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Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells
Neuroscience Letters, 77 (1987) 298 302 Elsevier Scientific Publishers Ireland Ltd
298
NSL 04637
Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells V. Bolotina, J. Boreck~,, V. Vlachov~, M. Baudy~ov~i and F. Vysko~il Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 15 December 1986; Revised version received and accepted 2 March 1987)
Key words: Chloride channel; High conductance; Substate; Neuroblastoma cell; Patch clamp Single channels in mouse neuroblastoma cells with a high condlaetance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels we~ selective for C1- as compared to cations and exhibited a linear I-V relationship between +40 and - 4 0 mV. These CI- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of CI- channels described was the dissipation of their conductance into several substates during the course of individual recordings.
Several types of voltage-dependent ionic channels have already been observed in a variety of cells using the patch-clamp technique. The highest unit conductance, of about 400 pS, was reported for CI- channels in rat myotubes [2], macrophages [3, 8], molluscan neurones [4] and epithelial cells [7]. The aim of our work was to ascertain whether neuroblastoma cells which are commonly used for ionic permeability studies also possess CI- channels and whether their features are similar to those observed in other preparations. The experiments were performed on mouse neuroblastoma clone E7 cells cultivated for 2-5 days [1, 9]. The patch clamp technique [5] was used to record the currents from single channels in the inside-out configuration. The currents were digitized from magnetic tape records into five 120-points data blocks at a sampling rate of 2.5 kHz and then processed by heuristic level recognition programme on DECLAB 11/03 laboratory computer. Different bathing solutions were used. The basic solution contained 140 mM KCI, Ca 2+ buffered by EGTA to 0.1 /tM, 10 mM HEPES. In the other solutions, KC1 was replaced by (a) 140 mM NaCI; (b) 140 mM choline chloride; (c) 70 mM KC1 and 70 mM Tris adjusted to pH 7.4 with HC1 (Tris/HC1) Correspondence: F. Vysko6il, Institute of Physiology, Czechoslovak Academy of Sciences, Prague 4, Videfiskfi 1083, Czechoslovakia. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
300
K + channels and - 4 . 6 mV for C1- selective channels. In the experiment presented in Fig. 1A, Erev shifted to - 2 . 9 mV, which is near the expected Erev for CI- ions. The substitution experiments indicated that cations do not contribute significantly to single channel currents. No substantial changes in g and Erev of the currents were observed when the KCI in the bath solution was replaced by 140 mM NaCI (compare, for example, circle curves in Fig. 1A,B). Moreover, when the cations in the bath solution were replaced by 140 mM choline chloride (see experiment presented in Fig. 1B), g and Erev of the high conductance channels were almost unchanged. These findings therefore indicate that the channels were preferentially selective for C1- and not to K + and Na+. These C1- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. Typically, the channels opened immediately after a potential step. Following depolarization, they remained open for a few seconds (in some experiments only for tens of milliseconds)
200ms pA
B°[ -10
C
-10 Fig. 2. Single channel currents recorded from t h e inside-out patch in 140 m M KCI bath solution at - 2 0 mV on the membrane. A, B - 1 min and C, D 30 min after the formation of the inside-out patch.
301
N 150 ~
100-
SO-
o
g,pS 0
100
200
300
400
500
Fig. 3. The multiplicity of C1 channel substates. Histogram of different conductance substates at - 2 0 mV for the patch described in Fig. 2C,D. Abscissa: conductance g in pS, ordinate: N, number of events, showing how m a n y times the channel enters the substate with the same conductance. Total time of the recording was 8 s. The signal was passed through a gaussian digital filter.
and then spontaneously closed entering the inactivated state. Hyperpolarization activated these channels for a much longer time; they remained open for tens of seconds up to minutes. The most interesting feature of the CI- channels observed was the gradual dissipation of their high conductance level into several substates in the course of an individual recording. This is illustrated in Figs. 2 and 3. Fig. 2 shows single channel currents at - 20 mV on the membrane immediately after the formation of an inside-out patch (traces A and B) and 30 rain after (traces C and D). In the former case (A, B) the channel was mostly fully opened and had a maximum conductance of 430 pS. Thirty rain later (C, D) several intermediate substates with different conductances appeared. From the amplitude histogram of the channel conductance (Fig. 3) at least 7 substates could be distinguished in this case. In other experiments 6-16 substates were commonly observed. A question remains, as to whether this type of channel is protein or lipid in nature. Their high conductance of about 400 pS, the non-specific appearance in many types of cells, and partial permeability to cations [8], may point to a phospholipid 'leakage' pore [6]. However, their potential dependence and the existence of substates favours a protein structure. It is not known yet whether the individual channels have a high conductance, or whether the responses represent the synchronous activity of a cluster of smaller channels and the appearance of substates represents their desynchronization [4]. The authors are grateful to Drs. Ruth Payne and Pavel Hnik for their helpful comments and to Mrs. Jarmila H)2ovfi for secretarial help.
302 1 Baudy~ov~i, M., Spurnh, V., Nebola, M., Vyklick~ Jr,, L. and Michl, J., Establishment of mouse neuroblastoma clone E-7 in serum-free medium, Physiol. Bohemoslov., 33 (1984) 155 162. 2 Blatz, A.L. and Magleby, K.L., Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle, Biophys. J., 43 (1983) 237-241. 3 Galkin, A.A. and Malyaev, A.A., A single chloride channels of high conductance in the membrane of mouse peritoneal macrophages, Biol. Membr. (Russian), (1985) 1242-1247. 4 Geletyuk, V.I. and Kazachenko, V.N., Single C1- channels in molluscan neurones: multiplicity of the conductance states, Membr. Biol., 86 (1985) 9-15. 5 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 6 Kaufmann, K., Protonic control of ion transport through lipid bilayer membranes. A mechanism for acetylcholine-induced ion channels, Funkt. Biol. Med~, 4 (1985) 215-221, 7 Krouse, M.E., Schneider, G.T. and Gage, P,W., A large anion-selective channels has seven conductance levels, Nature (London), 319 (1986) 584~0. 8 Schwarze, W. and Kolb, M.A., Voltage-dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes, Pfliigers Arch., 402 (1984) 281-291. 9 Vyklick~ Jr., L., Michl, J., Vlachov~i, V., Vyklick~, L. and Vysko(:il, F., Ionic currents in neuroblastoma clone E-7 cells, Neurosci. Lett., 55 (1985) 197 201.
298
NSL 04637
Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells V. Bolotina, J. Boreck~,, V. Vlachov~, M. Baudy~ov~i and F. Vysko~il Institute of Physiology, Czechoslovak Academy of Sciences, Prague (Czechoslovakia) (Received 15 December 1986; Revised version received and accepted 2 March 1987)
Key words: Chloride channel; High conductance; Substate; Neuroblastoma cell; Patch clamp Single channels in mouse neuroblastoma cells with a high condlaetance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels we~ selective for C1- as compared to cations and exhibited a linear I-V relationship between +40 and - 4 0 mV. These CI- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of CI- channels described was the dissipation of their conductance into several substates during the course of individual recordings.
Several types of voltage-dependent ionic channels have already been observed in a variety of cells using the patch-clamp technique. The highest unit conductance, of about 400 pS, was reported for CI- channels in rat myotubes [2], macrophages [3, 8], molluscan neurones [4] and epithelial cells [7]. The aim of our work was to ascertain whether neuroblastoma cells which are commonly used for ionic permeability studies also possess CI- channels and whether their features are similar to those observed in other preparations. The experiments were performed on mouse neuroblastoma clone E7 cells cultivated for 2-5 days [1, 9]. The patch clamp technique [5] was used to record the currents from single channels in the inside-out configuration. The currents were digitized from magnetic tape records into five 120-points data blocks at a sampling rate of 2.5 kHz and then processed by heuristic level recognition programme on DECLAB 11/03 laboratory computer. Different bathing solutions were used. The basic solution contained 140 mM KCI, Ca 2+ buffered by EGTA to 0.1 /tM, 10 mM HEPES. In the other solutions, KC1 was replaced by (a) 140 mM NaCI; (b) 140 mM choline chloride; (c) 70 mM KC1 and 70 mM Tris adjusted to pH 7.4 with HC1 (Tris/HC1) Correspondence: F. Vysko6il, Institute of Physiology, Czechoslovak Academy of Sciences, Prague 4, Videfiskfi 1083, Czechoslovakia. 0304-3940/87/$ 03.50 © 1987 Elsevier Scientific Publishers Ireland Ltd.
300
K + channels and - 4 . 6 mV for C1- selective channels. In the experiment presented in Fig. 1A, Erev shifted to - 2 . 9 mV, which is near the expected Erev for CI- ions. The substitution experiments indicated that cations do not contribute significantly to single channel currents. No substantial changes in g and Erev of the currents were observed when the KCI in the bath solution was replaced by 140 mM NaCI (compare, for example, circle curves in Fig. 1A,B). Moreover, when the cations in the bath solution were replaced by 140 mM choline chloride (see experiment presented in Fig. 1B), g and Erev of the high conductance channels were almost unchanged. These findings therefore indicate that the channels were preferentially selective for C1- and not to K + and Na+. These C1- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. Typically, the channels opened immediately after a potential step. Following depolarization, they remained open for a few seconds (in some experiments only for tens of milliseconds)
200ms pA
B°[ -10
C
-10 Fig. 2. Single channel currents recorded from t h e inside-out patch in 140 m M KCI bath solution at - 2 0 mV on the membrane. A, B - 1 min and C, D 30 min after the formation of the inside-out patch.
301
N 150 ~
100-
SO-
o
g,pS 0
100
200
300
400
500
Fig. 3. The multiplicity of C1 channel substates. Histogram of different conductance substates at - 2 0 mV for the patch described in Fig. 2C,D. Abscissa: conductance g in pS, ordinate: N, number of events, showing how m a n y times the channel enters the substate with the same conductance. Total time of the recording was 8 s. The signal was passed through a gaussian digital filter.
and then spontaneously closed entering the inactivated state. Hyperpolarization activated these channels for a much longer time; they remained open for tens of seconds up to minutes. The most interesting feature of the CI- channels observed was the gradual dissipation of their high conductance level into several substates in the course of an individual recording. This is illustrated in Figs. 2 and 3. Fig. 2 shows single channel currents at - 20 mV on the membrane immediately after the formation of an inside-out patch (traces A and B) and 30 rain after (traces C and D). In the former case (A, B) the channel was mostly fully opened and had a maximum conductance of 430 pS. Thirty rain later (C, D) several intermediate substates with different conductances appeared. From the amplitude histogram of the channel conductance (Fig. 3) at least 7 substates could be distinguished in this case. In other experiments 6-16 substates were commonly observed. A question remains, as to whether this type of channel is protein or lipid in nature. Their high conductance of about 400 pS, the non-specific appearance in many types of cells, and partial permeability to cations [8], may point to a phospholipid 'leakage' pore [6]. However, their potential dependence and the existence of substates favours a protein structure. It is not known yet whether the individual channels have a high conductance, or whether the responses represent the synchronous activity of a cluster of smaller channels and the appearance of substates represents their desynchronization [4]. The authors are grateful to Drs. Ruth Payne and Pavel Hnik for their helpful comments and to Mrs. Jarmila H)2ovfi for secretarial help.
302 1 Baudy~ov~i, M., Spurnh, V., Nebola, M., Vyklick~ Jr,, L. and Michl, J., Establishment of mouse neuroblastoma clone E-7 in serum-free medium, Physiol. Bohemoslov., 33 (1984) 155 162. 2 Blatz, A.L. and Magleby, K.L., Single voltage-dependent chloride-selective channels of large conductance in cultured rat muscle, Biophys. J., 43 (1983) 237-241. 3 Galkin, A.A. and Malyaev, A.A., A single chloride channels of high conductance in the membrane of mouse peritoneal macrophages, Biol. Membr. (Russian), (1985) 1242-1247. 4 Geletyuk, V.I. and Kazachenko, V.N., Single C1- channels in molluscan neurones: multiplicity of the conductance states, Membr. Biol., 86 (1985) 9-15. 5 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 6 Kaufmann, K., Protonic control of ion transport through lipid bilayer membranes. A mechanism for acetylcholine-induced ion channels, Funkt. Biol. Med~, 4 (1985) 215-221, 7 Krouse, M.E., Schneider, G.T. and Gage, P,W., A large anion-selective channels has seven conductance levels, Nature (London), 319 (1986) 584~0. 8 Schwarze, W. and Kolb, M.A., Voltage-dependent kinetics of an anionic channel of large unit conductance in macrophages and myotube membranes, Pfliigers Arch., 402 (1984) 281-291. 9 Vyklick~ Jr., L., Michl, J., Vlachov~i, V., Vyklick~, L. and Vysko(:il, F., Ionic currents in neuroblastoma clone E-7 cells, Neurosci. Lett., 55 (1985) 197 201.