BRE 21406 Detection of cell surface sodium channels by monoclonal antibodies -- could the channels become exposed to the external surface and 'down regulated' by binding to antibodies? HAMLITAL MEIRI Department of Physiology and Biophysics, Faculty of Medicine and The Rappaport Family lnstitute Jor Research in the Medical Science,;, Technion-lsrael Institute of Technology, Haifa 31096 (Israel) (Accepted October 29th, 1985) Key words: sciatic nerve - - sodium channel - - monoclonal antibody - - immunofluorescence - - mobility - - eel electroplax Different domains of the sodium channel were characterized in rat sciatic nerve according to the binding of antibodies to their anti- genic determinants. An extracellular domain, accessible to antibodies, modulates channel conductance. Another domain, which is not involved in the physiological activity, becomes accessible to externally applied antibodies only after prolonged exposure. This study also revealed mobile antigenic determinants whose internalization can be detected by immunofluorescence. A number of sodium channel-specific monoctonal antibodies (mAb's) have previously been characteri- zed 1-4,7-11,14. Many of these were generated against eel sodium channell,3, 4,7,9,l°. In preparation of the latter, two immunogens were used: the solubilized and partially purified tetrodotoxin (TTX)-recep- tor 3,4,10,11 and sodium channel rich membrane frag- ments of eel electroplaxS,10,11. The mAb's 5D~0 and 5F 3 belong to the first group. They were selected for their ability to bind 3,4 and immunoprecipitate 1°.11 the TTX-receptor. These mAb's appear to bind specifi- cally to the sodium channel in fish preparations 4,5, but do not affect their function 4. The mAb's SC-72- 14 and SC-72-38 belong to the second group and were selected for their ability to affect impulse propa- gation by modulating sodium channel conduc- tance1, 9. These mAb's bind to eel and mammalian channels in their natural membrane with similar po- tencies 9 and their binding is modified by sodium channel specific neurotoxins ~.9. In a previous study, we compared the ability of these 4 mAb's to depress the action potential in rat sciatic nerve and to label the channels at nodes of Ranvier 7,s. These experiments revealed a dose-de- pendent depression of the compound action potential by mAb SC-72-14 (K a --- 4.4 x 10-7 M) and mAb SC- 72-38 (K a = 6.6 x 10-8 M). The mAb 5D10 was com- pletely inactive, while mAb 5F 3 partially depressed the compound action potential in an irreversible manner but only when a high dose (100-300 ktg/ml) was applied for a prolonged (>1 h) incubation peri- od 7. In addition, under the standard staining condi- tions (i.e. 1 h incubation with mAb's at room temper- ature), the latter two mAb's did not stain the axolem- ma at the nodes of Ranvier, whereas mAb's SC-72-14 and SC-72-38 did 7. Based on these studies it was con- cluded that the antigenic determinants for mAb's 5D10 and 5F 3 were lost during the evolution from eel to mammals. This study investigates in more detail the interac- tion of mAb 5F 3 and 5D10 (the 'eel specific' mAb's) with the mammalian peripheral nerve. It seemed possible that the number of binding sites for the mAb's in mammalian peripheral nerve was too low for detection by the immunofluorescence and physio- logical assays used previously 7-9. Therefore, the highly sensitive radioimmuno assay was performed to determine if in homogenized tissue of peripheral nerve sites for the 'eel specific' mAb's could be de- tected and how many were present compared to the Correspondence: H. Meiri, Department of Physiology and Biophysics, Faculty of Medicine, The Rappaport Family Institute for Re- search in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9697, Haifa 31096, Israel. 0006-8993/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)