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Journal of Physiology (1992), 445, pp. 537-548 With 5 figures Printed in Great Britain

POTASSIUM CHANNELS AND REGULATION OF PROLIFERATION OF HUMAN MELANOMA CELLS

BY B. NILIUS AND W. WOHLRAB* Erfurt, Institute of Medical Physiology, Nordhduser the Medical Academy From Strasse 74, 0- 5010 Erfurt, Germany, and the *Department of Dermatology, Martin Luther University Halle-Wittenberg, Krohmeyer Str8sse, 0-4010 Halle (Saale), Germany

(Received 15 April 1991) SUMMARY

1. Ion channels and their possible relation to cell proliferation have been studied in a human melanoma cell line (IGR 1). Membrane currents were recorded by the patch-clamp technique using the cell-attached, cell-free and whole-cell mode. Cell growth was monitored by counting the number of cells at different days after seeding and [3H]thymidine incorporation. 2. A voltage-dependent 10 pS non-inactivating potassium channel (delayed rectifier) is the most commonly observed ion channel in this type of human cell. The channel is active at the normal resting potential and can be blocked by tetraethylammonium chloride (TEA) and also by a membrane-permeable cyclic adenosine monophosphate (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, cyclic AMP). A second type of potassium channel shows properties similar to voltage-dependent A-type potassium channels with complete inactivation. 3. A voltage-independent, non-selective cation channel with a single-channel conductance of approximately 20 pS could be seen in only 8% of the patches. Its properties of modulation are still unknown. 4. The incidence of the 10 pS, non-inactivated potassium channel was maximal at the fourth day after seeding (in 89% of the patches) and was significantly reduced at the seventh day (in 35% of the patches). 5. [3H]thymidine incorporation is maximal at the third day after seeding and is reduced when cells are grown in the presence of TEA or cyclic AMP. This peak of maximal [3H]thymidine incorporation correlated with the incidence of noninactivated potassium channels. 6. In the presence of TEA or cyclic AMP, growth of the cells is inhibited. We suppose that due to block of potassium channels, most of the melanoma cells are not able to enter the S-phase in the cell division cycle. 7. It is concluded that delayed rectifier potassium channels are involved in the control of melanoma cell proliferation. A similar finding has been reported for K+ channels in T-lymphocytes and human breast carcinoma cells. It is suggested that potassium channels may be involved in controlling the driving force for a calcium influx thereby interacting with Ca2+-dependent cell cycle control proteins. MS 9301

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B. NILIUS AND W. WOHLRAB INTRODUCTION

The control of proliferation of cells is believed to depend mainly on external signals such as growth factors and internal signals that modify the activity of cell cycle control proteins. Up until now, there have been few if any indications that ion channels can be critically involved in mechanisms leading to proliferation and differentiation of cells, although there is much evidence that at least calcium plays a dominant role in the control of the cell cycle (see Whitaker & Patel, 1990, for a review). An example has been given in T-lymphocytes where the density of functional potassium channels varies consistently with the degree of maturation and proliferation. Biological activation of these cells via antigenes or mitogenic lectines such as concanavalin A also activates these potassium channels (see Lewis & Cahalan, 1988, for a review). We have recently shown that a cell line derived from human melanocytes expresses voltage-dependent potassium channels (Nilius, Bohm & Wohlrab, 1990). In the melanoma cell line, IGR 1, the described 10 pS channel is thought to be modulated via cyclic AMP. Because an increase in the level of cyclic AMP seems to be a biologically important event to start differentiation of the cells and to start the synthesis of melanin (Aubert, Lagrange, Rorsman & Rosengren, 1976; Slominski, Moelmann & Kuklinska, 1989), we have argued that the potassium channels could also be involved in processes of cell proliferation and differentiation. Here, we describe evidence that modulation of potassium channels of the melanoma cell line IGR 1 influences cell proliferation and the incorporation of [3H]thymidine into these cells. Potassium channels might be involved in the controlling or fine-tuning of the driving force for transmembrane calcium movement thereby modulating cell proliferation. METHODS

Cells and culture conditions

Experiments were performed on the permanent melanin-producing melanoma cell line IGR 1 (kindly provided by Professor H. Rorsman, Lund). These cells have stable properties in respect to biochemical analysis and morphology (Aubert & Chrieceanu, 1973; Aubert, Rouge & Galindo, 1984). Cells from the same frozen sample arrested in G1 were separated into two aliquots. One aliquot was used for counting of cell numbers and for electrophysiological experiments; the other was used for measuring [3H]thymidine incorporation. Cells were grown as a monolayer on plastic culture dishes in Eagle medium (MEM, SIFIN, Berlin, Germany) with addition of 12% fetal calf serum (Serumwerk, Dessau, Germany) and 2 mM-glutamine. Penicillin (200 units/ml) was added to the culture medium. For use in electrophysiological experiments, cells were washed several times with normal HEPES solution. [3H]Thymidine incorporation was measured by using a scintillation counter (Pharmacia, Wallac 1410, Stockholm, Sweden). A 4 h period of incubation with 74 kBq/ml [3H]thymidine ([6-3H]thymidine, specific activity 1030 GBq/mmol, Radioisotopes Prague, CFSR) was applied at days 2,4 and 8 after seeding. Thereafter, the cells were incubated with a 0-125 % solution of trypsin (SPOFA, Prague, CSFR) for about 10-15 min. The trypsination of the cells was stopped by addition of the used culture medium. Aliquots of cell suspensions were then pipetted onto filter dishes, the cells were washed with a solution of 150 mM-NaCl, 5 % trichloroacetic acid and methanol each for 5 min, and then the samples were put in scintillation vials. To these were added 3 ml methanol and 6 ml scintillation cocktail (40 g PPO (2,5-diphenyloxazole) + 01 g POPOP (1,4-bis-2-(5-phenyloxazolyl)-benzene) + 1000 ml toluene). The activity of the cells was measured in six different

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samples from a culture dish. Counts per sample were normalized according to the maximal number of counts in the controls with no treatment of the cells with a potassium channel blocker. For quantification of cell growth, we counted the number of cells within the culture dish before electrophysiological measurements. For monitoring of numbers of cells, a grid was defined as the area in which cells were counted by scanning the bottom of the dish. The method used allows a statistical analysis of the number of cells counted in a probe.

Electrophysiological studies Single- and whole-cell currents were measured by use of the patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The patch clamp device was standard and is described in detail elsewhere (Nilius et al. 1990). Single-channel activity in cell-attached and cell-free patches as well as whole-cell currents were recorded. Measurements were performed on cells which adhere to the bottom of the chamber. We found that the density of the currents measured was maximal 3 days after seeding. This corresponds with the maximal incorporation of [3H]thymidine. Most of the studies to characterize the channel were done on cells between 2 and 4 days old. In total, eightyfour cells were studied with stable giga-ohm seals. We always used a sampling rate of 1 kHz. Traces of currents were filtered with an eight-pole Bessel filter set to 0-5 kHz. 1024 samples per trace were taken (12-bit, 100 kHz A/D converter, Tecmar Labmaster, Stoelting, Chicago, USA) and stored in an IBM-compatible PC (Olivetti 240 M, Ivrea, Italy). Details of the experimental protocol are given elsewhere (Nilius et al. 1990). All experiments were done at room temperature.

Solutions and chemicals In most of the single-channel experiments on cell-attached patches, pipettes were filled with the following 'extracellular' solution (in mM): NaCl, 140; KCI, 4; CaCl2, 2-5; MgCl2, 0 5; glucose, 11; HEPES, 5; pH 7*4. In these experiments, a bath solution with the following composition was used (in mM): KCI, 140; MgCl2, 1; HEPES, 5; EGTA (ethylene glycol-bis(,-aminoethylether)N,N'tetraacetic acid), 0-1; pH 7-2 with KOH. The same extracellular solution as described was used in whole-cell experiments as external solution. In these experiments the internal solution in the pipette contained (in mM): KCI, 140; MgCl2, 5; HEPES, 5; EGTA, 2; ATP (adenosine triphosphate), 4; pH 7-2 with KOH. To block potassium channels, we used TEA (tetraethylammonium chloride, Fluka, Switzerland) at a concentration of 1, 10 and 20 mM. Block of wholecell potassium currents was performed by application of the membrane-permeable cyclic AMP analogue (8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate, called 'cyclic AMP' in the text; Sigma Chemicals, St Louis, USA) used at a concentration of 5 and 20 /SM. At 20 mM-TEA and 20juM-cyclic AMP, potassium currents nearly completely disappeared. For experiments on cell growth (counting of cells) and measurement of thymidine incorporation, cells were grown in the presence of 20 mM-TEA and 5 or 20 ,SM-cyclic AMP. Statistics Pooled data are given as means + S.E.M if not mentioned otherwise. For statistical significance we used an unpaired t test defining a level of significance for P < 0-05.

RESULTS

Ion channels in melanoma cells Human melanoma cells from the permanent cell line IGR 1 were investigated. The cells produce melanin and develop dendrite-like processes. When the synthesis of melanin starts (approximately between day four and five after seeding) dendrite-like processes grow out of the soma of the cell and the melanosomes can be detected. We have performed patch clamp experiments on these kind of cells between the 2nd and 9th day after seeding. Cells were attached to the bottom of a culture dish. If a resting cell in normal extracellular medium is studied without any experimental intervention, in approximately 90 % of all patches we see a steady-

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state activity of an ion channel that results in outward currents. This current shows a strong voltage dependence. Steps from negative holding potentials to positive test potentials increase the open probability of the channel as seen in Fig. 1 A. The channel has a conductance of approximately 10 pS (q = 10-2 ± 0-2 pS, n = 11) that can be A i (pA)

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Fig. 1. Voltage dependence of a potassium channel in human melanoma cells. A, response of single-channel activity to a voltage step from a holding potential of -100 mV to a test potential of +50 mV. The averaged current is from fifty-nine sweeps. B, instantaneous current-voltage relationship of single-channel currents as obtained from a single linear voltage ramp from -100 (also holding potential) to + 100 mV. The linearly fitted slope conductance is 10-5 pS. The extrapolated reversal potential is -80-1 mV (140 mM-NaCl in the pipette, cells were incubated in 140 mM-KCl solution to zero the membrane potential). C, current-voltage relationship for averaged currents as obtained from linear voltage ramps from -100 to + 100 mV (sixty-four ramps were averaged). The smooth line is fitted according to eqn (2). Potential for half-maximal activation (V.) in this example is 31-3 mV, and the slope parameter s is 15-8 mV, the reversal potential was held at -80-1 mV during the fitting. A single-channel conductance (g) of 79 pS was approximated from the fit. -

measured both from amplitude histograms or linear voltage ramps as shown in Fig. 1B. Conductance was calculated from linear fits with i =9(V-Vrev), (1) where g is the single-channel conductance and V, is the reversal potential. The Vrev of the unitary current is at -80-1 mV in Fig. 1 A indicating that the channel is rather selective for potassium, as will be shown later. Activation of the channel is voltage

ION CHANNELS AND CELL PROLIFERATION

541

dependent. From the response to linear voltage ramps, averaged currents can be constructed that reflect macroscopic current-voltage relationships (I-V curves). Such an I-V curve is shown in Fig. 1 C. It is described by I = Npi, (2a) where N is the number of channels in the patch (N = 1 in Fig. 1). p is the probability of the channels being open and can be written as a Boltzmann term by p = 1/[1i +exp (-(V-IV)/s], (2b) where V1 is the potential for half-maximal activation, and s is the slope parameter in millivolts. With eqns (1), (2a) and (2b) one obtains I = gN(V-Vrev)/[1 + exp (- (V- Vi)/s)]. (3) Equation (3) was used to fit the I-V curves. N and Vrev were obtained from ramp experiments and were held during fitting. Half-maximal activation was found at 28 + 5 mV and the slope parameter was 13-2 + 1-6 mV (n = 10). A single-channel conductance of 8-8 + 1-4 pS was obtained from these fits. Selectivity of the channel has already been tested (Nilius et al. 1990). Using the reversal potentials of single-channel currents, a selectivity ratio of the channel to sodium and potassium (PNa/PK) of 0-036 + 0-02 was measured (n = 7; for the methods used, see Nilius, 1990). Besides the 10 pS non-inactivating potassium channel, an inactivating A-type potassium channel was recorded that appeared with a much smaller incidence. The conductance of this channel was approximately 10 pS. It was activated at more negative potentials than the non-inactivating K+ channel (VI was at approximately 3 mV). A third channel found in melanoma cells was a non-selective cation channel. Figure 2 shows an example for steady-state currents at -40 (top, left) and + 20 mV (top, right). The channel opened without any stimulation. It could be found in only three out of thirty-five cell-attached patches, and showed long-lasting single-channel openings. I-V curves were fitted by a method described in detail elsewhere (Nilius, 1990). The channel depicted in Fig. 2 has a conductance of 18-7 pS. From four cells, the conductance of the single channel was 20-1 + 3-2 pS. We could not succeed in finding any tool to modulate the channel. The channel survives for a short time in excised patches, but as a rule it runs down in several minutes. In general, most of the non-selective cation channels are permeable to calcium. The channel described here could therefore provide a pathway for transmembrane Ca2+ influx driven along the electrochemical gradient for calcium. Modulation of the non-inactivating potassium channel The channel found in almost every patch was a 'delayed' rectifier potassium channel. Properties of the channel have been studied in thirty-seven cells. The incidence of the channel correlates with the age of the cells. If a stable seal had been formed channel incidence was greatest between days 2 and 4 and was reduced at day 7. Table 1 summarizes these results. The incidence gives the relative occurrence of the channel if a patch has been formed. P,/y gives the statistical significance between the incidence for day x and y

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as obtained from the t test. For this estimation, if a channel was detected it was given a value of 1 and if not a value of 0. In a few cells whole-cell measurement was performed to estimate the density of the channels. For steps from a holding potential of -70 mV to a test potential of +20 mV the maximal outward current was 632 + 56 pA (day 4, five cells) and 59 + 29 pA (day 7, three cells).

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V(mV) Fig. 2. Appearance of a non-selective cation channel in human melanoma cells. Top, traces at the top are selected from stationary test potentials at -40 mV (left) and + 20 mV (right). The completely different pattern of openings should be noted (excised patch, 140 mM-NaCl in the pipette, 140 mM-KCl in the bath). Amplitude histograms are from sixty-four sweeps at the same potentials and from the same patch as the traces shown on top. The histograms are normalized as probability density function (PDF) per pA and fitted by a Gaussian function. Bottom, current-voltage relationship obtained from the same patch shown on top of this figure. The smooth curves are fitted by the method for non-linear approximation of i-V curves used by Nilius (1990). The mean conductance of this non-selective cation channel was 18-1 +0-3 pS (n = 3).

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Modulation of the non-inactivating channel has already been demonstrated elsewhere (Nilius et al. 1990). Figure 3 gives a synopsis of modulation of the channel known for melanoma cells (Nilius et al. 1990). From experiments done in the wholecell configuration, it could be shown that the current is inhibited by tetraethylammonium (TEA) chloride (see also Nilius et al. 1990). Cell conductance was TABLE 1 Day after Incidence seeding (% of patches) P 2 0-75 (14/20) P2/4 = 0-35 P2/7 = 0-04 4 0-89 (17/19) P2/4 = 0 35 P4/7 < 0 01 7 0 35 (6/17) P4/7 =< 00-01 P2/7 04 The incidence gives the relative occurrence of the channel if a patch has been formed. the significance between the incidence for day x and y as obtained from the t test.

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measured between -20 and +70 mV by linear approximation of averaged I-V relationships. It decreased from 6 4 + 1P2 (n = 4) to 0 8 + 0 3 nS (n = 3) in the presence of 10 mM-TEA. At 1 mM-TEA in one experiment the cell conductance was 4 0 nS. At 20 mM-TEA the current was completely blocked. Using these data, a rough estimation of the Kd (dissociation constant) value for block by TEA is between 2 and 4 mm. As already reported, the delayed potassium rectifier is also blockable by ,adrenergic interventions. Figure 3 C shows an example in which after wash-in of 5 /,M-cyclic AMP (see Methods) there was a reduction of the whole-cell current to 14 %. After wash-out, the inhibition was nearly reversible. It faded within 25 min to 79 % of the initial control value. Modulation properties of the potassium channel are in agreement with those described in detail previously (Nilius et al. 1990).

Modulation of cell growth by block of potassium channels In several cells, channel expression changes with a stereotyped pattern during cell development. The data of Lewis & Cahalan (1988) provide some evidence that K+ conductance is involved in events which control proliferation of the cells. Because of the similarity of K+ channels found in melanoma cells with channels in Tlymphocytes, we have checked whether (i) [3H]thymidine incorporation into melanoma cells during growth can be influenced by interventions that modulate potassium channels, and (ii) proliferation of the cells can be altered by modulation of potassium channels. Figure 4 gives the time course of [3H]thymidine incorporation into melanoma cells. Incorporation was studied on three different days. [3H]thymidine incorporation is maximal in the cells studied between days 3 and 4 after seeding. It seems that most of the cells that were arrested in the Gl-phase of the cell cycle were in the S-phase of the cell division cycle 3 days after seeding. Seven days after seeding, we found no substantial [3H]thymidine incorporation. The cells now showed dendrite-like processes and synthesized melanin. Measured on the same days, we found that [31H]thymidine incorporation is

B. NILIUS AND W. WOHLRAB

544 A

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Fig. 3. Pharmacology of the non-inactivating potassium channels shown by whole-cell measurements. A, current-voltage relationship of whole-cell currents measured from melanoma cells. From a holding potential of -100 mV steps to the indicated potentials were applied. The smooth curve is from a polynomial fit. The straight line indicates the leakage current. B, potassium currents are blocked by 10 mM-TEA (same cell as in A). C, block of whole-cell outward currents by application of 5 /tM of a membrane-permeable analogue of cyclic AMP (bottom). The decrease after superfusion of the cell with cyclic AMP was nearly reversible (25 min wash) (test potential: +20 mV).

inhibited when the cells grow in the presence of the known potassium channel blockers, cyclic AMP and TEA (Fig. 5, right, top and bottom). When in another experiment cells were counted in the culture dishes, we saw an inhibition of the cell growth after application of both cyclic AMP and TEA (Fig. 5, left, top and bottom). With 5 LM-cyclic AMP (not shown) we have already found an inhibition of 0 11 + 0-05 (n = 3) of the control value without cyclic AMP treatment.

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B. NILIUS ANVD W. WOHLRAB DISCUSSION

Comparison of melanoma potassium channels with potassium channels in other nonexcitable cells Ion channels in pigmented cells have until now not been well characterized. A systematic search (via the 'Medline' information system) has only resulted in one paper that describes potassium channels in a melanoma cell line (MB-HM8, Zaharadnikova, Zaharadnik & Rydlova, 1988). In these cells a potassium channel has been shown that exhibited a conductance of 16 pS at positive but 37 pS at negative potentials as well as a maxi-K+ channel with a conductance of 240 pS. In keratinocytes, which are functionally related to melanocytes, calcium-dependent Clcurrents have been described (Mauro, Pappone & Isserhof, 1990). These currents seem to be related to the initiation of cell differentiation. In this paper we demonstrate two potassium currents: (i) a non-inactivating delayed potassium rectifier that has been described in detail elsewhere (Nilius et al. 1990), and (ii) a transient potassium current (A-current) that appears with a low incidence and is not described here in detail. In several non-excitable cells a mixture between both activating and non-inactivating potassium currents is observed, e.g. in pineal and pituitary cells (Lingle, Sombati & Freeman, 1986; Aguayo & Weight, 1988). Brown fat cells and also osteoclasts which are fast proliferating cells show a potassium channel with similar properties to the non-inactivating channel described here (Lucero & Pappone, 1989; Ravesloot, Ypey, Vrijheid-Lammers & Nijweide, 1989). The non-inactivating potassium current has a TEA sensitivity similar to ntype potassium channels in T-lymphocytes and also a similar single-channel conductance (12-18 pS) to n-type potassium channels in T-lymphocytes (Lewis & Cahalan, 1988). However, the potential of half-maximal inactivation, V1, is much more negative for n-, n'- and 1-type potassium channels in T-lymphocytes than in melanoma cells which indicates a big variety of potassium channels in non-excitable cells.

Possible functional significance of potassium channels For T-lymphocytes it has been shown that regulation of whole-cell potassium currents is involved in control of proliferation and differentiation (Lewis & Cahalan, 1988). Also in fibroblasts and muscle cells activity of ion channels seems to be involved in cell proliferation (Caffrey, Brown & Schneider, 1987, 1989; Chen, Corbley, Roberts & Hess, 1988). A 23 pS potassium channel has been described in a human breast carcinoma cell line (MCF-7). The incidence of this channel is also maximal in the exponential phase of cell growth (Wegman, Young & Cook, 1991). In this study, the potassium channel was not obligatory for cell division, but shows an apparent correlation to cell proliferation and could be a regulatory tool. Melanoma cells used in the present study are characterized by proliferation that shows a maximum on the 3rd day after seeding, paralleled by a maximum in the [H3]thymidine incorporation. We have demonstrated that application of potassium channel blockers such as TEA and membrane-permeable cyclic AMP (see Fig. 5) reduces both growth and [H3]thymidine incorporation. We cannot provide direct evidence that potassium channels are involved in biochemical processes that are

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specific for cell growth. Their role in the maintenance of a large driving force for calcium by hyperpolarizing the membrane potential may be crucial for proliferation. A possible pathway can be supposed from the cell cycle control points (Whitaker & Patel, 1990): the start of transition from G1 to S is initiated by calcium-dependent phosphorylation of the cell cycle control protein p34(cdc2). Our experiments show that [H13]thymidine incorporation is dramatically blocked by application of potassium channel blockers to the medium in which cells grow. This suggests a possible block of 'start S', an event that is triggered by an increased intracellular calcium concentration. A reduced driving force for Ca2+ could be effected by a block of potassium channels and therefore could prevent the cells from entering into the Sphase. For endothelial cells, it has been impressively demonstrated that the membrane potential and the resting level of free intracellular calcium can be regulated by potassium channels (Liickhoff & Busse, 1990). This finding is in agreement with the hypothesis discussed here. This work was supported by the Fritz Thyssen Stiftung (Grant IGRI, AZ 9 26/90). We are grateful to Frank Bretschneider for his help during analysis of cell growth. REFERENCES

AGUAYO, L. G. & WVEIGHT, F. F. (1988). Characterization of membrane currents in dissociated adult rat pineal cells. Journal of Physiology 405, 397-419. AUBERT, C. & CHRIECEANU, E. (1973). Cultures de melanomes humains: characterisation. Reviews of the Institute Pasteur Lyon 6, 265-273. AUBERT, C., LAGRANGE, C., RORSMAN, G, & ROSENGREN, E. (1976). Catechols in primary and metastatic human malignant cells in monolayer culture. European Journal of Cancer 12, 441-445. AUBERT, C., ROUGE, F. & GALINDO, J. R. (1984). Melanocytes and culture conditions. Journal of the National Cancer Institute 71, 3-9. CAFFREY, J. M., BROWN, A. M. & SCHNEIDER, M. D. (1987). Mitogens and oncogens can block the induction of specific voltage-gated ion channels. Science 236, 570-573. CAFFREY, J. M., BROWN, A. M. & SCHNEIDER, M. D. (1989). Ca2" and Na' currents in developing skeletal myoblasts are expressed in a sequential program: reversible suppression by transforming growth factor beta- 1, an inhibitor of myogenic pathway. Journal of NVeuroscience 9, 3443-3453. CHEN, C., CORBLEY, M. J., ROBERTS, T. M. & HESS, P. (1988). Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts. Science 239, 1024-1026. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp technique for high resolution current recording from cells and cell-free membrane patches. Pfiuigers Archiv 391, 85-100. LEWIS, R. S. & CAHALAN, M. D. (1988). The plasticity of ion channels: parallels between the nervous and immune systems. Trends in Neurosciences 11, 214-218. LINGLE, C. L., SOMBATI, S. & FREEMAN, M. E. (1986). Membrane currents in identified lactotrophs of the rat anterior pituitary. Journal of Neuroscience 6, 2995-3005. LUCERO, T. L. & PAPPONE, P. A. (1989). Voltage-gated potassium channels in brown fat cells. Journal of Physiology 93, 451-472. LUCKHOFF, A. & BUSSE, R. (1990). Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the membrane potential. Pfiiugers Archiv 416, 305-311. MAURO, T. M., PAPPONE, P. A. & ISSERHOF, R. R. (1990). Extracellular calcium affects the membrane currents of cultured human keratinocytes. Journal of Cell Physiology 143, 13-20. NILIUS, B.(1990). Permeation properties of a non-selective cation channel in human vascular endothelial cells. Eflugers Archiv 416, 609-61 1, 18-2

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NILIUS, B., B6HM, T, & WOHLRAB, W. (1990). Properties of a potassium-selective ion channel in human melanoma cells. Pflugers Archiv 417, 269-277. RAVESLOOT, J. H., YPEY, D. L., VRIJHEID-LAMMERS, T. & NIJWEIDE, P. J. (1989). Voltageactivated K+ conductances in freshly isolated embryonic chicken osteoclasts. Proceedings of the National Academy of Sciences of the USA 86, 6821-6825. SLOMINSKI, A., MOELMANN, G. & KUKLINSKA, E. (1989). MSH inhibits growth in a line of amelanotic hamster melanoma cells and induces increases in cAMP levels and tyrosinase activity without inducing melanogenesis. Journal of Cell Sciences 92, 551-559. WEGMAN, E. A., YOUNG, J. A. & COOK, D. I. (1991). A 23-pS Ca2+-activated K+ channel in MCF7 human breast carcinoma cells: an apparent correlation of channel incidence with the rate of cell proliferation. Pfligers Archiv 417, 562-570. WHITAKER, M. & PATEL, R. (1990) Calcium and cell cycle control. Development 108, 525-542. ZAHARADNIKOVA, A., ZAHARADNIK, I. & RYDLOVA, K. (1988). Single channel potassium currents in human melanoma cells. General Physiology and Biophysics 7, 109-112.