257
Journal of Physiology (1991), 444, pp. 257-268 With 6 figure8 Printed in Great Britain
Ca2l AND VOLTAGE INACTIVATE Ca2l CHANNELS IN GUINEA-PIG VENTRICULAR MYOCYTES THROUGH INDEPENDENT MECHANISMS
BY ROBERT W. HADLEY* AND W. J. LEDERER From the Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA
(Received 18 April 1991) SUMMARY
1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca2+-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca2+dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca2+-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca2+-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca2+-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca2+-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca2+-dependent inactivation is entirely sensitive to ,* To whom correspondence should be addressed. MS 9311
9-2
R. W. HADLEY AND W. J. LEDERER 258 adrenergic stimulation. These facts suggest that either Ca2+-dependent inactivation results from Ca2+-dependent dephosphorylation of the Ca2+ channel, or that Ca2+dependent inactivation is modulated by protein kinase A. INTRODUCTION
A rise in the concentration of intracellular Ca2+ ([Ca2+]i) inactivates Ca2+ channels in many cell types, providing control of Ca2+ influx through a negative feedback mechanism (Eckert & Chad, 1984). However, the molecular events underlying Ca2+dependent Ca2+ channel inactivation have been disputed and have proven challenging to study. A number of possible mechanisms have been considered, including allosteric regulation of Ca2+ channel function (Plant, Standen & Ward, 1983), modulation of voltage-dependent inactivation by Ca2+ (Lee, Marban & Tsien, 1985), and activation of Ca2+-dependent phosphatases (Chad & Eckert, 1986). While this last hypothesis is supported by repeated suggestions that only phosphorylated Ca2+ channels are functional (Sperelakis & Schneider, 1976; Reuter & Scholz, 1977; Bean, Nowycky & Tsien, 1984; Armstrong & Eckert, 1987; Hymel, Striessnig, Glossmann & Schindler, 1988; Nunoki, Florio & Catterall, 1989), an absolute requirement for channel phosphorylation has been disputed vigorously (Kameyama, Hescheler, Hofmann & Trautwein, 1986; Kameyama, Hescheler, Mieskes & Trautwein, 1986; Trautwein, Cavalie, Flockerzi, Hofmann & Pelzer, 1987). Furthermore, even if this point were conceded it would still leave open the question of whether a rise in [Ca2+]i promotes Ca2+ channel dephosphorylation (Byerly, Leung & Yazejian, 1988). We have therefore reinvestigated the mechanism of Ca2+dependent inactivation in isolated cardiac ventricular myocytes by combining the recently developed techniques of photolytic Ca2+ release and measurement of cardiac Ca2+ channel gating current. By activating the photorelease of Ca2+ independently of voltage-clamp steps we have been able to show that voltage and Ca2+ inactivate the Ca2+ channel through completely separate mechanisms. Furthermore, Ca2+dependent inactivation is entirely sensitive to fl-adrenergic stimulation. These studies therefore suggest that different regions of the Ca2+ channel protein are involved in the two types of Ca2+ channel inactivation. A preliminary note on these experiments has been published (Hadiey & Lederer, 1991 a). METHODS
Cell preparation Ventricular myocytes were enzymatically isolated using the procedure of Mitra & Morad (1985). Briefly, guinea-pigs were killed while under deep, pentobarbitone (100 mg kg-')-induced anaesthesia, and their hearts were removed and transferred to a Langendorff apparatus. The hearts were perfused with a solution containing (in mM): NaCl, 135; KCl, 5-4; MgCl2, 1; NaH2PO4, 0 33; glucose, 11 and HEPES, 10, at a room temperature of 37 'C. After a short perfusion period, collagenase (0 5 mg ml-') and protease (0 1 mg ml-') were added to the perfusing solution. After an additional 3-5 min, the hearts were removed and minced into small pieces, which were placed into the above solution with 0-2 mM-CaC12 added. Rod-shaped myocytes appeared in the solution shortly thereafter. Experimental procedures The isolated ventricular myocytes were voltage clamped using the whole-cell patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Pipette resistances ranged from 0 5 to 2 MQ. The standard pipette solution contained (in mM): caesium glutamate, 105; tetra-
Ca2+-DEPENDENT INACTIVATION OF Ca2+ CHANNELS
259
ethylammonium chloride, 10; CsCl, 20; HEPES, 20; K2ATP, 5. The pH was adjusted to 7-4 with CsOH. K2ATP was used to keep Mg2+ concentrations as low as possible for the DM-nitrophen experiments. K2ATP was usually replaced with MgATP (0-2 mM) for the nitr-5 experiments. Several external solutions were used. The 120 mM-Na+, 2-5 mM-Ca2+ solution contained (in mM): NaCl, 120; KCl, 4; CaCl2, 2-5; MgCI2, 0 5 and CsCl, 20. The 0 Na+, 1 mM-Ca2+ solution contained (in mM): N-methyl-D-glucamine chloride, (NMDG-Cl) 145; CaCl2, 1 and MgCl2, 1. The 0 Na+, 1 mM-Cd+ solution contained (in mM): NMDG-Cl, 145; CaCl2, 1; MgCl2, 1; CdCl2, 1 and LaCl3, 0 1. All solutions also contained (in mM): HEPES, 10; glucose, 10 and tetrodotoxin, 0-03. Experiments were done at room temperature (20-23 °C). Cell length was measured from a fixed point near the pipette to the left end of the cell with a video-dimension analyser. Series resistance compensation was used during the experiments, and current records were filtered at 5-10 kHz. Gating current mea8urements Gating current measurements were made in the 0 Na+, 1 mM-Cd+ external solution which isolates currents that arise from intramembrane charge movement in these cells (Bean & Rios, 1989, Hadley & Lederer, 1989a). Charge movement records were constructed by subtracting linear capacitative currents from the record of interest. The capacitative current records were obtained by a separate voltage-clamp pulse consisting of a hyperpolarization from a holding potential of -100 to -140 mV. The Ca2+ channel gating current was isolated by holding the cell at -50 mV for several seconds before applying test depolarizations. Charge movement records obtained in this way are judged to be predominantly those of L-type Ca2+ channel gating currents. Evidence indicating that this current is an L-type Ca2+ channel gating current includes the linear relationship between the amount of charge moved and the degree of Ca2+ channel activation, the linear relationship between the degree of charge immobilization and voltage-dependent inactivation, and the fact that the current can be completely inhibited by dihydropyridine Ca2+ channel blockers (Hadley & Lederer, 1989b, 1990, 1991 b). Qon, the gating charge moved during the test depolarization, was measured by defining the steady-state current level as baseline, and integrating over the time of the depolarization. The steady-state current level was defined as the current mean over the last few milliseconds of the depolarization.
Ca2+ photoreleawe Either 2 mM-DM-nitrophen or nitr-5 plus 05-1O0 mM-CaC12 was included in the pipette when Ca2+ photorelease was desired. Calcium release was induced by a 0-2 ms flash of ultraviolet (UV) light generated by a discharge through a xenon arc flashlamp (Chadwick Helmuth, El Monte, CA, USA) of between 40 and 230 W s. The flash intensity values reflect the energy discharged by the flashlamp before attenuation along the optical path. Dichroic mirrors were used to select wavelengths between 310 and 390 nm before the flash was focused on the cell with a 63 x objective. See Niggli & Lederer (1990) for further details. Exposure of these chelators to UV light results in the rapid cleavage of some of the molecules, and a decrease in their binding affinity for Ca2+ (Kaplan, 1990). Flash-induced twitches using DM-nitrophen were of comparable magnitude to those induced by electrical stimulation (Niggli & Lederer, 1990). In addition, preliminary experiments where [Ca2+]1 was measured with the fluorescent calcium indicator Indo-1 during Ca2+ photorelease from DM-nitrophen have indicated that [Ca2+]i rises to at least 500 nm after a 200 W s discharge under similar conditions (M. S. Kirby, R. W. Hadley and W. J. Lederer, unpublished). These measurements were made 20 ms after the flash in order to allow flash-induced artifacts to settle, and used an optical path whose efficiency was 30 % of that used for the present experiments. RESULTS
While photolytic Ca2+ release produces block or inactivation of Ca2+ channels in chick dorsal root ganglion (DRG) neurones (Morad, Davies, Kaplan & Lux, 1988), other work has shown that it primarily potentiates cardiac Ca2+ current (ICa) (Gurney, Charnet, Pye & Nargeot, 1989). Figure 1 shows three records of the Ltype ICa in guinea-pig ventricular myocytes, which are superimposed for comparison. The ICa record marked ' 1 ' was obtained under control conditions, while the ICa record marked '2' was obtained 20 ms after the photorelease of Ca2+ from intracellular DM-
R. W. HADLEY AND W. J. LEDERER
260
nitrophen. The sudden rise in [Ca2+]i produced by the photolysis led to considerable ICa inactivation, as peak ICa was diminished. Flashes in the absence of intracellular DM-nitrophen had no effect on 'Ca' nor did flashes have any effect when DMnitrophen was present, but not loaded with Ca2+ (data not shown). In addition, Ca + 1000
0
? -1000
-2000
2>
-3000
'50 ms Fig. 1. Effect of photolytic Ca2+ release from DM-nitrophen on Ic. in guinea-pig ventricular myocytes. The cell was flashed with UV light (200 W s flashlamp discharge) 20 ms before the second trace. The record marked '1' was obtained 5 s before the flash, while the record marked '3' was obtained 10 s after the flash. Ic, was recorded in the 120 mM-Na+, 2-5 mm-Ca2+ external solution and was elicited by 200 ms voltage-clamp steps from -50 to 0 mV at 0-2 Hz. -4000
photorelease from DM-nitrophen still produced considerable inactivation when the concentration of HEPES in the pipette solution was increased to 100 mm. Thus, the inactivation induced by the UV flash is Ca2+ dependent. Trace 3 shows that, under these conditions (120 mM-external Na+), ICa had completely recovered by 10 s after the flash. On average, ICa was reduced to 0-77 + 0-13% of control (n = 8) with flashlamp discharges of 200 W s. In an effort to understand the differences between these findings and those of Gurney et al. (1989) we carefully examined ICa for photorelease-dependent potentiation. We rarely observed potentiation of ICa when DM-nitrophen was used (one of fifteen cells in 120 mM-Na+, 2-5 mM-Ca2+ solution). However, potentiation was often observed when similar experiments were performed with nitr-5, the photolabile Ca2+ chelator used by Gurney et al. (1989). Figure 2 shows the effects of Ca2+ photorelease from nitr-5 on Ic at two different flash energies (thus varying the extent of photorelease). The weaker flash (panel A) produced a small potentiation of ICa' with no sign of inactivation. However, a stronger flash (panel B) produced a stronger potentiation, with a minor inactivation of Ic. occurring immediately after the flash. These experiments were repeated in eight cells, and the average maximal potentiation was 48 ± 29 %, which is significant as judged by a Student's one-tailed, paired t test (P < 0-05). It is also worth noting that in this experiment, the
F C_-DEPENDENT INACTIVATION OF Ca2 CHANNELS
261
potentiation of ICa was accompanied by some slowing of ICa decay. However, this particular effect was more prominent in this instance than it was in others. Figure 3 compares the effects of voltage- and Ca2+-dependent inactivation on the Ca2+ channel gating current. The two traces in panel A show Ca2+ channel gating A
Journal of Physiology (1991), 444, pp. 257-268 With 6 figure8 Printed in Great Britain
Ca2l AND VOLTAGE INACTIVATE Ca2l CHANNELS IN GUINEA-PIG VENTRICULAR MYOCYTES THROUGH INDEPENDENT MECHANISMS
BY ROBERT W. HADLEY* AND W. J. LEDERER From the Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA
(Received 18 April 1991) SUMMARY
1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca2+-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca2+dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca2+-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca2+-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca2+-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca2+-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca2+-dependent inactivation is entirely sensitive to ,* To whom correspondence should be addressed. MS 9311
9-2
R. W. HADLEY AND W. J. LEDERER 258 adrenergic stimulation. These facts suggest that either Ca2+-dependent inactivation results from Ca2+-dependent dephosphorylation of the Ca2+ channel, or that Ca2+dependent inactivation is modulated by protein kinase A. INTRODUCTION
A rise in the concentration of intracellular Ca2+ ([Ca2+]i) inactivates Ca2+ channels in many cell types, providing control of Ca2+ influx through a negative feedback mechanism (Eckert & Chad, 1984). However, the molecular events underlying Ca2+dependent Ca2+ channel inactivation have been disputed and have proven challenging to study. A number of possible mechanisms have been considered, including allosteric regulation of Ca2+ channel function (Plant, Standen & Ward, 1983), modulation of voltage-dependent inactivation by Ca2+ (Lee, Marban & Tsien, 1985), and activation of Ca2+-dependent phosphatases (Chad & Eckert, 1986). While this last hypothesis is supported by repeated suggestions that only phosphorylated Ca2+ channels are functional (Sperelakis & Schneider, 1976; Reuter & Scholz, 1977; Bean, Nowycky & Tsien, 1984; Armstrong & Eckert, 1987; Hymel, Striessnig, Glossmann & Schindler, 1988; Nunoki, Florio & Catterall, 1989), an absolute requirement for channel phosphorylation has been disputed vigorously (Kameyama, Hescheler, Hofmann & Trautwein, 1986; Kameyama, Hescheler, Mieskes & Trautwein, 1986; Trautwein, Cavalie, Flockerzi, Hofmann & Pelzer, 1987). Furthermore, even if this point were conceded it would still leave open the question of whether a rise in [Ca2+]i promotes Ca2+ channel dephosphorylation (Byerly, Leung & Yazejian, 1988). We have therefore reinvestigated the mechanism of Ca2+dependent inactivation in isolated cardiac ventricular myocytes by combining the recently developed techniques of photolytic Ca2+ release and measurement of cardiac Ca2+ channel gating current. By activating the photorelease of Ca2+ independently of voltage-clamp steps we have been able to show that voltage and Ca2+ inactivate the Ca2+ channel through completely separate mechanisms. Furthermore, Ca2+dependent inactivation is entirely sensitive to fl-adrenergic stimulation. These studies therefore suggest that different regions of the Ca2+ channel protein are involved in the two types of Ca2+ channel inactivation. A preliminary note on these experiments has been published (Hadiey & Lederer, 1991 a). METHODS
Cell preparation Ventricular myocytes were enzymatically isolated using the procedure of Mitra & Morad (1985). Briefly, guinea-pigs were killed while under deep, pentobarbitone (100 mg kg-')-induced anaesthesia, and their hearts were removed and transferred to a Langendorff apparatus. The hearts were perfused with a solution containing (in mM): NaCl, 135; KCl, 5-4; MgCl2, 1; NaH2PO4, 0 33; glucose, 11 and HEPES, 10, at a room temperature of 37 'C. After a short perfusion period, collagenase (0 5 mg ml-') and protease (0 1 mg ml-') were added to the perfusing solution. After an additional 3-5 min, the hearts were removed and minced into small pieces, which were placed into the above solution with 0-2 mM-CaC12 added. Rod-shaped myocytes appeared in the solution shortly thereafter. Experimental procedures The isolated ventricular myocytes were voltage clamped using the whole-cell patch clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Pipette resistances ranged from 0 5 to 2 MQ. The standard pipette solution contained (in mM): caesium glutamate, 105; tetra-
Ca2+-DEPENDENT INACTIVATION OF Ca2+ CHANNELS
259
ethylammonium chloride, 10; CsCl, 20; HEPES, 20; K2ATP, 5. The pH was adjusted to 7-4 with CsOH. K2ATP was used to keep Mg2+ concentrations as low as possible for the DM-nitrophen experiments. K2ATP was usually replaced with MgATP (0-2 mM) for the nitr-5 experiments. Several external solutions were used. The 120 mM-Na+, 2-5 mM-Ca2+ solution contained (in mM): NaCl, 120; KCl, 4; CaCl2, 2-5; MgCI2, 0 5 and CsCl, 20. The 0 Na+, 1 mM-Ca2+ solution contained (in mM): N-methyl-D-glucamine chloride, (NMDG-Cl) 145; CaCl2, 1 and MgCl2, 1. The 0 Na+, 1 mM-Cd+ solution contained (in mM): NMDG-Cl, 145; CaCl2, 1; MgCl2, 1; CdCl2, 1 and LaCl3, 0 1. All solutions also contained (in mM): HEPES, 10; glucose, 10 and tetrodotoxin, 0-03. Experiments were done at room temperature (20-23 °C). Cell length was measured from a fixed point near the pipette to the left end of the cell with a video-dimension analyser. Series resistance compensation was used during the experiments, and current records were filtered at 5-10 kHz. Gating current mea8urements Gating current measurements were made in the 0 Na+, 1 mM-Cd+ external solution which isolates currents that arise from intramembrane charge movement in these cells (Bean & Rios, 1989, Hadley & Lederer, 1989a). Charge movement records were constructed by subtracting linear capacitative currents from the record of interest. The capacitative current records were obtained by a separate voltage-clamp pulse consisting of a hyperpolarization from a holding potential of -100 to -140 mV. The Ca2+ channel gating current was isolated by holding the cell at -50 mV for several seconds before applying test depolarizations. Charge movement records obtained in this way are judged to be predominantly those of L-type Ca2+ channel gating currents. Evidence indicating that this current is an L-type Ca2+ channel gating current includes the linear relationship between the amount of charge moved and the degree of Ca2+ channel activation, the linear relationship between the degree of charge immobilization and voltage-dependent inactivation, and the fact that the current can be completely inhibited by dihydropyridine Ca2+ channel blockers (Hadley & Lederer, 1989b, 1990, 1991 b). Qon, the gating charge moved during the test depolarization, was measured by defining the steady-state current level as baseline, and integrating over the time of the depolarization. The steady-state current level was defined as the current mean over the last few milliseconds of the depolarization.
Ca2+ photoreleawe Either 2 mM-DM-nitrophen or nitr-5 plus 05-1O0 mM-CaC12 was included in the pipette when Ca2+ photorelease was desired. Calcium release was induced by a 0-2 ms flash of ultraviolet (UV) light generated by a discharge through a xenon arc flashlamp (Chadwick Helmuth, El Monte, CA, USA) of between 40 and 230 W s. The flash intensity values reflect the energy discharged by the flashlamp before attenuation along the optical path. Dichroic mirrors were used to select wavelengths between 310 and 390 nm before the flash was focused on the cell with a 63 x objective. See Niggli & Lederer (1990) for further details. Exposure of these chelators to UV light results in the rapid cleavage of some of the molecules, and a decrease in their binding affinity for Ca2+ (Kaplan, 1990). Flash-induced twitches using DM-nitrophen were of comparable magnitude to those induced by electrical stimulation (Niggli & Lederer, 1990). In addition, preliminary experiments where [Ca2+]1 was measured with the fluorescent calcium indicator Indo-1 during Ca2+ photorelease from DM-nitrophen have indicated that [Ca2+]i rises to at least 500 nm after a 200 W s discharge under similar conditions (M. S. Kirby, R. W. Hadley and W. J. Lederer, unpublished). These measurements were made 20 ms after the flash in order to allow flash-induced artifacts to settle, and used an optical path whose efficiency was 30 % of that used for the present experiments. RESULTS
While photolytic Ca2+ release produces block or inactivation of Ca2+ channels in chick dorsal root ganglion (DRG) neurones (Morad, Davies, Kaplan & Lux, 1988), other work has shown that it primarily potentiates cardiac Ca2+ current (ICa) (Gurney, Charnet, Pye & Nargeot, 1989). Figure 1 shows three records of the Ltype ICa in guinea-pig ventricular myocytes, which are superimposed for comparison. The ICa record marked ' 1 ' was obtained under control conditions, while the ICa record marked '2' was obtained 20 ms after the photorelease of Ca2+ from intracellular DM-
R. W. HADLEY AND W. J. LEDERER
260
nitrophen. The sudden rise in [Ca2+]i produced by the photolysis led to considerable ICa inactivation, as peak ICa was diminished. Flashes in the absence of intracellular DM-nitrophen had no effect on 'Ca' nor did flashes have any effect when DMnitrophen was present, but not loaded with Ca2+ (data not shown). In addition, Ca + 1000
0
? -1000
-2000
2>
-3000
'50 ms Fig. 1. Effect of photolytic Ca2+ release from DM-nitrophen on Ic. in guinea-pig ventricular myocytes. The cell was flashed with UV light (200 W s flashlamp discharge) 20 ms before the second trace. The record marked '1' was obtained 5 s before the flash, while the record marked '3' was obtained 10 s after the flash. Ic, was recorded in the 120 mM-Na+, 2-5 mm-Ca2+ external solution and was elicited by 200 ms voltage-clamp steps from -50 to 0 mV at 0-2 Hz. -4000
photorelease from DM-nitrophen still produced considerable inactivation when the concentration of HEPES in the pipette solution was increased to 100 mm. Thus, the inactivation induced by the UV flash is Ca2+ dependent. Trace 3 shows that, under these conditions (120 mM-external Na+), ICa had completely recovered by 10 s after the flash. On average, ICa was reduced to 0-77 + 0-13% of control (n = 8) with flashlamp discharges of 200 W s. In an effort to understand the differences between these findings and those of Gurney et al. (1989) we carefully examined ICa for photorelease-dependent potentiation. We rarely observed potentiation of ICa when DM-nitrophen was used (one of fifteen cells in 120 mM-Na+, 2-5 mM-Ca2+ solution). However, potentiation was often observed when similar experiments were performed with nitr-5, the photolabile Ca2+ chelator used by Gurney et al. (1989). Figure 2 shows the effects of Ca2+ photorelease from nitr-5 on Ic at two different flash energies (thus varying the extent of photorelease). The weaker flash (panel A) produced a small potentiation of ICa' with no sign of inactivation. However, a stronger flash (panel B) produced a stronger potentiation, with a minor inactivation of Ic. occurring immediately after the flash. These experiments were repeated in eight cells, and the average maximal potentiation was 48 ± 29 %, which is significant as judged by a Student's one-tailed, paired t test (P < 0-05). It is also worth noting that in this experiment, the
F C_-DEPENDENT INACTIVATION OF Ca2 CHANNELS
261
potentiation of ICa was accompanied by some slowing of ICa decay. However, this particular effect was more prominent in this instance than it was in others. Figure 3 compares the effects of voltage- and Ca2+-dependent inactivation on the Ca2+ channel gating current. The two traces in panel A show Ca2+ channel gating A
