Tetrodotoxin-Sensitive Sodium Channels in Normal Human Fibroblasts and Normal Human Glia-Like Cells (Sodium Ionophore/Veratridine/Scorpion Toxin) ROBERT MUNSON, JR
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Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6425-6429, December 1979 Cell Biology Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells (sodium ionophore/veratridine/scorpion toxin) ROBERT MUNSON, JR. *, BENGT WESTERMARKt, AND LUIS GLASER* *Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 63110; and tThe Wallenberg Laboratory, University of Uppsala, Uppsala, Sweden Communicated by William D. Phillips, September 10, 1979 ABSTRACT Tetrodotoxin-sensitive sodium channels are MATERIALS AND METHODS detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in Normal human lung fibroblasts (line IMR 91, Institute for established neuronal cell lines in culture. Two- to 3fold stimu- Medical Research, Camden, NJ) and normal human dermal lations of sodium influx are observed in the presence of 0.2 mM fibroblasts initiated from skin biopsies were obtained from W. veratridine and scorpion venom at 0.1 mg/mi. Tetrodotoxin (2 Sly (Washington University School of Medicine). Cultures were MM) inhibits the observed stimulation of sodium influx. Previous grown in minimal essential medium containing 15% fetal bo- work has indicated that these neurotoxins act on the voltage- vine serum, glutamine (0.1 mg/ml), sodium pyruvate (0.11 sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable mg/ml), and 100 units of penicillin and 100 jig of streptomycin raises questions regarding both the uniqueness of this ionophore per ml. Normal human glia-like cells (CG series) were isolated as a property of excitable cells and the origin of the cells gen- as described by Ponten and MacIntyre (5). Glial cells were erally described as fibroblasts. grown as above without pyruvate and with the substitution of 10% calf serum for the fetal calf serum. 3T3 and simian virus Neuronal cells are characterized by the presence of an action- 40-transformed 3T3 cultures were grown in Dulbecco's mod- potential Na+ ionophore or voltage-dependent Na+ channel. ified Eagle's medium containing 10% calf serum. C6 glioma This channel is by definition the structure responsible for the and N18 neuroblastoma cells were grown in this medium increase in permeability to Na+ during the depolarizing phase containing 10% fetal bovine serum. All cultures were supple- of the nerve action potential. This ionophore can be activated mented with glutamine and antibiotics as described above. by veratridine and synergistically with veratridine by scorpion Sodium uptake assays were performed as described by toxin. This activation is specifically blocked by the addition of Stallcup and Cohn (6) with the addition of bovine serum al- tetrodotoxin. The presence of this ionophore can be determined bumin (1 mg/ml) and scorpion (Lelurus quinquestriatus) electrophysiologically or by measurements of Na+ influx into venom (0.1 mg/ml) (Sigma) (7). Assays were routinely carried cells stimulated by veratridine (with or without the addition out on confluent cultures in 35-mm Falcon tissue culture dishes. of scorpion toxin) and by the inhibition of this effect by tetro- Uptake was terminated after 5 min, unless otherwise indicated, dotoxin. Where it has been examined, there has been an ex- by four washes at room temperature with the sodium-free wash cellent correlation between the presence of the Na+ ionophore medium described by Catterall (8). detected electrophysiologically and the measurements obtained Protein was determined by absorbance at 230 and 260 nm by veratridine stimulation of Na+ uptake (1, 2). as described by Kalb and Bernlohr (9) after solubilization of the We and others have therefore been interested in using such cell layer with 1% sodium dodecyl sulfate or by the method of measurements as a criterion for the presence of neuronal cells Lowry et al. (10). or neuronally derived cells in culture. In addition to muscle RESULTS cells, only two examples of non-neuronal cells with tetrodo- toxin-sensitive channels have been reported in the literature. Addition of veratridine and scorpion venom to IMR 91 cells, The Na+ ionophore has been detected in secretory cells from a normal human lung fibroblast cell line, in the presence of the pancreas (3), and voltage-independent, veratridine-stim- ouabain resulted in an increased rate of Na+ entry (Fig. 1). This ulated Na+ channels were detected in squid Schwann cells (4). surprising observation is not a unique property of IMR 91 cells. As part of our investigation we examined various cultured cells Veratridine and scorpion toxin also stimulated the entry of Na+ for the presence of the Na+ ionophore. In addition to con- into three other human fibroblast lines (Fig. 2). In all cases, the firming the results of others that several established non-neu- effect was abolished by tetrodotoxin, a specific inhibitor of the ronal lines do not contain the Na+ ionophore, we were surprised Na+ ionophore. By contrast, Swiss 3T3 cells, simian virus 40- to find this ionophore in a number of cell lines described as fi- transformed 3T3 cells, and C6 cells (rat glioblastoma) did not broblasts as well as in normal human "glia-like" cells when show this response; N18, an established neuronal cell line, assayed with the combination of veratridine and scorpion toxin. showed a strong response to the addition of veratridine and These results raise important questions about the uniqueness scorpion toxin (Fig. 3). We have also detected low levels of of this ionophore for excitable cells as well as about the possible stimulated Na+ influx in secondary chick embryo fibroblasts of these (data not shown). origin cells. Stallcup (11) has classified Na+ channels (Na+ ionophores) in cultured cell lines into three categories based on their re- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "ad- sponse to the combination of scorpion toxin and veratridine and vertisement" in accordance with 18 U. S. C. §1734 solely to indicate their sensitivity to tetrodotoxin inhibition. Type B and type C this fact. channels show poor response to veratridine alone but a marked 6425 Downloaded by guest on September 28, 2021 6426 Cell Biology: Munson et al. Proc. Natl. Acad. Sci. USA 76 (1979) Z-C ~0 E C eC z 5 10 Time, min FIG. 1. Time course of 22Na uptake by IMR 91 fibroblasts. Confluent cultures (6.8 X 105 cells per dish; 0.3 mg protein) on 35-mm Falcon tissue culture dishes were assayed for Na+ influx in the absence FIG. 3. 22Na influx in neuronal and non-neuronal cell lines. of neurotoxins (0) or in the presence of 0.2 mM veratridine and Confluent cultures in 35-mm dishes were assayed as described in the scorpion venom (0.1 mg/ml) (0) for the indicated times. After four text. Symbols are as in Fig. 2. The cell density was as follows: estab- washes with sodium-free medium (8), the cell layer was dissolved with lished neuronal cell line (N18), 1.3 X 106 cells per dish; rat glioblas- 1% sodium dodecyl sulfate and assayed in a gamma counter. toma (C6), 2.8 X 106; Swiss 3T3 (3T3), 4.3 X 105; and simian virus 40-transformed 3T3 (SV3T3), 2.7 X 106. response to the combination of veratridine and scorpion venom. The Km-for veratridine in type C channels is independent of high basal influx rate, we have not attempted to determine an scorpion venom concentration; the Km in type B channels can apparent Km for veratridine with scorpion venom at less than decrease as the concentration of venom is increased. Type B and 25 Mg/ml. Tetrodotoxin sensitivity of the IMR 91 channel is type C channels are also distinguished by tetrodotoxin sensi- shown in Fig. 6. Half-maximal inhibition of stimulated uptake tivity. Type B channels exhibit an average Ki of approximately occurred at 7 X 10-8 M tetrodotoxin. 3 X 10-8 M whereas type C channels are approximately 1/30th The results of Catterall (12) suggest that the methods used as sensitive to the drug. Type B channels have been described to measure Na+ influx underestimate the true Na+ perme- for a number of cell lines classified as neuronal (11). ability, particularly with respect to the maximally stimulated The Na+ channel in cell line IMR 91 most closely resembles the type B channel as described by Stallcup. The data in Fig. 4 indicate an apparent Km of 22 Atg/ml for scorpion venom (at 200 tiM veratridine). Apparent Km values of 37 ,uM (at 25 ,ug 150 of scorpion venom per ml) and 29 MiM (at 100Mg of scorpion 0.7 venom per ml) were found for veratridine (Fig. 5). Due to the C 100 5 00 E - CE C O 0~~~~~~~~~~~~~~0 Scorpion toxin,,ug/ml FIG. 2. 22Na uptake by several normal human fibroblast lines, FIG. 4. Determination of apparent Km for scorpion venom. IMR measured for 5 min as described in Fig. 1. O, No toxin addition; m, 0.2 91 cultures were assayed for Na+ influx as described in Fig. 1. Mea- mM veratridine; *, veratridine plus scorpion venom (0.1 mg/ml); 0, surements were made at 5-min time points in the presence of 0.2 mM both stimulatory toxins plus 2 ,uM tetrodotoxin. The cell number and veratridine and the indicated levels of scorpion venom. In the absence protein content were: IMR 91, 8.2 X 105 cells per dish, 0.28 mg; S468, of scorpion venom, 67 nmol of Na+ was taken up and this value has 7 X 105 cells per dish, 0.29 mg; S471, 3.1 X 105 cells per dish, 0.18 mg; been subtracted from the data presented.