Elsevier

Brain Research

Volume 1107, Issue 1, 30 August 2006, Pages 1-12
Brain Research

Research Report
Immunolocalization of NaV1.2 channel subtypes in rat and cat brain and spinal cord with high affinity antibodies

https://doi.org/10.1016/j.brainres.2006.05.090Get rights and content

Abstract

High titer polyclonal antibodies were produced in rabbit against a peptide unique to NaV1.2 sodium channels. NaV1.2 antibodies displayed 500,000-fold greater affinity for the NaV1.2 peptide compared with NaV1.1 or NaV1.3 peptides from the same region. These antibodies, when coupled to Sepharose beads, retained saxitoxin binding sites from solubilized rat brain membranes. Eluted protein from this antibody-affinity column was recognized by antibodies directed against neuronal voltage-gated sodium channels. Rabbit antibodies, which had been partially purified, were used in immunocytochemical localization of the NaV1.2 channel in 50 μm rat brain slices at dilutions of 1:1000 or 1:2000. NaV1.2 channels were predominately localized in unmyelinated fibers in the cortex, hippocampus, spinal cord and hypothalamus. Varicosities were seen in fiber staining which may reflect true varicosities in the fiber or simply varying densities of sodium channels along the fiber. Cell body staining with the NaV1.2 antibody was primarily observed in the hypothalamus. Antibody staining in the cerebellum was complex, with staining observed primarily in posterior lobes and considerably lower amounts of staining observed in anterior lobes. Specific staining was limited to fibers located in the granule and molecular layer, in an orientation consistent with granule cell unmyelinated axon labeling.

Introduction

Voltage-gated sodium channels are responsible for the rapid influx of sodium ions that underlies the rising phase of the action potential in electrically excitable cells. The primary component of the channel is a 260 kDa glycoprotein, the alpha subunit, which forms the pore of the channel. In the rat brain, at least four distinct isoforms of this alpha subunit have been isolated (Chioni et al., 2005, Goldin et al., 2000, Kayano et al., 1988, Noda et al., 1986, Schaller et al., 1995). Of these isoforms, NaV1.1, NaV1.2, and NaV1.3 share approximately 87% sequence homology (Kayano et al., 1988) and have been shown to be regulated differentially during development, with NaV1.3 expression highest in embryonic tissue. NaV1.6 has from 81 to 94% homology with the membrane spanning regions and cytoplasmic loop 3 of NaV1.1, NaV1.2, and NaV1.3, but lower homology (53–70%)with the other cytoplasmic loops (Schaller et al., 1995). It also appears that, in the adult rat, the four subtypes may be localized to very different areas of the neuron: NaV1.1 primarily in soma and apical dendrite (Gong et al., 1999, Westenbroek et al., 1989), NaV1.2 in unmyelinated axons (Gong et al., 1999, Westenbroek et al., 1989), NaV1.3 in some fiber tracts, soma and proximal dendrites (Lindia and Abbadie, 2003), and NaV1.6 in Nodes of Ranvier and dendrites (Caldwell et al., 2000).

The four channel subtypes also display regional variations in localization in the adult rat brain. Several studies have utilized polyclonal antibodies specific for sodium channel subtypes to examine the distribution of these subtypes within the rat brain. Gordon et al. (1987) used anti-peptide antibodies specific for subtypes NaV1.1 and NaV1.2/NaV1.3 to immunoprecipitate 32P-labeled sodium channels from various regions of the brain: they found that NaV1.1 was primarily located in the more caudal regions of the brain and in the spinal cord, while NaV1.2 was found in more rostral regions. This was confirmed by the study of Westenbroek et al. (1989), which further indicated that NaV1.2 channels were found in areas rich in unmyelinated nerve fibers, while NaV1.1 was located in cell bodies and exhibited much less dense staining. However, the NaV1.2 antibody used in both studies showed cross reactivity with the NaV1.3 subtype, so the exact pattern of NaV1.2 localization was not clear from these data. Gong et al. (1999), using antibodies directed against peptides from a different region of the channels which had little homology between NaV1.1 and NaV1.2 (as well as little homology with NaV1.3), determined that NaV1.1 was expressed in highest levels in the brainstem, cortex, substantia nigra, and caudate, where it was found predominately in the soma. In this study (Gong et al., 1999), NaV1.2 had highest expression in the globus pallidus, hippocampus, and thalamus, where it was preferentially localized to the axons. In both this study and the studies by Catterall's group, however, it was difficult to see the discrete labeling of the NaV1.2 channel in axons, due possibly to a high antibody concentration or lower antibody affinity. Other studies examining the distribution of mRNA for the three subtypes have also shown distinct subtype-specific patterns of localization within the brain (Black et al., 1994, Furuyama et al., 1993, Oh et al., 1994); the presence of mRNA in a neuron, however, does not give information about the ultimate subcellular targeting of that protein in either the cell body membrane or the axon. Therefore, while the evidence clearly suggests differential distribution of channel subtypes within the brain and possibly within the cell, a clear picture of channel protein distribution is still lacking for many regions of the brain.

To address this question, polyclonal antibodies were produced which are specific for a region in the carboxyl terminus of the sodium channel alpha subunit for channel subtype NaV1.2. These antibodies labeled fine varicose fibers and some cell bodies in both rat and cat brain, providing the first clear immunocytochemical localization of NaV1.2 to discrete individual fibers. The antibody staining was completely blocked by preincubation of the antibody with 100 nM NaV1.2 peptide but not an equal concentration of NaV1.3 peptide. There was no specific staining with preimmune serum, a control in many of the immunocytochemistry figures. A brief account of this work has appeared elsewhere (Jarnot et al., 1995a, Jarnot et al., 1995b).

Section snippets

Antibody characterization

In prior studies utilizing anti-peptide antibodies directed against specific regions of the NaV1.2 sodium channel subtype, a major drawback has been the crossreactivity of these antibodies between subtypes NaV1.2 and NaV1.3 and lack of discrete staining. The sequences chosen for antibody production in those studies shared over 60% sequence similarity, and the NaV1.2 antibody showed only about a 100-fold greater affinity for the peptide corresponding to NaV1.2 over the peptide corresponding to

Discussion

This study describes the production, characterization, and utilization of a highly specific antibody for the NaV1.2 sodium channel subtype from rat brain. Immunolocalization revealed NaV1.2 in fine fibers and varicosities, which we assume are presynaptic release sites, throughout the brain, and in cell bodies in the hypothalamic area. These experiments represent the first discrete localization of NaV1.2 to specific structures in the brain, combined with a clear characterization of antibody

Antibody production and purification

Synthetic peptides corresponding to carboxyl-terminal sequences (Kayano et al., 1988) of rat brain sodium channel subtypes NaV1.1 (RI) (FTYNKNKLKGGANLLV; amino acids 1939–1955), NaV1.2 (RII) (SIYKKDKGKEDEGTPI; amino acids 1929–1945), and NaV1.3 (RIII) (SKYDKETIKGRIDLPI; amino acids 1875–1891) were produced by Dr. Michael Russ at the University of Kentucky MSAF Center, Lexington, KY. All peptides had a terminal cysteine added to facilitate coupling to a carrier molecule. The NaV1.2 peptide was

Acknowledgments

This work was supported by NIH grant NS28377 to A.M.C. We would like to thank Dr. Robert Fyffe and Dr. Wayne Carmichael for the use of equipment in their laboratories. We would like to especially thank Dr. Francisco Alvarez for his guidance in the immunocytochemical localization work.

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