Paper review "Immunochemical properties and cyochemical localization of the voltage-sensitive sodium channel from the electroplax of the eel(Electrophorus electricus)

 This blog and all posts are part of an assignment for a histology course at Memorial University of Newfoundland

Immunochemical properties and cytochemical localization of the voltage-sensitive sodium channel from the electroplax of the eel(Electrophorus electricus)
Lawrence C. Fritz and Jeremy P. Brockes

Introduction

The paper specifically studied the distribution and structure of voltage-sensitive channels within Electrophorus electricus, by comparing them with suitable homologous channels in an animal model(rat tissue was used, among others), using two monoclonal antibodies and a rabbit antiserum as reagents in adsorption assays. The study of voltage-sensitive sodium channels is important for our understanding of biology, as it seems that their physiological abilities are very conserved, and channels from varied sources act similar in terms of activation, inactivation and ion selectivity. At the time, there was new biochemical evidence for these very similar properties of sodium channels from varying sources. Partially purified proteins from eel electroplax, rat brain synaptosomes and rat sarcolemma, each possess a high molecular weight glycoprotein, when treated with specific channel toxins: tetradotoxin(TTX) and saxitoxin(STX). While other low molecular weight subunits seem to be found in rat brain synaptosomes and rat sarcolemma, only a single subunit heavy protein(250,000 daltons) was clearly identified from electric eel electroplax.

Sodium channels are distributed in a highly regulated manner within excitable cells. Much physiological and pharmacological evidence seems to suggest that the distribution of these channels is not homogenous, and instead is localized to very particular regions of the plasmalemma. Electrophysiological recording from the eel electrocyte demonstrates that only one side of the flattened cell can produce an action potential, suggesting possible differences between the number and density of channels within the two faces.


The paper has three main questions it seems to address: 1) How similar, in immunological terms, are sodium channels between different sources? 2) Are the antigenic determinants that are present in the channel exposed in the membrane-embedded state? 3) Can Immunological reagents be used to visualize the sodium channel distribution in the eel electroplax? 

Summary of methods

The methods used in this paper seem reasonable, as the use of monoclonal antibodies for histological staining seems to be a good choice, as antibodies are very specific for their targets. If the  previous evidence holds true, that sodium channels have a highly regulated asymmetric distribution, a clear picture can be visualized-- as large accumulations of antibodies will adhere to sodium rich channels, and vice versa when the antibodies do not bind to their targets. Electric organs from the E. electricus  were obtained from World wide Scientific Animals and used for sodium channel purification. Western blots were performed, using separated electroplax proteins, in non-reducing SDS-PAGE(4-12% poly acrylamide content). The Sachs organ from E. electricus were used in immunocytochemistry. TTX-binding assays were performed, and Immunoprecipitation of STX-binding component with 3.5% polyetyhlene glycol. Proteins were moved by electrophoresis to introcellulose paper in a buff solution. Adsorption assays were performed used crude synaptsomal membrane from the brain, that was subsequently homogenized in very specific buffer. The supernatant was centrifuged, the supernatant was recovered and the pellet resuspended in a different buffer solution, to be stored at -70 degrees Celsius. Adsorption assays were performed using rat liver and kidney,  then a sepharose 6B assay (a solid phase RIA), was performed which had wells coated with the sodium channel purification results, and reacted with mAB, rabbit anti-mouse IgG, and ^125 I-protein A. Binding to rabbit antiserum anti-p250 was recorded in a solid phase assay for adherence to membranes. For the Immunocytochemistry that was performed, tissue from the caudal electroplax organ(Sachs) was put in very specific buffer, sectioned in 10 micrometer slits, using a cryostat , along the long axis of the electroplax. Sections were dried and then put on coverslips, incubated with hybridoma supernatant, affinity-purified rabbit anti-mouse IgG and rhodamine labeled goat anti-rabbit IgG. Sections were mounted in UV inert mountant and observed with a Zeiss fluorescence microscope using epi-illumination at X63 or X40 power. The methods seemed typical and appropriate of histological procedures of sectioning, staining and tissue preparation.

Results:
For mAb and rabbit anti-p250 serum, monoclonal antibody 5D10 had been previously shown to react with a part of the electroplax sodium channel by it's capability to immunoprecipitate the STX-binding component. It also binds exclusively to a 250,000 dalton component, visualized in SDS-PAGE. In related assays, mAb 5F3 also specifically reacts with the eel channel. To detect this immunoprecipitation, STX was added to solubilized electroplax membranes and treated with increasing portions of mAb(the hybridoma supernatant). This antibody was then precipitated by adding equal rabbit anti-mouse IgG antiserum and then 3.5% polyethylene glycol, before centrifugation of immune complexes through a glycerol cushion and quantified, as shown in fig. 1.

Antibody 5F3 demonstrated a very specific binding affinity to the p250 protein, visualized upon Western blot for electroplax proteins. For each of the four stages of purification of the voltage-sensitive sodium channels(membrane, detergent, extract, DEAE fraction and sepharose 6B fraction). Then the paper was then given doses of antibody 5F3, rabbit anti-mouse IgG and labeled protein A in sequential fashion. (see fig 2) An autoradiograph shows that a 250,000 dalton protein(p250 protein) reacted with 5F3 antibody, but not stained by the unrelated IgG1 mAb. No other polypeptide components present bound to 5F3.

The rabbit anti-serum was tested against an SDS-PAGE purified p250 protein, using Western blot analysis. The electroplax membrane proteins were reacted with rabit anti-p250 serum and I-protein A. The autoradiograph(fig. 3) showed that there was only specific reaction with the p250 protein. Preimmune serum had no binding under these conditions.

The results suggest that mAb 5F3 and rabbit anti-p250 serum, just as mAb 5D10, reacts specifically with the p-250 component of the eel sodium channel.

Immunological tests between sodium channels from the electroplax and sodium channels of external sources: mAb 5D10 and 5F3 were cross-reacted and recorded, in a quantitative adsorption. Varying amounts of membranes from different tissues and different species were treated with a limiting dilution of antibody. Membrane pellets from centrifugation and the resulting supernatant were tested for residual antibody. If the membrane has an antigen that is recognized by mAb, then that antibody should be depleted by adsorption to the target. The results of the mAb 5D10 binding to eel electroplax, crude synaptosomal membrane from rat or frog brain are demonstrated on fig. 4. From the results of this assay, it is clearly shown that 5D10 adhered to the electroplax membrane, but not to the rat brain or frog brain membranes in detectable amounts. This was also true when chick brain was tested. Essentially identical results were gathered when the antibody 5F3 was tested. The adsorption assays show that sodium channels from eel electroplax, rat, frog and chick brain are immunologically distinct , and that mAbs recognize the eel sodium channel in it's native membrane-integrated form. Similar experiments with rabbit anti-p250 showed that greater than 90% of these antibodies could be adsorbed by electroplax membranes.

Cross reactivity of the p250 antiserum was tested. Membranes from the test sources were coated onto the wells and reacted with anti-p250 and pre-immune serum from the same rabbit. Wells were washed, and reacted with I-protein A. (see fig. 5) binding to eel electroplax membranes is compared against rat crude synaptosomal membranes. Detectable binding to the rat brain membranes of the serum was seen, but only at a very low level, as indicated by the displacement between the two curves. No specific binding was seen in rat liver. Rabbit anti-p250 also bound to chick and frog brain synaptosomal membranes, but this too was at a very low, but detectable, level. Demonstrating, that even with a polyclonal antiserum, there is very limited immunological cross-reaction between sodium channels of the eel and other species.

Cross-reaction between monoclonal and polyclonal antibodies using rat skeletal muscle was observed using sodium channel adsorption assays. No cross-reaction was noted.

Immunocytochemical studies: mAbs 5D10, 5F3 and rabbit anti-p250 serum were all tested for their histological visualization of sodium channels in frozen eel electroplax. Sections were reacted with either monoclonal hybridoma supernatant, followed by affinity-purified rabbit anti-mouse IgG and goat anti-rabbit IgG coupled to rhodamine, or followed by goat anti-rabbit IgG coupled to rhodamine. Phase contrast or rhodamine fluorescence optics can then be used to view the sections.

When staining was done with mAb 5D10, no rhodamine flourescence was noted between fixed and unfixed tissue. Rabbit anti-p250 however, visualized sodium channels by it's reaction with the sodium channels and the subsequent reaction of the rhodamine-coupled second antibody. Antibody 5F3 however, produced clearer images when used as a primary antibody. Fig 6 shows photos taken of phase contrast microscopy slides and rhodamine optics, from an electrocyte stained with mAb 5F3. Sodium channels are indicated by intense fluorescence and seem to associate only with the caudal face of the electrocyte. This was true for rabbit anti-p250 serum as well. No fluorescence was observed when sections were reacted with IgG1 mAbs directed against unrelated antigens, showing the specificity that 5F3 has in terms of immunoreactivity (see fig 7)

While most of the caudal face membrane was stained by mAb 5F3, there are regions devoid of antibody activity(see Fig. 8). Filamentous material which is external to electrocytes localized on the caudal face, correlates with the immunoreactivity with 5F3. This filamentous material can be seen in fig. 6(left) and 7(left)

Fig. 1

Fig 2

fig. 3

fig. 4

fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig note: Since the images contain the specific figure titles, I thought it would not do the paper justice by paraphrasing the information in a less informative and inaccurate method; as such, I added only the figure number in my captions


Discussion
The two monoclonal antibodies(5F3 and 5D10) and the rabbit antiserum recognized the electroplax sodium channel. The specificity of binding was determined by two methods: 1) they were shown to precipitate the STX-binding component from detergent extracts of electroplax membranes, demonstrating the ability of these reagents to recognize the eel sodium channel 2) Western blot anaylsis showed that when the eel channel protein was separated by SDS-PAGE, a detectable reaction was only seen in the p250 band. 
adsorption assays showed that mAbs 5D10 and 5F3 have very little affinity for rat, frog and chick brain. Rabbit antiserum cross-reacts on a very minor but detectable level. The STX binding component assay showed no immunoprecipitation of brain sodium channels could be detected. Eel electroplax membranes are immunologically distinct from brain sodium channels, as tested in this paper. Limited cross-reaction was noted in radiolabeled antibody binding assays, is in accord with the failure to cytochemically visualize any binding of the reagents to a variety of excitable issues. Only visual binding of the anti-electroplax sodium channel antibodies was seen only in the electroplax(see fig 6 and 8). See the paper for more details on the discussion.

In response to the 3 questions posed at the beginning of the paper: 1) The immunological similarity between sodium channels between differing sources is not so great as expected, as shown by experiments between antibody binding to other sources(eg. rat brain synaptosome) and the eel electroplax sodium channel; although foreign antibodies could indeed identify the eel electroplax, these antibodies specific for the electroplax did not have the same binding affinity for differing channel sources. 2) Yes these antigenic determinants were present during the membrane embedded state, and 3) yes, the eel electroplax sodium channel could indeed be visualized by certain specific antibodies.  

Opinions of the paper

Personally, I liked the paper and the use of antibodies as histological stains. They were very thorough in their introduction about sodium channel distribution, similarities between purified protein structure and size, etc. The figures were adequate, abundant and had appropriate titles, and clear results to be drawn from them. They seemed quite thorough in their testing of antibody binding affinity in this paper. The results supported the authors claims about the binding of p250 protein to the antibodies and about recognition of the sodium channel by foreign antibodies.

References:
http://www.jneurosci.org/content/3/11/2300.full.pdf+html

Disclaimer: all of the information here and images used are not belonged to me in any way shape or form, and were provided only by the hard work and dedication of the authors of the paper, who deserve all the credit.