Ultrastructural localization of voltage-sensitive sodium channels using [125I]α scorpion toxin

Brain Research. 334 ( 198519- 17

9

Elsevier BRE 107,17

Ultrastructural Localization of Voltage-Sensitive Sodium Channels Using [nsI]a Scorpion Toxin* PIERRt- ('AU. ANNICK MASSACRIER. JEAN-LOUIS BOUDIER, FRANq.'OIS ( ' O U R A U I ) t with technical assistance of JANINE BOTI'INI and PAUI_E I)EPREZ 1 l, ahoratoire d'ths'tologie IERA ('NRS 322L eLaboratotre de Biochimie (INSERM U 172. I',RA ( N R 5 bl 7j bacult(; de MOdecine.'Secteur Nord. 13326 Mar,seilh' ('edex 15 (FranceJ

(Accepted August 10th, 1984) Key wr;rds: neuroblastoma cell - - ¢~scorpion toxin receptors - - quantitativc autoradiography - - ultrastrt,ctural localization

The distribution ot~z scorpion toxin (a-ScTx) receptors was examined in differentiated mouse neuroblastoma cell cultures (N IE 115 clone) bv electron microscope autoradiography using [k:51]wScTx. This neurotoxin binds specifically to voltagc-sensitive sodium channels, slowing down the inacti~ation of the sodium permeability. Quantitative analvsis demonstrated that onl} plasma membranes werc labelled. The u-Scl x receptors seemed to be randomly dispersed on both cell bodies and cell processes. Microvilli protruding from the cell bodies carried more sodium channels than other parts of the membrane. Fhc specific binding site density for ~z-ScTxvaried trom 4 (cell body membrane) to 13 (cell process membrane) per square micrometer. IN'I'ROI)U("I'I()N

The alkaloids veratridine and batrachotoxin inducc persistent activation of sodium ionophore by binding

Voltage-sensitive sodium channels are m e m b r a n e proteins present in electrically excitable cells such as neurons, skeletal muscle and heart muscle cells ~'. "Fhev are involved in thc rapid change of the sodium

to neurotoxin site 21.L Polypeptide toxins from Buthi-

t r a n s m e m b r a n e permeability associated with an action potential ~'~. Until recently the histological localization of sodium channels was analyzed using indirect methods such as cytochemical (for review see ref. 48) or freeze-fracturc >,2z,23 studies in the Ranvier node and in initial axonal segments "'~. known to carry a high density of sodium ionophorcs. Neurotoxins that bind specifically to voltage-sensitive sodium channels have been extensively used to study their molecular properties, to monitor the purification of their polypeptide components and to check their functional reconstitution2.>-~1. Voltage-sensitive sodium channels carry 4 separate neurotoxin receptor sites. Tetrodotoxin and saxitoxin bind to site 1 and block sodium transportS.3~.

nae scorpion venoms tot-scorpion toxin: rz-ScTx) and

sea a n e m o n e nematocysts bind to site 3 and slow down the inactivation of the sodium channel. The affinity of u-ScTx for its receptor depends on the membrane potential~e.vL Centrurinae scorpion venom contains fl-scorpion toxins which bind to site 4 and induce an abnormal sodium permeability which can be blocked by tetrodotoxin. The binding of a fl-ScTx to its specific site is i n d e p e n d e n t of the m e m b r a n e potential kv. [l->I]a-ScTx has been used to study the distribution of sodium ionophores in neuronal and neuroblastoma cell cultures at the light microscope level'~ and in neurohypophysial axons at the ultrastructural level >. In the present work, differentiated mouse neuroblastoma cells, in which the binding of [L>lla-ScTx has been well-documented ~a.l
• Part of this work has been presented at the 1st European Congress for (_'ellBiology. Paris 190;2. Correspondence: P. Cau, I,aboratoire d'Histologie. FacultY'de M~decine.'Secteur Nord, Boulevard Pierre Dramard. 13326 Marscillc

Codex 15. France. ()006-8993.'S5.$03.30© 1985 Elsevier Science Publishers B.V. (Biomedical Division)

nels by quantitative ultrastructural autoradiography. MATERIALS AND METHODS

('ell cultures Mouse neuroblastoma (NIE 115 clone) were differentiated by DMSO treatment as previously described33. Cells were grown in Dulbecco modified Eagle's medium containing 5% fetal calf serum. To induce morphological differentiation, the culture medium was supplemented with 2% DMSO for 48 h. then replaced by medium without DMSO but containing 0.2% serum. The cells were used after 48 h. when more than 90% of the cells had developed long processes.

c~-Sc 7"x bin ding An ct-ScTx, toxin II from Androctonus australis Hector, was purified and iodinated as previously described 35.39. Binding experiments were done in a Nafree medium containing 140 mM choline chloride, 5 mM KC1, 1.8 mM CaCI 2, 0.8 mM MgSO4, 10 mM glucose, 0.1% bovine serum albumin, 25 mM Hepes and Tris base to obtain pH 7.233. The medium prevented toxin-induced depolarization, since Na + channels will not be functional in the absence of Na +. Cells were incubated at 37 °C for 30 rain in 3 conditions: (1) in the presence of 1 nM [125I]a-ScTx to measure the total binding; (2) in the presence of 1 nM [12Sl]a-ScTx and 100 nM native a-ScTx to determine the non-specific binding; and (3) without any toxin as a blank experiment. At the end of incubation, the cell layers were washed 3 times with the Na-free medium at 4 °C.

Preparing autoradiograms The cells were processed in the culture dishes for standard electron microscopy. Fixation by 2.5% glutaraldehyde in sodium phosphate buffer (0.177 M, pH 7.2) was followed by 2% osmium tetroxide in the same buffer, dehydration and embedding in Epon. The loss of radioactivity at each step was checked by counting aliquots from the solutions used (fixatives, alcohols . . . ). Less than 10% of the initial bound radioactivity was lost mainly after glutaraldehydc fixation. Five blocks were randomly chosen from the 38 prepared for each culture dish and re-embedded to obtain a section plane parallel to the culture surface.

Ultrathin sections were collected on collodion coaled slides and autoradiograms were prepared tcfllowin~: the flat-substratc method I using llford 14 emuls)~)n and Microdol X development after 45 dax,~ c,l cxp¢)sure.

Quantitative analysis This was carried out in 4 successive stages using specially designed BASIC microcomputer programs~. The background density was first measured in tissue-free sections from the blank experiments using the quadrat methodSl and found to be very low: 0.14 silver grains per 100~m 2 of section surface. The localization of silver grains over cell compartments was then studied using the circle method ~ . Micrographs were submitted to systematic sampling 5° and printed at a final magnification of 10,0(X). Calibration of the microscope and of the printer was checked using a replica of lines. Circle diameter was 3.8 mm corresponding to a half-distance (HD) value of 1(30 nm (the half distance is the distance from a radioactive line source containing 50c,~ of the silver grains produced by disintegrations at the line source4~ ). Using the same set of micrographs, the plasma membrane surface-area and the volume of cell compartments were then measured by stereological methodsS0. The cell membrane perimeter was calculated by counting intercepts of membrane profiles with a curvilinear grid34. The areas of cell compartment cross-sections were calculated by point counting. Then, the cell membrane surface area and the volume of cell compartments were obtained by multiplying both the cell perimeter and the cross-sectional area of cell compartments by the thickness of the sections (0.1/~m) estimated using the fold method ~2. For both quantitative autoradiographic and stereological analysis the same multipurpose test system was used: 40 circles, 40 points, distance of test points 28 ram. The silver grain density per,um2 of cell membrane surface or per ~m 3 of cell compartment volume was then calculated. Its value was used to calculate the a-ScTx binding site density from the formula given by Fertuck and SalpetereL The value obtained was corrected for the fact that only silver grains within a distance of 170 nm from the membrane were considered (69%), and for the percentage occupancy of czScTx sites: the latter was estimated to be 80% from

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& Fig. 1. Differentiated mouse neuroblastoma clone NIE 115. Total binding experiment, x 75C)0. N u m e r o u s silver grains (arrows) cain be seen over the plasma m e m b r a n e of the cell body (B) and of the cell processes (P). N u m e r o u s microvilli protruding trom the cell bodies (1~) and the cell processcs (L>) can be observed. (Monocrystalline emulsion of Ilford I. 4 . 4 5 days exposure using Microdol X as the developer).

12 saturation experiments done in the same culture conditions. The efficiency of the llford 1.4 emulsion was specified as 15% (M. A. Williams, personal communication). About 1000 perpendicular distances bet~.een the center of silver grains and the cell mctnbrane were measured at high magnification (x 911,0011), and plotted as a histogram ~1. RESUI/IS

Fig. 1 shows differentiated neuroblastoma cells processed for autoradiography (Total binding experiment). Numerous silver grains can be observed over plasma membrane of cell bodies and cell processes. ['he total number of sider grains counted was about 10× higher than the background density: 1.52 vs (I. 14 silver grains per 100 l('m 2 of tissue section. More silver grains (× 1.63) were detected in the cultures incubated with iodinatcd a-ScTx (Total binding: specific plus non-specific binding) than in those with iodinated plus native a-ScTx (non-specific binding) (l-able 1). A comparison, using the chi square test, of the number of silver grains and the number of (reguhtrlv distributed) systematic circles falling over each culture compartment revealed a significantly higher

chi square value in the total binding experiment, l'hc difference could be attributed to all p l a s m a n'~embrane-containing compartmenls in which the numbe~ of silver grains exceeded the normalized nmnber ol systematic circles sampled, thus detnonstratmg ',t marked concentration of labelled c,-,ScIx in these cotnpartments. On the contrary, no difference was detected between the silver grains and the %slen'lalic circle distribution in the non-specific binding experiment, which showed a random distribution amongst all cell culture compartments, i.e. the nutnber of silver grains encountered was only related I~, the crosssectional area of each compartment. Estimation of ct-ScTx binding site density was calculated using data from the circle method amdysis and a stereological study (Table II). If cell men> branes were divided into two compartments only (cell bodies and cell processes), no marked differcncc was observed in the density of specific binding sites (total binding sites minus non-specific binding sites): 6.4 vs 7.4 perlzm z. Llowcvcr, an important difference was revealed by separating in each cell con> partmcnt the smooth region of the membrane l rom the microvilli linked to it. Microvilli, which appeared as thin but fairly long digitations from both cell bodies and processes (Fig. 1). rcpresentcd abont two

TABLE I Circle analysis. Silver grains falling over cell c o m p a r t m e n t s were sampled using circles concentric to them. The circle diameter (3.8 mm) was chosen to give a 50% probability that the radioactive source producing the silver grain lay inside the circle ~7. The localization

of 40 circles of the same diameter systematically distributed over each micrograph was also recorded using a transparent overlay. Comparisons between the number of silver grains and the number of systematic circles were made using the chi square tesl after normalizing the n u m b e r of circles for each cell c o m p a r t m e n t .

(ell compartment

Cell body cytoplasm Cell process cytoplasm Nucleoplasm Extracellular space Cell body plasma m e m b r a n e Cell body microvilli Cell process plasma m e m b r a n e Cell process microvilli Isolated microvilli Nuclear m e m b r a n e Total counted N u m b e r s of micrographs Chi square test

Total binding experiment

Non-specific binding experiment

Silver grains

Systernati( circles

Normalized circles

Silver grains

Systematic circles

Normali,.ed ctrcle.~

51 21 ~" 158 27 ~2 3t} 19 g "~ 412

518 93 ,"9 ,"~ 2220 60 ltJ8 37 46 53 53 348(I

61.33 11.01 34.57 262.83 7.10 12.79 4.38 5.45 6.27 ",-7 6._, 412

41 1 ~" 101 6 lt) 1

476 25 ~26 153g 52 22 ~ !H I :, 54 2 #'2

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13 TABLE II Estimation of ¢~-ScTx binding site density in differentiated neuroblastoma cells: the membrane area (j~m2) was calculated by a stereological method, the a-ScTx binding site density per j~m: of cell membrane arca was calculated from the formula given by Fertuck and Salpetcr 25. (Mean _+ S.D.: number of measurements 87 (total binding cxperimcnt), 63 (non-specific binding experiment). ('ell compartments

Total binding experiment

Cell body membrane Cell body microvilli Cell process membrane Cell process microvilli Nuclear membrane Total cell body membranes Total cell process membranes Total plasma membranes

Membrane area (~m 2)

Silver grains/.um: (x 10 2)

Sites: um 2 A

149+26 242+44 81+14 152+27 94+_17 391-+70 231_+42 757+134

18.04+ 3.24 5.7+1.0 25.52_+4.59 8.14_:1.5 47.65+8.59 15.1_+.2.7 12.45+2.26 3.9_+0.7 5.33+_0.96 1.7+0.3 22.74_+4.(18 7.2+1.3 25.05+4.53 7.9-'-1.4 20.47+3.67 6.5+1.2

Non.specific binding experiment

aSc T~ specific

Membrane area (l~m2)

Silver grains(urn2 (× 10 -2)

Site~. .urn2 B

binding sites (,/am: ) = A- B

149+__27 4 1"+ ,_7 17-__0.2 30_+6 100_18 560_+10 47_+9 633_+113

3.78+0.67 ."~. .28+0 . 41 5.58_+0.01 0 7.45+1.34 2.68+0.48 "~ + _.00_0.37 2.82_+0.51

1.,_0., 0.7+0.1 1.8-+0.3 I) 2.4z0.4 0.9+0.1 0.6-+0.1 0.9_+0.1

4 "~+ 1.0 7.4_+ 1.5 13.3--2.7 3.9=0.7

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6.4-,i.3 7.~, ± 1.4 5.6_+1.2

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Fig. 2. Histograms of perpendicular distances between the center of silver grains and the plasma membranes. (Number ol clas',e',: 31. class width: IlX) nm). Hatched columns represent the non-specific binding experiments. The central class (-50: ~-50 nm) correspoml,, to the plasma membrane and contains 179 measurements ( 18c;~ of the total 990 measurements plotted ).

14 linked to thc cell processes (× l.t)). The average value for the total plasma membrane was about 6 a-ScTx-specific sites per l~m:, resulting in a distance of about 400 nm between two sites, assuming a random distribution. No clusters of silver grains were observed even over microvilli. Thus. (t-Sc'Ix binding sites seemed to be randomly dispersed. Histograms of the perpendicular distances between the center of silver grains and the cell membrane confirmed the specific binding of (,-ScTx on membranes. In cultures incubated with iodinated a-ScFx alone, the histogram was bell-shaped (Fig. 2), with a maximum corresponding to a large number of silver grains ( 18~7~) distributed m the vicinity of the plasma membrane ( - 5 0 : + 5 0 nm). In cultures incubated with iodinatcd (,-Sc'Ix in the presence of an excess of native toxin, the histogram was very flat and showed no particular localization of silver grains, in agreement with data collected from the circle analysis (Table 1). DIS('USSION This report describes the first ultrastructural localization of wfltage-sensitive sodium channels in a cellular system in vitro using [leSl]a-ScTx as a specific molecular probc. The polypeptide scorpion toxin appeared to be a useful tool for histological studies. Tissue proccssing for histology kept thc polypeptide toxin in situ after binding to specific sites which cannot be done with non-peptide neurotoxins such as tetrodotoxin and saxitoxin. In our experiments, the loss of radioactivity during tissue processing was less than 10% of the total radioactivity bound to the cultured cells, A n o t h e r advantage of scorpion toxins is that they can be labelled with radioiodide to a high specific activity (2(X)0 Ci/mmol in this study) which allows their use in autoradiographic experiments. The only other probe which has been used before to localize sodium channels is an antibody against the tetrodotoxin binding protein 2a. Ultrastructural quantitative autoradiography gave data on localization of a-ScTx binding sites and allowed an estimation of their density. First. it is clear that the saturable binding of [12Sl]a-Sc'l'x to the N 115 cell line is related to the voltage-sensitive sodium channels since it is inhibited by depolarization of the cell membrane (data not shown) and it is absent in

the non-excitable neuroblastonra cell line N 1(J3~5.~: The specificity of the label was demonstrated by the low number of silver grains in neuroblastoma cells incubated with both iodinated toxin and :m excess of native toxin. The ratio between tire silver grain dcnstties in the two experimental conditions ~lotal bit> ding/non-specific binding) varied from "~-8,1 depending upon the membrane compartment considered. Similar values have been reported trom a light microscope autoradiographic study using another a-Sc'l'x (from Leiurus quinquestriams v e n o m ) i n a different neuroblastoma clone". Both circle analysis and the study of histograms of silver g r a i n - m e m b r a n e distances demonstrated that ,-ScTx-specific binding sites were located exclusiveIv on cell plasma membranes. No sites were detected on nuclear membranes nor elsewhere reside the cell. The silver grain density inside the cell (l!. 1 +_ 0.02 per um3, mean + S.D.) was not different from that in the extracellular space (0.08 _+ 0.(/l per um~). v,.ith the exception of cell process cytoplasm ((I. 1~ z 0.03 per umr). In the latter case, the higher silver grain densitv could be explained by the superposition ol scattered radioactive emissions from two near membrane sources. These results confirmed previously published data about the turnover of sodium channels using saxitoxin as molecular probe 4:. ()nly 1.3% of the saxitoxin binding sites have been removed from the cell surface during an incubation of 30 rain at 37 :'('. Internalization of Na ~ channels seemed to be a very slow phenomenon which cannot be observed after a short incubation with the ¢~-Sc'l'x. The microvilli of cell bodies carried more ~z-Scl'x sites than other cell body membranes. All the compartments containing microvilli were 'junctional compartments "sl and also contained small regions of "smooth' non-microvilli membrane. "l'he circle method, we used, leads to a slight overestimation of the number of silver grains attributed to thc microvilli themselves. But cell body microvilli carried about two times more a-ScTx sites than cell body membrane contrasting with the fact that cell process microvilli carried 3 x less ,-ScTx-specific sites than the cell process membrane itself. Previous studies have demonstrated exclusive or initial binding of various ligands to microvilli, for example immunoglobulins on B lymphocytes 3~,, Semliki Forest virus on B H K cells >~-~-~, insulin on lymphocytesL The histological

15 localization of few ligands on neuroblastoma cells has been previously reported. Fluorescence microscopy studies demonstrated that opiate receptors 25.27.2~ and concanavalin A receptors26 form immobile clusters on neuroblastoma cell surface without internalization. Light microscope autoradiographic studies have suggested x that there was no difference between the silver grain density over cell bodies and cell processes in neuroblastoma N 18 cells. This can be contrasted with a marked difference in cultured spinal cord neurons, in which the grain density was higher on cell processes than on cell bodies. Similar observations have been made in cultured embryonic nerve cells a. Electrophysiological and neurotoxin binding studies as well as cytochemistry also support the general hypothesis that Na + channel distribution is non-homogeneous along neuronal membranes (for review see ref. 40). The difference in ct-ScTx site density between neuroblastoma cell bodies and cell processes re-appeared when the contribution from the microvilli was omitted (see Table II), resulting in a ratio of 2.9 between the two main membrane compartments. An important question is to know whether the density of c~-ScTx binding sites is identical to the density of Na + channels. An estimation of the stoichiometry between a-ScTx- and saxitoxin binding sites has given a ratio of 1 to 3 in the N 18 neuroblastoma cell line 15, whereas the ratio is 1 to 1 in reconstituted voltagesensitive Na + channels from rat brain a6. The affinity of (z-ScTx to its binding sites was higher in neuroblastoma cells than in reconstituted channels -~6. These results favor the hypothesis of the presence in neuroblastoma cells of two types of Na- channels, one bearing high affinity binding sites for ct-ScTx (1/3 of the Na* ionophores) and the other type low affinity a-ScTx binding sites which were not detectable (2/3), both types bearing high affinity binding sites for saxitoxin. According to this hypothesis, the total Na + channel density should be 3 x higher than the density of ct-ScTx binding sites, ct-ScTx being a marker of a subclass of Na + channels. In an alternative hypothesis, each ct-ScTx binding site could be associated with 3 saxitoxin binding sites~5. This question is still open as it is not known if the stoichiometry of saxitoxin to ct-ScTx binding sites is the same in neuroblastoma cells as in other nerve cell membranes. Another difficulty in estimating the Na* channel

density arises from the fact that both biochemical and electrophysiological experiments underestimate the cell surface area, thus overestimating the N a - channel density. Geometrical approximations of cell shape neglect all cell surface irregularities whereas the present study and other stereological experiments 4s show that there is a ratio of about 3.5 to 1 between the total membrane area and the membrane area excluding microvilli or other membrane folds which cannot be detected by light microscopy. After correction for cell surface underestimation, the density of ct-ScTx binding sites in neuroblastoma N 18 clone 14. in neuroblastoma N 115 clone (F. Couraud. unpublished results), and the density of Na- ionophores in N 115 neuroblastoma cells as estimated patch-clamp experiments 37 were in the same range as that reported in the present study. Furthermore: electrophysiological studies can only analyze open Na* channels for example after batrachotoxin binding37. Finally it is important to know if the low density in ct-ScTx binding sites as reported in the paper is compatible with cell excitability. Previous studies using N 115 neuroblastoma clone have demonstrated that differentiated cells exhibit the electrical properties of excitable cells3.31,a4 (and review in ref. 43). However, the neuroblastoma cell population is heterogeneous with regards to both morphological and electrophysiological properties. Even if all cells are able to be activated using neurotoxins such as c~-ScTx and bear high-affinity a-ScTx binding sites, only a few cells, the largest ones, show well-developed action potentials, the others being inexcitablc probably because they carry silent or latent sodium channels 3. Fhe (~-ScTx binding site density reported here represented an average value for the whole cell population. It seems to be likely that the a-ScTx site density of excitable neuroblastoma cells is higher than this average value. Moreover, the ct-ScTx binding site density reported here is in the same order of magnitude as the density of other Na" channel related neurotoxin binding sites in small non-myelinated nerve fibers 4~ and in dissociated fetal mouse brain cells ~ In conclusion, quantitative ultrastructural autoradiography using radio-iodinated ct-ScTx appeared to be a useful method with which to analyze the localization of Na + ionophores and to estimate their density in cell systems in vitro.

ACKNOWI.ED(iEMENTS

e m u l s i o n . W e t h a n k P r o t . B. l ) r o z tor helpful discussions.

T h e a u t h o r s t h a n k Dr. M. S e a g a r for r e v i e w i n g the text. W e t h a n k P r o f . M. A . W i l l i a m s for advice a b o u t

This

work

was

supported

by

(iranls

I)(IRSI

81 1 571 a n d I N S E R M C R I , 81 60(19.

m e t h o d s a n d the e f f i c i e n c y o f the a u l o r a d i o g r a p h i c

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