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Journal of Physiology (1990), 425, pp. 117-132 With figures Printed in Great Britain
TETRAETHYLAMMONIUM BLOCKADE OF APAMIN-SENSITIVE AND INSENSITIVE Ca2l-ACTIVATED K+ CHANNELS IN A PITUITARY CELL LINE
BY DANIEL G. LANG* AND AILEEN K. RITCHIE From the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77550-2779, USA
(Received 22 August 1989) SUMMARY
1. The pharmacological sensitivities and physiological contributions of two types of Ca2+-activated K+ channels (BK and SK) in GH3 cells were examined by the outside-out, whole-cell and cell-attached modes of the patch-clamp technique. 2. BK channels (250-300 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external tetraethylammonium (TEA) and by 50 nm-charybdotoxin (CTX), but were not blocked by apamin. 3. SK channels (9-14 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM-CTX. 4. The dissociation constant (Kd) for TEA block of SK channels (31 +0-37 mM) was 12-fold greater than the Kd for the BK channels (260 + 21 gM). The TEA blockade of both channels was not strongly voltage dependent; for both channels the TEA binding site sensed less than 20 % of the membrane electric field. 5. Application of blockers of the BK channels (1 mM-TEA and 50 nM-CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after-hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30 % of the spontaneously pacing cells. 6. It is suggested that BK channels contribute to action potential repolarization; whereas SK channels contribute to the regulation of action potential firing rate. INTRODUCTION
Numerous varieties of Ca2+-activated K+ channels have been described that differ widely in unit conductance, Ca2+ sensitivity, voltage sensitivity and pharmacology (Blatz & Magleby, 1987; Reinhart, Chung & Levitan, 1989). In view of this diversity in channel properties and its widespread occurrence in nearly all cell types, it is not surprising that Ca2+-activated K+ channels have been proposed to serve many different functional roles including repolarization of action potentials, regulation of resting membrane potential and spike discharge frequency, salt and water secretion, Present address: Department of Otolaryngology, The University of Texas Medical Branch, Galveston, TX 77550-2778, USA. *
MS 7896
D. G. LANG AND A. K. RITCHIE 118 and cell volume regulation. In order to assess the functional roles of these channels we have begun a pharmacological study, at the single-channel level, of two types of Ca2+-activated K+ channels that co-exist in the GH3 anterior pituitary cell line. The most extensively studied and widely occurring channel is the largeconductance BK channel (200-300 pS in 150 mM-symmetrical K+) that is activated by both internal calcium and depolarizing voltages (Blatz & Magleby, 1987). In GH3 cells, the BK channel requires high concentrations of internal Ca2+, much greater than 10 /M, for activation at -50 mV (Lang & Ritchie, 1987). However, the sensitivity to Ca2+ increases greatly as the cell is depolarized. This is similar to the calcium sensitivity of BK channels from skeletal muscle (Barrett, Magleby & Pallotta, 1982), but is nearly two orders of magnitude less sensitive to Ca2+ than the BK channels in rat lacrimal gland (Marty, Tan & Trautmann, 1984). In most (Blatz & Magleby, 1984; Iwatsuki & Petersen, 1985; Blatz & Magleby, 1987), but not all (Benham, Bolton, Lang & Takewaki, 1985; Wong & Adler, 1986), cells the BK channel is blocked by submillimolar concentrations of external TEA. The channel is also blocked by nanomolar concentrations of CTX (Miller, Moczydlowski, Latorre & Phillips, 1985; Reinhart et al. 1989), but is insensitive to apamin (Romey & Lazdunski, 1984; Pennefather, Lancaster, Adams & Nicoll, 1985; Reinhart et al. 1989). Far less is known about a distinctly different type of Ca2+-activated K+ channel of small unitary conductance (9-20 pS in 150 mM-symmetrical K+) that is referred to as the SK channel. This channel is activated by 1 /SM-internal Ca2+ at negative membrane potentials, exhibits a very weak voltage sensitivity (Blatz & Magleby, 1986; Lang & Ritchie, 1987), and is inhibited by apamin (Blatz & Magleby, 1986; Capiod & Ogden, 1989). The SK channel (Blatz & Magleby, 1986) and the apaminsensitive conductance in whole-cell recording (Romey & Lazdunski, 1984; Pennefather et al. 1985; Ritchie, 1987a, b) is reported to be relatively insensitive to inhibition by TEA. Its sensitivity to CTX is unknown. In this report, we confirm that SK channels in GH3 cells, like those described in rat skeletal muscle (Blatz & Magleby, 1986) and guinea-pig hepatocytes (Capiod & Ogden, 1989), are inhibited by apamin, we establish its insensitivity to CTX, and examine the mechanism and sensitivity of SK channel blockade by TEA. These results are compared with companion studies performed on the BK channels in GH3 cells. Finally, this information on pharmacological specificity is used to investigate the contribution of BK and SK channels to the duration, the after-hyperpolarization, and frequency of discharge of spontaneous action potentials in GH3 cells. A preliminary account of these studies has appeared (Lang & Ritchie, 1988). METHODS
Cell culture. The GH, cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). The GH3 cells were grown at 36 °C in a 5% CO2 atmosphere in Ham's F10 media supplemented with 15 % heat-inactivated horse serum and 2-5 % fetal calf serum. Cells used for electrophysiological experiments were plated on polylysine-coated glass cover-slips and were used within 2-10 days after plating. Cells from passages 27-40 were used in the experiments described in this paper. Electrophysiological recordings. The whole-cell, cell-attached and outside-out modes of the patchclamp technique were utilized in this study (Hamill, Marty, Neher, Sakmann & Sigworth, 1981).
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Electrodes used for cell-attached patches were made from Corning 7052 glass (Friedrick & Dimmock; Millville, NJ, USA) in two steps on a David Kopf 700C electrode puller. Electrodes used for outside-out patches were made from TW-150 glass (World Precision Instruments, New Haven, CO, USA) and were pulled in a single step. The electrodes were coated with Sylgard and firepolished. The resistances ranged from 12 to 20 Mg; however, electrodes pulled from TW- 150 glass had much smaller diameter bores at the tip than those pulled from 7052 glass. Electrodes used for whole-cell current-clamp studies were made with TW- 150 glass and had resistances of 2-5 MQ. Seal resistances were in the range of 40-90 GQ. Recordings were performed at room temperature with a List model EPC-7 patch-clamp amplifier (Darmstadt, FRG). Analysis. The data were stored on FM tape (Racal Store 4DS) with a frequency response of DC to 2-5 kHz (-3 dB, 4-pole Tchebychef). The single-channel records were filtered at 1 kHz (-3 dB, 8-pole Bessel) and digitized at a sampling rate of 10 kHz. The digitized single-channel data were analysed on a 80286 computer by using the ANALYSIS program developed by H. Affolter (courtesy of Dr Roberto Coronado). Single-channel amplitudes were measured directly from data segments displayed on the computer screen using cursors or from amplitude histograms generated by the ANALYSIS program. The amplitude histograms consisted of 256 bins with each bin containing the number of sample points falling within the bin width; the amplitude of the singlechannel current was taken as the difference between the peaks for opened and closed current levels. Channel amplitudes in the presence of TEA were obtained from cursor measurements of data segments displayed on the computer screen since the histograms tended to underestimate the amplitude during rapid flicker block. Channel openings were detected as events that fell within a window set by the user. The open probability was taken as the total time the current fell within the window divided by the total duration of the data segment. Patches that contained two channels were analysed in two passes. During the first pass, the window was set to include events when only one channel was open and when two channels were open simultaneously; during the second pass, the window was set to include only events when two channels were open simultaneously. The open probabilities from each pass were summed and divided by two to yield the average open probability for a single channel. We have verified amplitude and open-probability measurements obtained from the ANALYSIS program by comparing with results obtained by manual measurements from chart paper recordings of taped data replayed at 1/4 speed (effective bandwidth of 360 Hz). Dose-response curves were fitted to data points by a least-squares minimization algorithm (Minsq, Micromath Scientific Software, Salt Lake City, UT, USA). The goodness of fit is given by the coefficient of determination (CD). All currents and voltages for the single-channel records are expressed with the same polarity conventions as used in whole-cell voltage clamp. Membrane potentials are expressed with the intracellular relative to the extracellular side. Inward currents are shown to open in the downward direction and are defined as a positive charge moving from the extracellular to the intracellular side of the membrane. The closed state of the channel, except when obvious, is indicated by the letter 'C' and the open state by the letter 'O'. Solutions. The solutions used in the cell-attached-patch experiments were (mM): KCl, 150; MgCl2, 2; N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 10; 4-aminopyridine (4-AP), 5; pH 7 30 for the pipette and KCl, 150; CaCl2, 0 01; HEPES, 15; pH 7-50 for the bath. The solutions used in outside-out patch experiments were (mM): KCl, 150; MgCl2, 05; HEPES, 10; ethyleneglycol-bis(/J-aminoethylether)-N,N,',N'-tetraacetic acid (EGTA), 2; free Ca2 , 15 UM; pH 7 20 for the pipette; TEA-Cl, x; NaCl, 75-X ; KCl, 75; MgCl2, 2; HEPES, 10; 4-AP, 5; pH 7 30 for bath A; and KCl, 150; MgCl2, 2; HEPES, 10; 4-AP, 5; pH 730 for bath B. Free Ca2+ concentrations in the range of 1-3 /tM were buffered with 2 mM-EGTA and were calculated using an apparent Kd of 150 nm (Thomas, 1982). The Mg2+ concentration had a negligible effect on the buffered free Ca2+ concentration. This was verified by the method of Fabiato & Fabiato (1979) assuming an apparent Kd of EGTA for Mg2+ of 84 mm (Thomas, 1982). Calcium concentrations greater than 3 jtm were achieved by simply adding the desired amount of free Ca2+. The solutions used in whole-cell experiments were (mM): KCl, 150; HEPES, 3; pH 7-20 for the pipette and NaCl, 140; KCl, 5-6; CaCl2, 2; MgCl2, 1; HEPES, 10; glucose, 10; pH 7 30 for the bath. Apamin was obtained from Sigma Chemical Company (St Louis, MO, USA). Ionomycin (free acid) was obtained from Calbiochem (La Jolla, CA, USA). Charybdotoxin (CTX) was a generous gift from the laboratory of Dr Christopher Miller. Whole cells were perfused by pressure ejection (70 kPa) of solutions from a large-bore pipette (diameter 10-15 jum) placed 50-100 ,um from the cell. The cells were superperfused with normal
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D. G. LANG AND A. K. RITCHIE
saline during the control segments to reduce K+ accumulation. The effects of pharmacological agents on whole-cell activity were tested by first lowering a pipette containing saline plus drug into the bath near the cell, and then switching the pressure from the control pipette to the drug pipette. The control pipette was then raised out of the bath to prevent contamination of its contents by the drug. Excised, outside-out patches were placed in a small-volume recording chamber (approximately 0-2 ml) and perfused at a rate of approximately 1 ml/min. The solutions were gravity fed, and the solution changes required approximately 30 s to reach equilibrium. For the CTX experiments on outside-out patches, the perfusion was stopped and CTX was injected into the recording chamber. RESULTS
The effects of apamin, TEA and CTX on Ca2+-activated K+ channels in GH3 cells were mainly tested on outside-out patches. Since the internal face of the patch membrane was exposed to the pipette solution, the BK and SK channels could not be identified on the basis of sensitivity to changes in the internal free Ca2+ concentrations. However, the BK channels were readily identified on the basis of their large unitary conductance. The SK channels were identified by their small unitary conductance, lack of voltage dependence of the open probability, and by relatively long-duration openings at negative membrane potentials. In GH3 cells, the unit conductance of the SK channel in symmetrical 150 mM-K+ is 9-14 pS, and slightly smaller (about 6 pS) in experiments performed with 75 mM-external and 150 mM-internal K+. Infrequently, a channel with a similar unit conductance as SK channels was seen in inside-out patch experiments. This channel is insensitive to changes in internal calcium and at negative membrane potentials it exhibits very brief duration openings that occur in bursts. SK channels could be distinguished from the Ca2+-insensitive channels by differences in channel open durations at negative membrane potentials.
Apamin sensitivity The sensitivity of BK channels to inhibition by apamin was tested by using outside-out patches exposed to either 75 mM-external and 150 mM-internal K+ or symmetrical 150 mM-K+ gradients. Neither the single-channel amplitude nor the open probability was affected by 20 min incubations with 5 JM-apamin. Thus, apamin does not block the BK channels. The apamin sensitivity of the SK channels was tested by two different methods. First, the frequency of observing SK channels in cell-attached patches was compared without and with apamin in the patch pipette solution. In these experiments, the patch pipette contained 150 mM-K+ and was held at -50 mV so that unitary currents were in the inward direction. Calcium influx in the cell was stimulated by applying the Ca2+ ionophore ionomycin to the extrapatch, whole-cell membrane. Under these conditions, ionomycin causes a transient elevation of the intracellular free Ca2+ concentration. We have verified this by fluorescent measurements of cytosolic calcium levels in GH3 cells loaded with the calcium-sensitive dye Fura-2 (D. G. Lang & A. K. Ritchie, unpublished observation). In the absence of apamin, SK channel activity was stimulated by application of 50,uM-ionomycin (in 1% DMSO) in twelve out of sixteen patches (Fig. 1). Application of 1 % DMSO had no
121 TEA BLOCK OF BK AND SK CHANNELS effect. Stimulation of SK channel activity by ionomycin did not occur when 500 nmapamin was included in the pipette solution in eight out of eight patches. The second method was to test the effect of apamin on SK channel activity in outside-out patches. SK channel openings were observed at negative holding lonomycin
). The different symbols represent data obtained from five different patches containing BK channels and four different patches containing SK channels. The curves represent best-fit theoretical dose-response curves for a one-to-one drug-to-receptor binding scheme.
where i and ib are the unitary currents in the presence and absence of TEA, respectively. Bo and z are the blocker concentration and valency, respectively. The value of a can be determined from the slope, and the value of Kd(O mV) can be determined from the Y-intercept of the plot of ln (i/ib -1) against voltage (V). These plots are shown in Figs 3C and 4C. For both the BK and SK channels of GH3 cells, the block by external TEA was not strongly voltage dependent (6 < 0-2). The Kd(O mV)s for the BK channels and for the SK channels were 210 ,UM and 4-4 mM, respectively. We have also measured the dose-response characteristics of the block by external TEA by a different method. The concentration dependence of TEA inhibition was measured as the percentage reduction of the single-channel amplitude (Fig. 5). The continuous curves represent theoretical dose-response curves for a one-to-one drugto-receptor binding scheme: Percentage inhibition =1+Kd/B' where Kd is the dissociation constant and Bo is the concentration of external TEA. The theoretical dose-response curves were fitted to the data points by a least-squares minimization algorithm. The Kd values obtained from these fits, 260 + 21 ftM
126
D. G. LANG AND A. K. RITCHIE B
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80 s after apamin application
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Fig. 6. Effect of charybdotoxin on BK and SK channels. These outside-out patches were exposed to a symmetrical 150 mM-K' gradient (bath B). A, the BK channel activity in this patch was obtained with 3 gM-internal free Ca2+ at a holding potential of + 40 mV. Records are shown for control solution, in the presence of 50 nM-CTX, and after wash-out of CTX. The records were low-pass filtered at 1 kHz (-3 dB, 8-pole Bessel). B, the open probability for the channel in A is plotted against voltage for controls (@), in the presence of 50 nM-CTX (0), and after washing (A). CTX reversibly reduced the fractional open time of this BK channel but did not affect its unitary conductance (270 ps). C, the SK channel activity in this patch was obtained with 1 gM-internal free Ca2+ at a holding potential of -50 mV. In the control segment, the mean current level was approximately 2-5 pA indicating that at least five SK channels were present in the patch. The mean current remained high in the presence of 50 nM-CTX, although CTX may have blocked one of the five channels in this patch. In two other patches, 50 nM-CTX had no effect on SK channel activity. Apamin (500 nM) reduced the number of open SK channels and, after 80 s, completely blocked all SK channel activity in this patch. The records were lowpass filtered at 360 Hz (-3 dB, 8-pole Bessel).
TEA BLOCK OF BK AND SK CHANNELS
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(estimate + standard deviation; CD = 0-95) for the BK channels and 31+ 0-37 mm (CD = 0 95) for the SK channels, are in good agreement with that obtained from the Woodhull method. Charybdotoxin sensitivity The CTX sensitivity of the BK channels was examined by using outside-out patches exposed to a symmetrical 150 mM-K+ gradient with internal free Ca2+ A 0 mV-
CTX Control B 0 mV -
/Apamin Control I
20 mV
200 ms
Fig. 7. Effects of BK and SK channel blockers on action potentials. These currentclamped cells were continually superfused with either control saline or saline containing inhibitor. A, superfusion with 50 nM-CTX prolonged action potential duration, enhanced the slow after-hyperpolarization, and slowed the firing rate. B, application of apamin (500 nM) caused a decrease in the slow after-hyperpolarization and an increase in the spontaneous firing rate of this cell. Apamin had little effect on the duration of the action potential.
concentrations of either 1 or 3 /M. Charybdotoxin (50 nM), applied to the external face of the membrane patch, caused a reversible decrease of the open probability of BK channels but did not affect the single-channel amplitude (Fig. 6A and B). Thus, in contrast to the rapid flicker block by external TEA, the block of BK channels by CTX shows very slow kinetics. This inhibition of the BK channels was seen in all eight patches examined. The voltage dependence for the block by CTX was not investigated. It is concluded that the BK channels of GH3 cells are inhibited by CTX. The CTX sensitivity of the SK channels was also tested under a symmetrical 150 mM-K+ gradient with 1 /tM-internal free Ca2 . SK channel activity could be detected at a holding potential of -50 mV. Application of 50 nM-CTX had no effect on SK channel amplitude but may have caused a weak reduction of open probability, as seen in the patch illustrated in Fig. 6C. However, in two other patches, 50 nm-CTX had no effect on SK channel activity. These channels were confirmed to be SK
D. G. LANG AND A. K. RITCHIE
128
channels by inhibition with 500 nM-apamin. Thus, it is concluded that SK channels are relatively insensitive to 50 nM-CTX. The effect of higher concentrations of CTX was not tested. Physiological contributions of the BK and SK channels We have examined the effects of the above-described inhibitors of the BK and SK channels on action potential behaviour under current-clamp conditions. A voltageApamin-sensitive cells Control
"F"T--r7
F
Apamin-insensitive cells Control
PIF
500 mM-apamin
500 mm-apain 1 pA 10 s
3
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'U~~~~~~~~~~~~~~~~~~~~~~~~~_ A
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=
2 |~~~~~~~~~A
0
0 Control Apamin Control Apamin Fig. 8. Effect of apamin on patch action current firing frequency. Patch action current activity of apamin-sensitive (left) and -insensitive (right) cells are shown before and after application of 500 nM-apamin to the extra-patch membrane. The records were low-pass filtered at 360 Hz (-3 dB, 8-pole Bessel). The action potential firing rate in fourteen spontaneously pacing cells was manually tallied from 100 s data segments before and after 500 nM-apamin (bottom panels). Application of apamin caused an increase in firing rate of greater than 30 % in five cells, while nine showed no change in firing rate. The mean increase in firing rate for the apamin-sensitive cells was 90 % which was a significant increase (P < 0 025). The different symbols represent data obtained from different cells.
dependent, rapidly inactivating K+ current in GH3 cells is not affected by 30 mmTEA, 200 nM-apamin (Ritchie, 1987 a) or 50 nM-CTX (D. G. Lang & A. K. Ritchie, unpublished observations). Charybdotoxin (50 nM) prolonged action potential duration in both cells that were tested (Fig. 7A), presumably as a consequence of BK channel blockade. An enhancement of the after-hyperpolarization and a slowing of the action potential firing rate were also observed. These effects are presumably due to the increased entry of Ca2+ that occurs when the duration of the action potential is prolonged and
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subsequent activation of Ca2+-activated K+ channels that are resistant to CTX. A qualitatively similar effect to that produced by CTX was observed when 1 mM-TEA (five cells) was applied to GH3 cells (Ritchie & Lang, 1989). This concentration of TEA causes reductions in single-channel current amplitudes of about 80 % for BK channels and only 25 % for SK channels. Application of 500 nm-apamin to cells under current clamp had little effect on the duration of the action potential but caused an increase in the action potential firing rate and a reduction of the slow after-hyperpolarization (Fig. 7B). We have further examined the contributions of SK channels to the regulation of action potential firing rate by monitoring the effect of apamin on the frequency of occurrence of patch action currents. Patch action currents are small, outwardly directed transient currents that can be recorded from the cell-attached patch each time the cell fires an action potential. This method offers the advantage that the cell interior is left undisturbed. Three classes of cells were routinely observed: silent cells that did not fire action potentials; irregular cells that sporadically fired action potentials; and pacing cells (approximately 60 % of the cells examined) that fired action potentials at rates of 0'2-3 Hz. Application of 500 nM-apamin to either silent cells or irregular cells did not cause the cells to begin pacing; however, apamin did affect the firing rate of some of the pacing cells (Fig. 8). In five out of fourteen pacing cells, apamin caused an increase of the action potential firing rate by greater than 30%; the other nine cells showed no significant change in action potential firing rate. Thus, the apaminsensitive SK channels, by contributing to the slow after-hyperpolarizations, can slow action potential firing rates in some GH3 cells. DISCUSSION
External TEA blocked the BK channels of GH3 cells (Kd = 260 mM) by reducing the single-channel current amplitude. The block was not strongly voltage dependent; the TEA binding site sensed less than 20 % of the membrane electric field. These values are within the range of Kds (200-300 #M) and voltage sensitivities (0-31 %) that similar methods of analysis have yielded for BK channels in skeletal muscle (Blatz & Magleby, 1984; Vergara, Moczydlowski & Latorre, 1984), adrenal chromaffin cells (Yellen, 1984) and sympathetic ganglion cells (Smart, 1987). Charybdotoxin (50 nM) greatly reduced the open probability of BK channels from GH3 cells but did not change the single-channel amplitude. This is consistent with the mechanism of CTX inhibition of BK channels from rat skeletal muscle in lipid bilayers (Miller et al. 1985; Anderson, MacKinnon, Smith & Miller, 1988; MacKinnon & Miller, 1988). The BK channels in GH3 cells were insensitive to apamin. The SK channels in GH3 cells were inhibited by apamin and high concentrations of external TEA, but were not blocked by 50 nm-CTX. The block of SK channels by apamin was expressed as a cessation of single-channel openings. As is the case for the BK channels, the TEA block of the SK channels was expressed as an apparent reduction of single-channel current amplitude. The Kd was 3'1 mm, and the TEA binding site sensed less than 20 % of the membrane electric field. Thus, in GH3 cells, we find that the apamin-sensitive SK channel is inhibited by TEA but with a Kd that is twelve times that of the BK channels. This difference partially accounts for 5
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D. G. LANG AND A. K. RITCHIE
previous reports that the apamin-sensitive Ca2l-activated K+ conductance in rat skeletal muscle (Romey & Lazdunski, 1984; Blatz & Magleby, 1986), frog sympathetic neurones (Pennefather et al. 1985; Kawai & Watanabe, 1986), and GH3 cells (Ritchie, 1987a) is relatively insensitive to TEA. However, it is curious that a substantial fraction of the apamin-sensitive current in GH3 cells under whole-cell recording is still present in 30 mM-TEA (Ritchie, 1987 a), while in the present study this concentration of TEA decreased single-channel amplitude by more than 95 %. Likewise, in rat myotubes (Romey & Lazdunski, 1984), a large fraction of the apamin-sensitive Ca2+-activated K+ conductance in intact cells was still present in 20-25 mM-external TEA. We do not know if the apparently greater affinity of SK channels for TEA in excised patches is somehow related to the recording conditions (e.g. high external K+ concentrations), or if GH3 cells have an additional type of apamin-sensitive Ca2+-activated K+ channel that is more resistant to TEA than the SK channel. The possibility also exists that SK channels in GH3 cells may have a greater sensitivity to TEA than the apamin-sensitive channels of other cell types. Calcium-activated K+ channels are known to be important in action potential repolarization since chelation of intracellular Ca2+ greatly prolongs action potential duration (Ritchie, 1987 b). Furthermore, we have previously shown that BK channels are active in cell-attached patches during a brief 20 ms period of time that coincides with action potential repolarization (Lang & Ritchie, 1987). If BK channels are the primary contributors to repolarization, then selective BK channel inhibition should prolong action potential duration. Relatively selective inhibition of the BK channel in GH3 cells can be achieved by 1 mM-TEA or 50 nM-CTX. Both 50 nM-CTX and 1 mM-TEA (Ritchie & Lang, 1989) prolong action potential duration of GH3 cells under current clamp. These concentrations of TEA or CTX do not significantly inhibit the SK channels. Recently, CTX has been found to block voltage-dependent K+ currents in human and murine T lymphocytes (Sands, Lewis & Cahalan, 1989), cloned IA K+ channels from Drosophila Shaker mutants (MacKinnon, Reinart & White, 1988), and a 35 pS Ca2+-activated K+ channel in Aplysia neurones (Herman & Erxleben, 1987). In GH3 cells, a transient voltage-dependent K+ current is not blocked by 30 mM-TEA (Ritchie, 1987 a) or by 50 nM-CTX (D. G. Lang & A. K. Ritchie, unpublished observations). A 38 pS Ca2+-activated K+ channel is present in GH3 cells (Ritchie & Lang, 1989); however, due to its infrequent occurrence, we have been unable to determine its sensitivity to CTX. Since the 38 pS channel is not affected by 1 mM-TEA (D. G. Lang & A. K. Ritchie, unpublished observations), the ability of either 1 mM-TEA or 50 nm-CTX to prolong action potential duration strongly suggests that, as in neurones (Pennefather et al. 1985; Lancaster & Nicoll, 1987), BK channels play an important role in repolarization of the action potential. Under current-clamp conditions, apamin (500 nM) reduced the slow afterhyperpolarization by about 3-5 mV but had little effect on action potential duration. Apamin has a similar effect in cultured skeletal muscle (Romey & Lazdunski, 1984) and sympathetic ganglion cells (Pennefather et al. 1985; Kawai & Watanabe, 1986). We investigated the possibility that the slow after-hyperpolarizations may help regulate the firing rate of spontaneously pacing cells by monitoring patch action currents in cell-attached-patch electrodes. When apamin was applied to the extrapatch whole-cell membrane, it increased the firing rate of five out of fourteen
131 TEA BLOCK OF BK AND SK CHANNELS spontaneously pacing cells. The mean increase in firing rate in these five cells was 90%. Thus, SK channels can help to regulate the rate of cell firing presumably by affecting the duration of the slow after-hyperpolarization. This is consistent with our previous observation that SK single-channel activity in cell-attached patches occurs during the 0-3-5 s period of time associated with the interspike interval, with opening probability being highest immediately after the action potential (Lang & Ritchie, 1987). The difference in the functional roles of the BK vs. SK channels is a consequence of the higher sensitivity of SK channels to Cal+ at negative membrane potentials; in contrast, BK channels are mainly sensitive to Ca2+ at depolarized potentials (Pennefather et al. 1985; Lang & Ritchie, 1987). Apamin increased spike frequency in only 30% of pacing cells. This is probably related to the fact that not all GH3 cells have apamin-sensitive Ca2+-activated K+ currents (Ritchie, 1987 a). Clearly, channels other than SK channels must be contributing to the slow after-hyperpolarization. GH3 cells have a 38 pS Ca2+activated K+ channel that is active during the slow after-hyperpolarization (Ritchie & Lang, 1989) but it is observed very infrequently. Thus, more studies are needed to define the ionic conductances that regulate firing rate. Such information is important since the firing rate helps control Ca2+ influx, and hence the rate of prolactin secretion, in GH3 cells. The authors are grateful to Dr Christopher Miller for the generous gift of the charybdotoxin and to Dr Roberto Coronado for providing the ANALYSIS program. This work was supported by grants from the NIH (DK 33898) and the Muscular Dystrophy Association. REFERENCES
ANDERSON, C. S., MACKINNON, R., SMITH, C. & MILLER, C. (1988). Charybdotoxin block of single Ca2+-activated K+ channels: Effects of channel gating, voltage, and ionic strength. Journal of General Physiology 91, 317-333. BARRETT, J. N., MAGLEBY, K. L. & PALLOTTA, B. S. (1982). Properties of single calcium-activated potassium channels in cultured rat muscle. Journal of Physiology 331, 211-230. BENHAM, C. D., BOLTON, T. B., LANG, R. J. & TAKEWAKI, T. (1985). The mechanism of action of Ba2+ and TEA on single Ca2+-activated K+ channels in arterial and intestinal smooth muscle cell membranes. Pfluigers Archiv 403, 120-127. BLATZ, A. L. & MAGLEBY, K. L. (1984). Ionic conductance and selectivity of single calciumactivated potassium channels in cultured rat muscle. Journal of General Physiology 84, 1-23. BLATZ, A. L. & MAGLEBY, K. L. (1986). Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. Nature 323, 718-720. BLATZ, A. L. & MAGLEBY, K. L. (1987). Calcium-activated potassium channels. Trends in Neurosciences 10, 463-467. CAPIOD, T. & OGDEN, D. C. (1989). The properties of calcium-activated potassium ion channels in guinea-pig isolated hepatocytes. Journal of Physiology 409, 285-295. FABIATO, A. & FABIATO, F. (1979). Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. Journal de physiologic 75, 463-505. HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Archiv 391, 85-100. HERMAN, A. & ERXLEBEN, C. (1987). Charybdotoxin selectively blocks small Ca-activated K channels in Aplysia neurons. Journal of General Physiology 90, 27-48. IWATSUKI, N. & PETERSEN, 0. H. (1985). Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell recording. Journal of Membrane Biology 86, 139-144. 5-2
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KAWAI, T. & WATANABE, M. (1986). Blockade of Ca-activated K conductance by apamin in rat sympathetic neurones. British Journal of Pharmacology 87, 225-232. LANCASTER, B. & NICOLL, R. A. (1987). Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. Journal of Physiology 389, 187-203. LANG, D. G. & RITCHIE, A. K. (1987). Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line. Pftugers Archiv 410, 614-622. LANG, D. G. & RITCHIE, A. K. (1988). Pharmacological sensitivities of large and small conductance Ca2+-activated K' channels in the GH3 anterior pituitary cell line. Biophysical Journal 53, 144 a. MACKINNON, R. & MILLER, C. (1988). Mechanism of charybdotoxin block of the high-conductance, Ca2+-activated K+ channel. Journal of General Physiology 91, 335-349. MACKINNON, R., REINHART, P. H. & WHITE, M. W. (1988). Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features. Neuron 1, 997-1001. MARTY, A., TAN, Y. P. & TRAUTMANN, A. (1984). Three types of calcium-dependent channel in rat lacrimal glands. Journal of Physiology 357, 293-325. MILLER, C., MOCZYDLOWSKI, E., LATORRE, R. & PHILLIPS, M. (1985). Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313, 316-318. PENNEFATHER, P., LANCASTER, B., ADAMS, P. R. & NICOLL, R. A. (1985). Two distinct Cadependent K currents in bullfrog sympathetic ganglion cells. Proceedings of the National Academy of Sciences of the USA 82, 3040-3044. REINHART, P. H., CHUNG, S. & LEVITAN, I. B. (1989). A family of calcium-dependent potassium channels from rat brain. Neuron 2, 1031-1041. RITCHIE, A. K. (1987a). Two distinct calcium-activated potassium currents in a rat anterior pituitary cell line. Journal of Physiology 385, 591-609. RITCHIE, A. K. (1987 b). Thyrotropin-releasing hormone stimulates a calcium-activated potassium current in a rat anterior pituitary cell line. Journal of Physiology 385, 611-625. RITCHIE, A. K. & LANG, D. G. (1989). Activity of single ion channels that regulate membrane excitability in GH3 anterior pituitary cells. In Neural Control of Reproductive Function, ed. LAKOSKI, J. M., PEREZ-POLO, J. R. & RASSIN, D. K., pp. 463-479. Alan R. Liss, Inc., New York. ROMEY, G. & LAZDUNSKI, M. (1984). The coexistence in rat muscle cells of two distinct classes of Ca2+-dependent K+ channels with different pharmacological properties and different physiological functions. Biochemical and Biophysical Research Communications 118, 669-674. SANDS, S. B., LEWIS, R. S. & CAHALAN, M. D. (1989). Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes. Journal of General Physiology 93, 1061-1074. SMART, T. G. (1987). Single calcium-activated potassium channels recorded from cultured rat sympathetic neurones. Journal of Physiology 389, 337-360. THOMAS, M. V. (1982). Techniques in Calcium Research, p. 46. Academic Press, San Diego. VERGARA, C., MOCZYDLOWSKI, E. & LATORRE, R. (1984). Conduction, blockade and gating in a Ca2+-activated K+ channel incorporated into planar lipid bilayers. Biophysical Journal 45, 73-76. WONG, B. S. & ADLER, M. (1986). Tetraethylammonium blockade of calcium-activated potassium channels in clonal anterior pituitary cells. Pflugers Archiv 407, 279-284. WOODHULL, A. M. (1973). Ionic blockage of sodium channels in nerve. Journal of General Physiology 61, 687-708. YELLEN, G. (1984). Ionic permeation and blockade in Ca2+-activated K+ channels of bovine chromaffin cells. Journal of General Physiology 84, 157-186.
Journal of Physiology (1990), 425, pp. 117-132 With figures Printed in Great Britain
TETRAETHYLAMMONIUM BLOCKADE OF APAMIN-SENSITIVE AND INSENSITIVE Ca2l-ACTIVATED K+ CHANNELS IN A PITUITARY CELL LINE
BY DANIEL G. LANG* AND AILEEN K. RITCHIE From the Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77550-2779, USA
(Received 22 August 1989) SUMMARY
1. The pharmacological sensitivities and physiological contributions of two types of Ca2+-activated K+ channels (BK and SK) in GH3 cells were examined by the outside-out, whole-cell and cell-attached modes of the patch-clamp technique. 2. BK channels (250-300 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external tetraethylammonium (TEA) and by 50 nm-charybdotoxin (CTX), but were not blocked by apamin. 3. SK channels (9-14 pS in symmetrical 150 mM-K') in outside-out patches were blocked by external TEA and by apamin, but were not blocked by 50 nM-CTX. 4. The dissociation constant (Kd) for TEA block of SK channels (31 +0-37 mM) was 12-fold greater than the Kd for the BK channels (260 + 21 gM). The TEA blockade of both channels was not strongly voltage dependent; for both channels the TEA binding site sensed less than 20 % of the membrane electric field. 5. Application of blockers of the BK channels (1 mM-TEA and 50 nM-CTX) to whole cells under current clamp prolonged action potential duration; whereas application of apamin, a selective blocker of the SK channel, inhibited a slowly decaying after-hyperpolarization and had little effect on action potential duration. Apamin also increased the firing rate in 30 % of the spontaneously pacing cells. 6. It is suggested that BK channels contribute to action potential repolarization; whereas SK channels contribute to the regulation of action potential firing rate. INTRODUCTION
Numerous varieties of Ca2+-activated K+ channels have been described that differ widely in unit conductance, Ca2+ sensitivity, voltage sensitivity and pharmacology (Blatz & Magleby, 1987; Reinhart, Chung & Levitan, 1989). In view of this diversity in channel properties and its widespread occurrence in nearly all cell types, it is not surprising that Ca2+-activated K+ channels have been proposed to serve many different functional roles including repolarization of action potentials, regulation of resting membrane potential and spike discharge frequency, salt and water secretion, Present address: Department of Otolaryngology, The University of Texas Medical Branch, Galveston, TX 77550-2778, USA. *
MS 7896
D. G. LANG AND A. K. RITCHIE 118 and cell volume regulation. In order to assess the functional roles of these channels we have begun a pharmacological study, at the single-channel level, of two types of Ca2+-activated K+ channels that co-exist in the GH3 anterior pituitary cell line. The most extensively studied and widely occurring channel is the largeconductance BK channel (200-300 pS in 150 mM-symmetrical K+) that is activated by both internal calcium and depolarizing voltages (Blatz & Magleby, 1987). In GH3 cells, the BK channel requires high concentrations of internal Ca2+, much greater than 10 /M, for activation at -50 mV (Lang & Ritchie, 1987). However, the sensitivity to Ca2+ increases greatly as the cell is depolarized. This is similar to the calcium sensitivity of BK channels from skeletal muscle (Barrett, Magleby & Pallotta, 1982), but is nearly two orders of magnitude less sensitive to Ca2+ than the BK channels in rat lacrimal gland (Marty, Tan & Trautmann, 1984). In most (Blatz & Magleby, 1984; Iwatsuki & Petersen, 1985; Blatz & Magleby, 1987), but not all (Benham, Bolton, Lang & Takewaki, 1985; Wong & Adler, 1986), cells the BK channel is blocked by submillimolar concentrations of external TEA. The channel is also blocked by nanomolar concentrations of CTX (Miller, Moczydlowski, Latorre & Phillips, 1985; Reinhart et al. 1989), but is insensitive to apamin (Romey & Lazdunski, 1984; Pennefather, Lancaster, Adams & Nicoll, 1985; Reinhart et al. 1989). Far less is known about a distinctly different type of Ca2+-activated K+ channel of small unitary conductance (9-20 pS in 150 mM-symmetrical K+) that is referred to as the SK channel. This channel is activated by 1 /SM-internal Ca2+ at negative membrane potentials, exhibits a very weak voltage sensitivity (Blatz & Magleby, 1986; Lang & Ritchie, 1987), and is inhibited by apamin (Blatz & Magleby, 1986; Capiod & Ogden, 1989). The SK channel (Blatz & Magleby, 1986) and the apaminsensitive conductance in whole-cell recording (Romey & Lazdunski, 1984; Pennefather et al. 1985; Ritchie, 1987a, b) is reported to be relatively insensitive to inhibition by TEA. Its sensitivity to CTX is unknown. In this report, we confirm that SK channels in GH3 cells, like those described in rat skeletal muscle (Blatz & Magleby, 1986) and guinea-pig hepatocytes (Capiod & Ogden, 1989), are inhibited by apamin, we establish its insensitivity to CTX, and examine the mechanism and sensitivity of SK channel blockade by TEA. These results are compared with companion studies performed on the BK channels in GH3 cells. Finally, this information on pharmacological specificity is used to investigate the contribution of BK and SK channels to the duration, the after-hyperpolarization, and frequency of discharge of spontaneous action potentials in GH3 cells. A preliminary account of these studies has appeared (Lang & Ritchie, 1988). METHODS
Cell culture. The GH, cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). The GH3 cells were grown at 36 °C in a 5% CO2 atmosphere in Ham's F10 media supplemented with 15 % heat-inactivated horse serum and 2-5 % fetal calf serum. Cells used for electrophysiological experiments were plated on polylysine-coated glass cover-slips and were used within 2-10 days after plating. Cells from passages 27-40 were used in the experiments described in this paper. Electrophysiological recordings. The whole-cell, cell-attached and outside-out modes of the patchclamp technique were utilized in this study (Hamill, Marty, Neher, Sakmann & Sigworth, 1981).
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Electrodes used for cell-attached patches were made from Corning 7052 glass (Friedrick & Dimmock; Millville, NJ, USA) in two steps on a David Kopf 700C electrode puller. Electrodes used for outside-out patches were made from TW-150 glass (World Precision Instruments, New Haven, CO, USA) and were pulled in a single step. The electrodes were coated with Sylgard and firepolished. The resistances ranged from 12 to 20 Mg; however, electrodes pulled from TW- 150 glass had much smaller diameter bores at the tip than those pulled from 7052 glass. Electrodes used for whole-cell current-clamp studies were made with TW- 150 glass and had resistances of 2-5 MQ. Seal resistances were in the range of 40-90 GQ. Recordings were performed at room temperature with a List model EPC-7 patch-clamp amplifier (Darmstadt, FRG). Analysis. The data were stored on FM tape (Racal Store 4DS) with a frequency response of DC to 2-5 kHz (-3 dB, 4-pole Tchebychef). The single-channel records were filtered at 1 kHz (-3 dB, 8-pole Bessel) and digitized at a sampling rate of 10 kHz. The digitized single-channel data were analysed on a 80286 computer by using the ANALYSIS program developed by H. Affolter (courtesy of Dr Roberto Coronado). Single-channel amplitudes were measured directly from data segments displayed on the computer screen using cursors or from amplitude histograms generated by the ANALYSIS program. The amplitude histograms consisted of 256 bins with each bin containing the number of sample points falling within the bin width; the amplitude of the singlechannel current was taken as the difference between the peaks for opened and closed current levels. Channel amplitudes in the presence of TEA were obtained from cursor measurements of data segments displayed on the computer screen since the histograms tended to underestimate the amplitude during rapid flicker block. Channel openings were detected as events that fell within a window set by the user. The open probability was taken as the total time the current fell within the window divided by the total duration of the data segment. Patches that contained two channels were analysed in two passes. During the first pass, the window was set to include events when only one channel was open and when two channels were open simultaneously; during the second pass, the window was set to include only events when two channels were open simultaneously. The open probabilities from each pass were summed and divided by two to yield the average open probability for a single channel. We have verified amplitude and open-probability measurements obtained from the ANALYSIS program by comparing with results obtained by manual measurements from chart paper recordings of taped data replayed at 1/4 speed (effective bandwidth of 360 Hz). Dose-response curves were fitted to data points by a least-squares minimization algorithm (Minsq, Micromath Scientific Software, Salt Lake City, UT, USA). The goodness of fit is given by the coefficient of determination (CD). All currents and voltages for the single-channel records are expressed with the same polarity conventions as used in whole-cell voltage clamp. Membrane potentials are expressed with the intracellular relative to the extracellular side. Inward currents are shown to open in the downward direction and are defined as a positive charge moving from the extracellular to the intracellular side of the membrane. The closed state of the channel, except when obvious, is indicated by the letter 'C' and the open state by the letter 'O'. Solutions. The solutions used in the cell-attached-patch experiments were (mM): KCl, 150; MgCl2, 2; N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), 10; 4-aminopyridine (4-AP), 5; pH 7 30 for the pipette and KCl, 150; CaCl2, 0 01; HEPES, 15; pH 7-50 for the bath. The solutions used in outside-out patch experiments were (mM): KCl, 150; MgCl2, 05; HEPES, 10; ethyleneglycol-bis(/J-aminoethylether)-N,N,',N'-tetraacetic acid (EGTA), 2; free Ca2 , 15 UM; pH 7 20 for the pipette; TEA-Cl, x; NaCl, 75-X ; KCl, 75; MgCl2, 2; HEPES, 10; 4-AP, 5; pH 7 30 for bath A; and KCl, 150; MgCl2, 2; HEPES, 10; 4-AP, 5; pH 730 for bath B. Free Ca2+ concentrations in the range of 1-3 /tM were buffered with 2 mM-EGTA and were calculated using an apparent Kd of 150 nm (Thomas, 1982). The Mg2+ concentration had a negligible effect on the buffered free Ca2+ concentration. This was verified by the method of Fabiato & Fabiato (1979) assuming an apparent Kd of EGTA for Mg2+ of 84 mm (Thomas, 1982). Calcium concentrations greater than 3 jtm were achieved by simply adding the desired amount of free Ca2+. The solutions used in whole-cell experiments were (mM): KCl, 150; HEPES, 3; pH 7-20 for the pipette and NaCl, 140; KCl, 5-6; CaCl2, 2; MgCl2, 1; HEPES, 10; glucose, 10; pH 7 30 for the bath. Apamin was obtained from Sigma Chemical Company (St Louis, MO, USA). Ionomycin (free acid) was obtained from Calbiochem (La Jolla, CA, USA). Charybdotoxin (CTX) was a generous gift from the laboratory of Dr Christopher Miller. Whole cells were perfused by pressure ejection (70 kPa) of solutions from a large-bore pipette (diameter 10-15 jum) placed 50-100 ,um from the cell. The cells were superperfused with normal
120
D. G. LANG AND A. K. RITCHIE
saline during the control segments to reduce K+ accumulation. The effects of pharmacological agents on whole-cell activity were tested by first lowering a pipette containing saline plus drug into the bath near the cell, and then switching the pressure from the control pipette to the drug pipette. The control pipette was then raised out of the bath to prevent contamination of its contents by the drug. Excised, outside-out patches were placed in a small-volume recording chamber (approximately 0-2 ml) and perfused at a rate of approximately 1 ml/min. The solutions were gravity fed, and the solution changes required approximately 30 s to reach equilibrium. For the CTX experiments on outside-out patches, the perfusion was stopped and CTX was injected into the recording chamber. RESULTS
The effects of apamin, TEA and CTX on Ca2+-activated K+ channels in GH3 cells were mainly tested on outside-out patches. Since the internal face of the patch membrane was exposed to the pipette solution, the BK and SK channels could not be identified on the basis of sensitivity to changes in the internal free Ca2+ concentrations. However, the BK channels were readily identified on the basis of their large unitary conductance. The SK channels were identified by their small unitary conductance, lack of voltage dependence of the open probability, and by relatively long-duration openings at negative membrane potentials. In GH3 cells, the unit conductance of the SK channel in symmetrical 150 mM-K+ is 9-14 pS, and slightly smaller (about 6 pS) in experiments performed with 75 mM-external and 150 mM-internal K+. Infrequently, a channel with a similar unit conductance as SK channels was seen in inside-out patch experiments. This channel is insensitive to changes in internal calcium and at negative membrane potentials it exhibits very brief duration openings that occur in bursts. SK channels could be distinguished from the Ca2+-insensitive channels by differences in channel open durations at negative membrane potentials.
Apamin sensitivity The sensitivity of BK channels to inhibition by apamin was tested by using outside-out patches exposed to either 75 mM-external and 150 mM-internal K+ or symmetrical 150 mM-K+ gradients. Neither the single-channel amplitude nor the open probability was affected by 20 min incubations with 5 JM-apamin. Thus, apamin does not block the BK channels. The apamin sensitivity of the SK channels was tested by two different methods. First, the frequency of observing SK channels in cell-attached patches was compared without and with apamin in the patch pipette solution. In these experiments, the patch pipette contained 150 mM-K+ and was held at -50 mV so that unitary currents were in the inward direction. Calcium influx in the cell was stimulated by applying the Ca2+ ionophore ionomycin to the extrapatch, whole-cell membrane. Under these conditions, ionomycin causes a transient elevation of the intracellular free Ca2+ concentration. We have verified this by fluorescent measurements of cytosolic calcium levels in GH3 cells loaded with the calcium-sensitive dye Fura-2 (D. G. Lang & A. K. Ritchie, unpublished observation). In the absence of apamin, SK channel activity was stimulated by application of 50,uM-ionomycin (in 1% DMSO) in twelve out of sixteen patches (Fig. 1). Application of 1 % DMSO had no
121 TEA BLOCK OF BK AND SK CHANNELS effect. Stimulation of SK channel activity by ionomycin did not occur when 500 nmapamin was included in the pipette solution in eight out of eight patches. The second method was to test the effect of apamin on SK channel activity in outside-out patches. SK channel openings were observed at negative holding lonomycin
). The different symbols represent data obtained from five different patches containing BK channels and four different patches containing SK channels. The curves represent best-fit theoretical dose-response curves for a one-to-one drug-to-receptor binding scheme.
where i and ib are the unitary currents in the presence and absence of TEA, respectively. Bo and z are the blocker concentration and valency, respectively. The value of a can be determined from the slope, and the value of Kd(O mV) can be determined from the Y-intercept of the plot of ln (i/ib -1) against voltage (V). These plots are shown in Figs 3C and 4C. For both the BK and SK channels of GH3 cells, the block by external TEA was not strongly voltage dependent (6 < 0-2). The Kd(O mV)s for the BK channels and for the SK channels were 210 ,UM and 4-4 mM, respectively. We have also measured the dose-response characteristics of the block by external TEA by a different method. The concentration dependence of TEA inhibition was measured as the percentage reduction of the single-channel amplitude (Fig. 5). The continuous curves represent theoretical dose-response curves for a one-to-one drugto-receptor binding scheme: Percentage inhibition =1+Kd/B' where Kd is the dissociation constant and Bo is the concentration of external TEA. The theoretical dose-response curves were fitted to the data points by a least-squares minimization algorithm. The Kd values obtained from these fits, 260 + 21 ftM
126
D. G. LANG AND A. K. RITCHIE B
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Fig. 6. Effect of charybdotoxin on BK and SK channels. These outside-out patches were exposed to a symmetrical 150 mM-K' gradient (bath B). A, the BK channel activity in this patch was obtained with 3 gM-internal free Ca2+ at a holding potential of + 40 mV. Records are shown for control solution, in the presence of 50 nM-CTX, and after wash-out of CTX. The records were low-pass filtered at 1 kHz (-3 dB, 8-pole Bessel). B, the open probability for the channel in A is plotted against voltage for controls (@), in the presence of 50 nM-CTX (0), and after washing (A). CTX reversibly reduced the fractional open time of this BK channel but did not affect its unitary conductance (270 ps). C, the SK channel activity in this patch was obtained with 1 gM-internal free Ca2+ at a holding potential of -50 mV. In the control segment, the mean current level was approximately 2-5 pA indicating that at least five SK channels were present in the patch. The mean current remained high in the presence of 50 nM-CTX, although CTX may have blocked one of the five channels in this patch. In two other patches, 50 nM-CTX had no effect on SK channel activity. Apamin (500 nM) reduced the number of open SK channels and, after 80 s, completely blocked all SK channel activity in this patch. The records were lowpass filtered at 360 Hz (-3 dB, 8-pole Bessel).
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(estimate + standard deviation; CD = 0-95) for the BK channels and 31+ 0-37 mm (CD = 0 95) for the SK channels, are in good agreement with that obtained from the Woodhull method. Charybdotoxin sensitivity The CTX sensitivity of the BK channels was examined by using outside-out patches exposed to a symmetrical 150 mM-K+ gradient with internal free Ca2+ A 0 mV-
CTX Control B 0 mV -
/Apamin Control I
20 mV
200 ms
Fig. 7. Effects of BK and SK channel blockers on action potentials. These currentclamped cells were continually superfused with either control saline or saline containing inhibitor. A, superfusion with 50 nM-CTX prolonged action potential duration, enhanced the slow after-hyperpolarization, and slowed the firing rate. B, application of apamin (500 nM) caused a decrease in the slow after-hyperpolarization and an increase in the spontaneous firing rate of this cell. Apamin had little effect on the duration of the action potential.
concentrations of either 1 or 3 /M. Charybdotoxin (50 nM), applied to the external face of the membrane patch, caused a reversible decrease of the open probability of BK channels but did not affect the single-channel amplitude (Fig. 6A and B). Thus, in contrast to the rapid flicker block by external TEA, the block of BK channels by CTX shows very slow kinetics. This inhibition of the BK channels was seen in all eight patches examined. The voltage dependence for the block by CTX was not investigated. It is concluded that the BK channels of GH3 cells are inhibited by CTX. The CTX sensitivity of the SK channels was also tested under a symmetrical 150 mM-K+ gradient with 1 /tM-internal free Ca2 . SK channel activity could be detected at a holding potential of -50 mV. Application of 50 nM-CTX had no effect on SK channel amplitude but may have caused a weak reduction of open probability, as seen in the patch illustrated in Fig. 6C. However, in two other patches, 50 nm-CTX had no effect on SK channel activity. These channels were confirmed to be SK
D. G. LANG AND A. K. RITCHIE
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channels by inhibition with 500 nM-apamin. Thus, it is concluded that SK channels are relatively insensitive to 50 nM-CTX. The effect of higher concentrations of CTX was not tested. Physiological contributions of the BK and SK channels We have examined the effects of the above-described inhibitors of the BK and SK channels on action potential behaviour under current-clamp conditions. A voltageApamin-sensitive cells Control
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Apamin-insensitive cells Control
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0 Control Apamin Control Apamin Fig. 8. Effect of apamin on patch action current firing frequency. Patch action current activity of apamin-sensitive (left) and -insensitive (right) cells are shown before and after application of 500 nM-apamin to the extra-patch membrane. The records were low-pass filtered at 360 Hz (-3 dB, 8-pole Bessel). The action potential firing rate in fourteen spontaneously pacing cells was manually tallied from 100 s data segments before and after 500 nM-apamin (bottom panels). Application of apamin caused an increase in firing rate of greater than 30 % in five cells, while nine showed no change in firing rate. The mean increase in firing rate for the apamin-sensitive cells was 90 % which was a significant increase (P < 0 025). The different symbols represent data obtained from different cells.
dependent, rapidly inactivating K+ current in GH3 cells is not affected by 30 mmTEA, 200 nM-apamin (Ritchie, 1987 a) or 50 nM-CTX (D. G. Lang & A. K. Ritchie, unpublished observations). Charybdotoxin (50 nM) prolonged action potential duration in both cells that were tested (Fig. 7A), presumably as a consequence of BK channel blockade. An enhancement of the after-hyperpolarization and a slowing of the action potential firing rate were also observed. These effects are presumably due to the increased entry of Ca2+ that occurs when the duration of the action potential is prolonged and
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subsequent activation of Ca2+-activated K+ channels that are resistant to CTX. A qualitatively similar effect to that produced by CTX was observed when 1 mM-TEA (five cells) was applied to GH3 cells (Ritchie & Lang, 1989). This concentration of TEA causes reductions in single-channel current amplitudes of about 80 % for BK channels and only 25 % for SK channels. Application of 500 nm-apamin to cells under current clamp had little effect on the duration of the action potential but caused an increase in the action potential firing rate and a reduction of the slow after-hyperpolarization (Fig. 7B). We have further examined the contributions of SK channels to the regulation of action potential firing rate by monitoring the effect of apamin on the frequency of occurrence of patch action currents. Patch action currents are small, outwardly directed transient currents that can be recorded from the cell-attached patch each time the cell fires an action potential. This method offers the advantage that the cell interior is left undisturbed. Three classes of cells were routinely observed: silent cells that did not fire action potentials; irregular cells that sporadically fired action potentials; and pacing cells (approximately 60 % of the cells examined) that fired action potentials at rates of 0'2-3 Hz. Application of 500 nM-apamin to either silent cells or irregular cells did not cause the cells to begin pacing; however, apamin did affect the firing rate of some of the pacing cells (Fig. 8). In five out of fourteen pacing cells, apamin caused an increase of the action potential firing rate by greater than 30%; the other nine cells showed no significant change in action potential firing rate. Thus, the apaminsensitive SK channels, by contributing to the slow after-hyperpolarizations, can slow action potential firing rates in some GH3 cells. DISCUSSION
External TEA blocked the BK channels of GH3 cells (Kd = 260 mM) by reducing the single-channel current amplitude. The block was not strongly voltage dependent; the TEA binding site sensed less than 20 % of the membrane electric field. These values are within the range of Kds (200-300 #M) and voltage sensitivities (0-31 %) that similar methods of analysis have yielded for BK channels in skeletal muscle (Blatz & Magleby, 1984; Vergara, Moczydlowski & Latorre, 1984), adrenal chromaffin cells (Yellen, 1984) and sympathetic ganglion cells (Smart, 1987). Charybdotoxin (50 nM) greatly reduced the open probability of BK channels from GH3 cells but did not change the single-channel amplitude. This is consistent with the mechanism of CTX inhibition of BK channels from rat skeletal muscle in lipid bilayers (Miller et al. 1985; Anderson, MacKinnon, Smith & Miller, 1988; MacKinnon & Miller, 1988). The BK channels in GH3 cells were insensitive to apamin. The SK channels in GH3 cells were inhibited by apamin and high concentrations of external TEA, but were not blocked by 50 nm-CTX. The block of SK channels by apamin was expressed as a cessation of single-channel openings. As is the case for the BK channels, the TEA block of the SK channels was expressed as an apparent reduction of single-channel current amplitude. The Kd was 3'1 mm, and the TEA binding site sensed less than 20 % of the membrane electric field. Thus, in GH3 cells, we find that the apamin-sensitive SK channel is inhibited by TEA but with a Kd that is twelve times that of the BK channels. This difference partially accounts for 5
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previous reports that the apamin-sensitive Ca2l-activated K+ conductance in rat skeletal muscle (Romey & Lazdunski, 1984; Blatz & Magleby, 1986), frog sympathetic neurones (Pennefather et al. 1985; Kawai & Watanabe, 1986), and GH3 cells (Ritchie, 1987a) is relatively insensitive to TEA. However, it is curious that a substantial fraction of the apamin-sensitive current in GH3 cells under whole-cell recording is still present in 30 mM-TEA (Ritchie, 1987 a), while in the present study this concentration of TEA decreased single-channel amplitude by more than 95 %. Likewise, in rat myotubes (Romey & Lazdunski, 1984), a large fraction of the apamin-sensitive Ca2+-activated K+ conductance in intact cells was still present in 20-25 mM-external TEA. We do not know if the apparently greater affinity of SK channels for TEA in excised patches is somehow related to the recording conditions (e.g. high external K+ concentrations), or if GH3 cells have an additional type of apamin-sensitive Ca2+-activated K+ channel that is more resistant to TEA than the SK channel. The possibility also exists that SK channels in GH3 cells may have a greater sensitivity to TEA than the apamin-sensitive channels of other cell types. Calcium-activated K+ channels are known to be important in action potential repolarization since chelation of intracellular Ca2+ greatly prolongs action potential duration (Ritchie, 1987 b). Furthermore, we have previously shown that BK channels are active in cell-attached patches during a brief 20 ms period of time that coincides with action potential repolarization (Lang & Ritchie, 1987). If BK channels are the primary contributors to repolarization, then selective BK channel inhibition should prolong action potential duration. Relatively selective inhibition of the BK channel in GH3 cells can be achieved by 1 mM-TEA or 50 nM-CTX. Both 50 nM-CTX and 1 mM-TEA (Ritchie & Lang, 1989) prolong action potential duration of GH3 cells under current clamp. These concentrations of TEA or CTX do not significantly inhibit the SK channels. Recently, CTX has been found to block voltage-dependent K+ currents in human and murine T lymphocytes (Sands, Lewis & Cahalan, 1989), cloned IA K+ channels from Drosophila Shaker mutants (MacKinnon, Reinart & White, 1988), and a 35 pS Ca2+-activated K+ channel in Aplysia neurones (Herman & Erxleben, 1987). In GH3 cells, a transient voltage-dependent K+ current is not blocked by 30 mM-TEA (Ritchie, 1987 a) or by 50 nM-CTX (D. G. Lang & A. K. Ritchie, unpublished observations). A 38 pS Ca2+-activated K+ channel is present in GH3 cells (Ritchie & Lang, 1989); however, due to its infrequent occurrence, we have been unable to determine its sensitivity to CTX. Since the 38 pS channel is not affected by 1 mM-TEA (D. G. Lang & A. K. Ritchie, unpublished observations), the ability of either 1 mM-TEA or 50 nm-CTX to prolong action potential duration strongly suggests that, as in neurones (Pennefather et al. 1985; Lancaster & Nicoll, 1987), BK channels play an important role in repolarization of the action potential. Under current-clamp conditions, apamin (500 nM) reduced the slow afterhyperpolarization by about 3-5 mV but had little effect on action potential duration. Apamin has a similar effect in cultured skeletal muscle (Romey & Lazdunski, 1984) and sympathetic ganglion cells (Pennefather et al. 1985; Kawai & Watanabe, 1986). We investigated the possibility that the slow after-hyperpolarizations may help regulate the firing rate of spontaneously pacing cells by monitoring patch action currents in cell-attached-patch electrodes. When apamin was applied to the extrapatch whole-cell membrane, it increased the firing rate of five out of fourteen
131 TEA BLOCK OF BK AND SK CHANNELS spontaneously pacing cells. The mean increase in firing rate in these five cells was 90%. Thus, SK channels can help to regulate the rate of cell firing presumably by affecting the duration of the slow after-hyperpolarization. This is consistent with our previous observation that SK single-channel activity in cell-attached patches occurs during the 0-3-5 s period of time associated with the interspike interval, with opening probability being highest immediately after the action potential (Lang & Ritchie, 1987). The difference in the functional roles of the BK vs. SK channels is a consequence of the higher sensitivity of SK channels to Cal+ at negative membrane potentials; in contrast, BK channels are mainly sensitive to Ca2+ at depolarized potentials (Pennefather et al. 1985; Lang & Ritchie, 1987). Apamin increased spike frequency in only 30% of pacing cells. This is probably related to the fact that not all GH3 cells have apamin-sensitive Ca2+-activated K+ currents (Ritchie, 1987 a). Clearly, channels other than SK channels must be contributing to the slow after-hyperpolarization. GH3 cells have a 38 pS Ca2+activated K+ channel that is active during the slow after-hyperpolarization (Ritchie & Lang, 1989) but it is observed very infrequently. Thus, more studies are needed to define the ionic conductances that regulate firing rate. Such information is important since the firing rate helps control Ca2+ influx, and hence the rate of prolactin secretion, in GH3 cells. The authors are grateful to Dr Christopher Miller for the generous gift of the charybdotoxin and to Dr Roberto Coronado for providing the ANALYSIS program. This work was supported by grants from the NIH (DK 33898) and the Muscular Dystrophy Association. REFERENCES
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