James S. Trimmer

Caspr2, a new member of the neurexin superfamily, is localized at the juxtaparanodes of myelinated axons and associates with K+ channels.

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Here, we describe the identification of Caspr2, a mammalian homolog of Drosophila Neurexin IV (Nrx-IV), and show that this neurexin-like protein and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. We further show that Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains a putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains.

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

Dependence of Nodal Sodium Channel Clustering on Paranodal Axoglial Contact in the Developing CNS

Na+ channel clustering at nodes of Ranvier in the developing rat optic nerve was analyzed to determine mechanisms of localization, including the possible requirement for glial contactin vivo. Immunofluorescence labeling for myelin-associated glycoprotein and for the protein Caspr, a component of axoglial junctions, indicated that oligodendrocytes were present, and paranodal structures formed, as early as postnatal day 7 (P7). However, the first Na+ channel clusters were not seen until P9. Most of these were broad, and all were excluded from paranodal regions of axoglial contact. The number of detected Na+ channel clusters increased rapidly from P12 to P22. During this same period, conduction velocity increased sharply, and Na+ channel clusters became much more focal. To test further whether oligodendrocyte contact directly influences Na+ channel distributions, nodes of Ranvier in the hypomyelinating mouse Shiverer were examined. This mutant has oligodendrocyte-ensheathed axons but lacks compact myelin and normal axoglial junctions. During development Na+ channel clusters in Shiverer mice were reduced in numbers and were in aberrant locations. The subcellular location of Caspr was disrupted, and nerve conduction properties remained immature. These results indicate that in vivo, Na+ channel clustering at nodes depends not only on the presence of oligodendrocytes but also on specific axoglial contact at paranodal junctions. In rats, ankyrin-3/G, a cytoskeletal protein implicated in Na+ channel clustering, was detected before Na+ channel immunoreactivity but extended into paranodes in non-nodal distributions. In Shiverer, ankyrin-3/G labeling was abnormal, suggesting that its localization also depends on axoglial contact.

A Novel Targeting Signal for Proximal Clustering of the Kv2.1 K+ Channel in Hippocampal Neurons

The discrete localization of ion channels is a critical determinant of neuronal excitability. We show here that the dendritic K+ channels Kv2.1 and Kv2.2 were differentially targeted in cultured hippocampal neurons. Kv2.1 was found in high-density clusters on the soma and proximal dendrites, while Kv2.2 was uniformly distributed throughout the soma and dendrites. Chimeras revealed a proximal restriction and clustering domain on the cytoplasmic tail of Kv2.1. Truncations and internal deletions revealed a 26-amino acid targeting signal within which four residues were critical for localization. This signal is not related to other known sequences for neuronal and epithelial membrane protein targeting and represents a novel cytoplasmic signal responsible for proximal restriction and clustering.

Temporal and regional expression of NMDA receptor subunit NR3A in the mammalian brain

NR3A is a developmentally regulated N‐methyl‐D‐aspartate receptor (NMDAR) subunit that was previously known as NMDAR‐L or χ‐1. Unlike other NMDAR subunits, NR3A inhibits the NMDAR‐associated ion channel in a novel manner, and a role in synaptogenesis has been suggested for this subunit. Here, we report a comprehensive study to delineate the temporal and anatomic expression of NR3A protein in the mammalian brain by using a monoclonal anti‐NR3A antibody. NR3A protein was found to peak at postnatal day (P) 8, and to decrease gradually from P12 to adulthood in the rat central nervous system. Moreover, NR3A protein was heavily expressed in all areas of the isocortex, portions of the amygdaloid nuclei, and selective cell layers and nuclei of the hippocampus, thalamus, hypothalamus, brainstem, and spinal cord. NR3A protein was also expressed in the cerebellar cortex, whereas only weak signal was detected in the previous in situ studies by using riboprobes. At an ultrastructural level, NR3A was associated specifically with asymmetrical synapses and localized to postsynaptic membranes. This information will facilitate future research on NMDARs by providing clues to possible inclusion of the NR3A subunit in NMDARs in many brain regions. J. Comp. Neurol. 450:303–317, 2002.

K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve.

The localization of Shaker-type K+ channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic β-subunit Kvβ2 were colocalized in juxtaparanodal zones. During development, clustering of K+ channels lagged behind that for nodal Na+ channels by about 5 days. In contrast to the PNS, K+ channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K+ channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na+ channel clusters, and little or no detectable clustering of K+ channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K+ channel clustering and localization.

Dexamethasone rapidly induces Kv1.5 K+ channel gene transcription and expression in clonal pituitary cells

Glucocorticoids specifically increase Kv1.5 K+ channel mRNA in normal and clonal (GH3) rat pituitary cells. Here, we demonstrate that dexamethasone, a glucocorticoid agonist, rapidly induces Kv1.5 gene transcription, but does not affect Kv1.5 mRNA turnover (t1/2 approximately 0.5 hr) in GH3 cells. Immunoblots indicate that the steroid also increases the expression of the 76 kd Kv1.5 protein approximately 3-fold within 12 hr without altering its half-life (t1/2 approximately 4 hr). In contrast, Kv1.4 protein expression is unaffected. Finally, we find that the induction of Kv1.5 protein is associated with an increase in a noninactivating component of the voltage-gated K+ current. Our results indicate that hormones and neurotransmitters may act within hours to regulate excitability by controlling K+ channel gene expression.

Generation and Characterization of Subtype-specific Monoclonal Antibodies to K+ Channel α- and β-subunit Polypeptides

Abstract Molecular characterization of mammalian voltage-sensitive K + channel genes and their expression became possible with the cloning of the Shaker locus of Drosophila . However, analysis of the expression patterns and subunit composition of native K + channel protein complexes requires immunological probes specific for the individual K + channel gene products expressed in excitable tissue. Here, we describe the generation and characterization of monoclonal antibodies (mAbs) against eight distinct mammalian K + channel polypeptides; the Kv1.1, Kv1.2, Kv1.4, Kv1.5 and Kv1.6 Shaker -related α-subunits, the Kv2.1 Shab -related α-subunit, and the kvβ1 and Kvβ2 β-subunits. We characterized the subtype-specificity of these mAbs against native K + channels in mammalian brain and against recombinant K + channels expressed in transfected mammalian cells. In addition, we used these mAbs to investigate the cellular and subcellular distribution of the corresponding polypeptides in rat cerebral cortex, as well as their expression levels across brain regions. Copyright

Regulation of ion channel expression by cytoplasmic subunits

Neuronal and cardiac voltage-gated ion channels contain auxiliary subunits that can profoundly affect the gating of the pore-forming and voltage-sensing alpha subunits. Recent studies on the structurally similar cytoplasmic beta subunits of Ca2+ and K+ channels reveal that these subunits can also exert profound effects on the expression of the integral membrane protein channel components. The mechanisms by which these effects occur are now being elucidated through a combined approach that employs biophysical, pharmacological, cell biological and biochemical techniques.

Subunit composition and novel localization of K+ channels in spinal cord.

Axonal K+ channels involved in normal spinal cord function are candidate targets for therapeutics, which improve sensorimotor function in spinal cord injury. To this end, we have investigated the expression, localization, and coassociation of Kv1 α and β subunits in human, rat, and bovine spinal cord. We find that Kv1.1, Kv1.2, and Kvβ2 form heteromultimeric complexes at juxtaparanodal zones in myelinated fibers. However, these same complexes are also present in paranodal regions of some spinal cord axons, and staining with antibodies against Caspr, a component of the paranodal axoglial junction, overlaps with these paranodal K+ channels. This latter observation suggests a unique role for these channels in normal spinal cord function and may provide an explanation for the sensitivity of spinal cord to K+ channel blockers. Moreover, the conservation of these characteristics between human, rat, and bovine nodes of Ranvier suggests an essential role for this defined channel complex in spinal cord function. J. Comp. Neurol. 429:166–176, 2001.