants lacking a key catalytic tyrosine residue found in the active site of all aldo-keto reductases have wild-type trafficking characteristics. However, mutations in residues within the NADP ؉ binding pocket eliminated effects on Kv1.2 trafficking. In cultured hippocampal neurons, Kv␤ subunit coexpression led to axonal targeting of Kv1.2, recapitulating the Kv1.2 localization observed in many brain neurons. Similar to the trafficking results in COS-1 cells, mutations within the cofactor binding pocket reduced axonal targeting of Kv1.2, whereas those in the catalytic tyrosine did not. Together, these data suggest that NADP ؉ binding and/or the integrity of the binding pocket structure, but not catalytic activity, of Kv␤ subunits is required for intracellular trafficking of Kv1 channel complexes in mammalian cells and for axonal targeting in neurons.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

Generating recombinant monoclonal antibodies (R-mAbs) from mAb-producing hybridomas offers numerous advantages that increase the effectiveness, reproducibility, and transparent reporting of research. We report here the generation of a novel resource in the form of a library of recombinant R-mAbs validated for neuroscience research. We cloned immunoglobulin G (IgG) variable domains from cryopreserved hybridoma cells and input them into an integrated pipeline for expression and validation of functional R-mAbs. To improve efficiency over standard protocols, we eliminated aberrant Sp2/0-Ag14 hybridoma-derived variable light transcripts using restriction enzyme treatment. Further, we engineered a plasmid backbone that allows for switching of the IgG subclasses without altering target binding specificity to generate R-mAbs useful in simultaneous multiplex labeling experiments not previously possible. The method was also employed to rescue IgG variable sequences and generate functional R-mAbs from a non-viable cryopreserved hybridoma. All R-mAb sequences and plasmids will be archived and disseminated from open source suppliers.