91
Journal of Physiology (1990), 423, pp. 91 -110 With 8 figures Printed in Great Britain
THE REGULATION OF ATP-SENSITIVE K+ CHANNEL ACTIVITY IN INTACT AND PERMEABILIZED RAT VENTRICULAR MYOCYTES BY C. G. NICHOLS* AND W. J. LEDERER From the Department of Physiology, University of Maryland, 660 West Redwood Street, Baltimore, MD 21201, USA
(Received 24 April 1989) SUMMARY
1. In isolated rat heart ventricular myocytes exposed to 2 mM-cyanide in the presence of 10 mM-2-deoxyglucose (complete metabolic blockade), there is a timedependent increase in ATP-sensitive potassium (KATP) channel activity. The increase in KATP channel activity accompanies the decline of twitch amplitude. Channel activation and decline of the twitch amplitude precede the development of a ' rigor' contracture. 2. We measured KATP channel activity in permeabilized cells using the open-cell attached (O-C-A) patch configuration (by establishing a cell-attached patch and then permeabilizing the cell by exposure to saponin). The apparent ATP dependence of KATP channel activity could be described by a sigmoid curve with ki ATP (ATP concentration required for half-maximum inhibition of channel activity) = 122 ,JM and H (Hill coefficient) = 1'225. 3. In the O-C-A patch configuration, 10 mM-creatine phosphate (CrP) decreased the apparent ki, ATP from 122 IBM to about 10 /tm, and the maximal activity (in zero ATP) was decreased to about 30 % of the maximal activity in the absence of CrP. 4. In isolated inside-out (1-0) patches, ATP inhibited KATP channel activity at much lower [ATP] than in the O-C-A patch configuration (ki ATP = 25 /,M, H = 2). CrP was without effect on 1-0 patches. 5. These results are consistent with the hypothesis that the difference in the ATP dependence of KATP channel activity in the O-C-A and 1-0 patch configurations arises because of ATP consumption in the O-C-A patch configuration. The results suggest that hydrolysis of ATP to ADP by endogenous ATPases leads to the development of gradients of [ATP] and [ADP] between the bath and the 'inside' of the open cell. By re-phosphorylating ADP, CrP is able to dissipate these gradients, revealing the 'true' ATP dependence of channel activity, which is the same as that in the 1-0 patch configuration. 6. In order to estimate the contribution of KATP channel activity to the rat cardiac action potential at different [ATP] we have made the following measurements. Using electrodes of resistance 2-8 MCI the density of KATP channels was 10-3 + 0-1 channels per patch (n = 162). The single KATP channel current-voltage relationship in 4 mMexternal K+ is approximately linear (between -80 and + 20 mV) with a conductance of 23 pS. In the presence of 200 /ZM-ADP, 50 ptm-GDP and 1 mM-free Mg2+, the ATP MS 7648
C. G. NICHOLS AND W. J. LEDERER dependence of channel activity was well fitted with ki,ATP = 1OOUM and Hill coefficient = 2. 7. We have incorporated these relationships into a rat ventricular cell action potential model. The model predicts that the action potential duration will shorten as KATP channel activity increases and that significant shortening of the action potential may result from changes of [ATP] in the millimolar range. 92
INTRODUCTION
K+-selective channels that are inhibited by intracellular ATP (KATP channels) have been implicated in the shortening of the cardiac action potential under conditions of metabolic blockade (Noma, 1983; Trube & Hescheler, 1984; Allen & Orchard, 1987; Belles, Hescheler & Trube, 1987; Fosset, De Weille, Green, SchmidAntomarchi & Lazdunski, 1988; Elliott, Smith & Allen, 1989). Evidence has recently been provided to show that, by shortening the action potential, KATP currents are largely responsible for early failure of contraction in rat ventricular myocytes exposed to complete metabolic blockade (Lederer, Nichols & Smith, 1989) or substrate-free hypoxia (Stern, Silverman, Houser, Josephson, Capogrossi, Nichols, Lederer & Lakatta, 1988), and in ferret hearts exposed to metabolic blockade (Elliott et al. 1989). The mechanism by which the current is activated in these situations remains a matter of controversy. In isolated patches, KATP channels are blocked by micromolar levels of ATP (Noma, 1983; Findlay, 1988 a, b). Biochemical and nuclear magnetic resonance measurements of [ATP] in the whole heart suggest that in metabolic blockade, mechanical failure and action potential shortening occur at millimolar levels of ATP (Williamson, 1966; Hearse, 1979; Matthews, Radda & Taylor, 1981; Allen, Morris, Orchard & Pirolo, 1985; Elliott et al. 1989) and it has therefore been argued that this [ATP] is too high for decreased [ATP] to be the mechanism underlying action potential shortening and twitch failure (Trube & Hescheler, 1984; Allen et al. 1985; Elliott et al. 1989). A possible solution to this controversy is that the ki, ATP ([ATP] causing halfmaximal inhibition of channel activity) in vivo is actually much higher than that measured in isolated patches, i.e. some modulator of ATP sensitivity is lost on isolation of the patch. Kakei, Noma & Shibasaki (1985) and Noma & Shibasaki (1985) reported that the ATP sensitivity in the open-cell attached (0-C-A) patch, and whole-cell configurations (where ki, ATP = 500 pM) is greater than in inside-out patches (ki, ATP = 20-100 ,M; Noma, 1983; Findlay, 1988a; Lederer & Nichols, 1989). This has been taken as suggestive evidence that the ATP sensitivity of KATP channels in vivo may be altered by some cytosolic, or cyto-skeletal, element that is lost on patch excision (Noma & Shibasaki, 1985; Elliott et al. 1989). The difference between results obtained in open-cell attached and inside-out patches needs explanation if we are to understand the regulation of KATP channels in vivo. In this study, we have investigated the ATP sensitivity of KATP channels in permeabilized rat heart cells using the O-C-A patch configuration (Kakei et al. 1985). Our results suggest that measurements of ATP sensitivity in this preparation are confounded by [ATP] gradients, and that the presence of [ATP] gradients may be shifting the true ATP dependence (Lederer & Nichols, 1989). The true ATP
KATP CHANNEL ACTIVITY IN RAT HEART 93 dependence of channel activity in the O-C-A patch configuration appears to be the same as that found in isolated inside-out membrane patches, with channel blockade occurring at micromolar [ATP]. However, channel density is sufficiently high that even given this sensitivity to ATP, we predict that channel activity will affect membrane potential when ATP falls below, or ADP rises above, normal levels. Preliminary reports of some of the findings of this study has been given to the Physiological Society (Lederer & Nichols, 1988) and to the Biophysical Society (Nichols & Lederer, 1989 a). METHODS
Preparation of single rat ventricular myocytes Single ventricular myocytes were obtained from adult rat hearts by established enzymatic dissociation techniques (see Lederer & Nichols, 1989). Experimental and analysis Experiments were performed at room temperature, in one of two experimental chambers that were mounted on the stage of an inverted microscope (Nikon, Inc., Garden City, NY, USA). The first chamber (Cannell & Lederer, 1986), used in whole-cell, cell attached and open-cell attached patch experiments, allowed rapid changes of the solution bathing the cells. In some of these experiments, cell images were recorded throughout the experiment with a CCD video camera (Pulnix Inc., Sunnyvale, CA, USA) mounted on the microscope. Cell length could be measured from the video signal by a video dimension analyser (Steadman, Moore, Spitzer & Bridge, 1988). Microelectrodes (2-8 MO) were pulled from thin-walled filamented borosilicate glass (1-5 mm o.d., TW150F-6, WPI Inc., New Haven, CT, USA) on a horizontal puller (BB-CH Mechanex, Geneva, Switzerland). The open-cell attached patch configuration was obtained as follows. After formation of an on-cell patch, a second (large-bore) microelectrode filled with high-K+, zero-Na+ solution containing 1 % saponin (w/v) was brought up to the downstream end of the cell. Positive pressure was applied to the rear of the saponin-containing pipette to eject solution onto the surface of the cell. Permeabilization was assessed by the development of a slight swelling of the cell within 1-5 s of exposure to saponin (Kakei et al. 1985). The saponin-containing pipette was then withdrawn. A second type of chamber (Qin & Noma, 1988) was used in the isolated patch experiments. The experimental details of the fabrication, and use, of this 'oil-gate' chamber are described in Lederer & Nichols (1989). Patch-clamp currents were recorded using a Dagan Model 8900 Patch Clamp (Dagan Corp., Minneapolis, MN, USA) with a 10 GOQ headstage, and filtered at 1 or 10 kHz. Signals were digitized at 22 kHz (Neurocorder, Neurodata, New York, NY, USA) and stored on videotape. Experiments were replayed through an 8-pole Bessel filter (at 500 or 1000 Hz) and digitized at 20 kHz into a microcomputer (Compaq Computer Corp., Houston, TX, USA; Data Translation, Marlborough, MA, USA) using Vacuum Mk II software (M.B.C. Systems, Baltimore, MD, USA) for subsequent analysis using Lotus 1-2-3 (Lotus Development Corp., Cambridge, MA, USA).
Pipette solutions Pipettes were filled with either high-K+ (extracellular) solution containing (mM): 140 KCl; 1 KEGTA, 10 K-HEPES,; or with low-K+ (extracellular) solution containing (mM): 140 NaCl, 4 KCl, 1 CaCl2, 5 Na-HEPES, each buffered to pH 7-25.
Bathing solutions Intact cells. During experiments the cells were bathed in a solution of the following composition (mM): 135 NaCl, 4 KCl, 1 MgCl2, 0 or 2 CaCl2, and 10 glucose. The solution pH was 7-4. Glucose was replaced by 10 mM-2-deoxyglucose at least 20 min before exposure to cyanide. Sodium cyanide (2 mM) was added from a concentrated solution (200 mM-NaCN, 670 mM-HEPES) immediately before use. Deoxyglucose and cyanide were added to inhibit glycolysis and oxidative phosphorylation respectively (cf. Allen et al. 1985). Permeabilized cells. Before permeabilization, cells were perfused with an extracellular solution
C. G. NICHOLS AND W. J. LEDERER containing (mm): 135 NaCi, 4 KCl, 1 MgCl2, 10 HEPES, and 10 glucose. The pH was 7-3 and the solution was nominally Ca2+ free. This prevented the development of [Ca2+], overload when perfusion was switched to the following (intracellular) solution (mm): 140 KCl, 10 K-HEPES, 0-5 MgCl2, 4 or 2 K-ATP, 1 K-EGTA. Solution pH was 7-25. inside-out patches, the bath (intracellular) InIide-out isolated patches. In the experiments using Mg2+, 1 K-EGTA. Solution pH was 7-25. solution contained (mm): 140 KCl, 10 HEPES, 10-5 free 2 Patches were isolated in solution containing an additional 2mM-K-ATP andmM-MgCl2.
94
94
RESULTS
KATP channel activation in rat ventricular myocytes
high-K+
In normal (low-K+) extracellular solutions, with a pipette solution at the outer surface of the patch membrane, KATP channel activity was very rarely to bathing solutions observed in on-cell patches. After addition of 2 of K+ channels a increase in the activity 10 containing mm-2-deoxyglucose, large of after the addition cyanide (Fig. could eventually be recorded, typically 1-15 The onset of channel activity preceded the onset of contracture development by 1A). 1-2 min. Assuming that the number of functional channels in the patch was approximated by the maximum observed number of overlaps of channel openings (in this case seven), then the maximum observed open-state probability at 0 mV (given byI/[ni], where I = mean patch current, n = number of channels observed in patch, i = single-channel current) varied between 0 4 and 0-9 (n = 5 experiments). In the experiment shown in Fig. 1B, field stimulation was used to initiate twitch contractions. This caused capacitative current surges that are recorded as regular spikes on the patch current record. It is apparent that the activation of KATP channels begins as the twitch amplitude begins to decline. The disappearance of the before the onset of contracture. twitch (lower panel) begins approximately This result is consistent with the results of Lederer et al. (1989) who showed that decline of the twitch under these conditions was due principally to a decrease in action potential duration caused by the activation of a large K+ conductance. The increase in K+ conductance is attributable to activation of KATP channels.
mM-cyanide
min
1 min
ATP dependence of KATP channels in open-cell attached patches Figure 2A shows typical records of patch membrane current and cell length from an experiment using the open-cell attached patch technique. At the start of the and a cell-attached patch was formed experiment, the cell was bathed in 2 approximately one-third of the way from one end of the cell. Immediately preceding the onset of the record, approximately half of the cell (away from the patch) was permeabilized by exposure to saponin (Kakei et al. 1985). At the start of the record, the bathing solution was switched to one containing 0 1 Approximately twenty-five KATP channels were activated. Lowering the bath concentration of ATP ([ATP]) to less than mm generally caused no change or increased, channel activity but always increased the rate of run-down of channel activity. In order to minimize the rate of channel run-down, exposure to was always used to obtain a control measure of channel activity (Kakei et al. 1985). The experiment of Fig. 2A illustrates the protocol used to measure the ATP dependence of open-cell attached channel activity. The cell was exposed to 01 again. in Fig. 2A) and then to ATP, then to a test solution (0 4
mM-ATP
mM-ATP.
0.1
mM-ATP
in, 0-1 mM-ATP
041 mM-ATP
mM-
95 KATP CHANNEL ACTIVITY IN RAT HEART Between exposures to low-ATP solutions, the preparation was exposed to high (2 or 4 mM) ATP for at least 2 min in a further attempt to minimize channel run-down (Ohno-Shosaku, Zunkler & Trube, 1987; Findlay, 1988 b). The patch current was averaged over 1-2 min after attaining a steady level, and the average current in the A
CN-
Cal
0
E
_0
'D
135e 1 min
CN-
B
-E 100 r /
_
120L 1 min
Fig. 1. Activation of KATP channels in rat ventricular myocytes exposed to complete metabolic blockade. A and B, slow time-base records of on-cell patch current (above), and cell length (below). 2-Deoxyglucose (10 mM) was present throughout. Cyanide (2 mM) was applied for the time indicated by the bar. In B, note that externally applied stimuli trigger twitch contractions, and induce a large artifact on the current record. The onset of KATP channel activity precedes the onset of contracture (A) and accompanies the decline of the twitch (B). Channel activity reversed on removal of cyanide (A).
test solutions was expressed as a fraction of that measured in the steady state at 041 mM-ATP. Where more than 50% run-down occurred between initial and final exposure to 0-1 mM-ATP, the data were rejected from analysis. For test solution = zero ATP, channel run-down was rapid and these acceptance criteria were not employed. In these experiments (n = 8), post-control channel activity was 23-6 + 5-5 % of precontrol activity. Thus, the data obtained in zero ATP may be less reliable than the data obtained with other test solutions.
C. G. NICHOLS AND W. J. LEDERER
96 A
Journal of Physiology (1990), 423, pp. 91 -110 With 8 figures Printed in Great Britain
THE REGULATION OF ATP-SENSITIVE K+ CHANNEL ACTIVITY IN INTACT AND PERMEABILIZED RAT VENTRICULAR MYOCYTES BY C. G. NICHOLS* AND W. J. LEDERER From the Department of Physiology, University of Maryland, 660 West Redwood Street, Baltimore, MD 21201, USA
(Received 24 April 1989) SUMMARY
1. In isolated rat heart ventricular myocytes exposed to 2 mM-cyanide in the presence of 10 mM-2-deoxyglucose (complete metabolic blockade), there is a timedependent increase in ATP-sensitive potassium (KATP) channel activity. The increase in KATP channel activity accompanies the decline of twitch amplitude. Channel activation and decline of the twitch amplitude precede the development of a ' rigor' contracture. 2. We measured KATP channel activity in permeabilized cells using the open-cell attached (O-C-A) patch configuration (by establishing a cell-attached patch and then permeabilizing the cell by exposure to saponin). The apparent ATP dependence of KATP channel activity could be described by a sigmoid curve with ki ATP (ATP concentration required for half-maximum inhibition of channel activity) = 122 ,JM and H (Hill coefficient) = 1'225. 3. In the O-C-A patch configuration, 10 mM-creatine phosphate (CrP) decreased the apparent ki, ATP from 122 IBM to about 10 /tm, and the maximal activity (in zero ATP) was decreased to about 30 % of the maximal activity in the absence of CrP. 4. In isolated inside-out (1-0) patches, ATP inhibited KATP channel activity at much lower [ATP] than in the O-C-A patch configuration (ki ATP = 25 /,M, H = 2). CrP was without effect on 1-0 patches. 5. These results are consistent with the hypothesis that the difference in the ATP dependence of KATP channel activity in the O-C-A and 1-0 patch configurations arises because of ATP consumption in the O-C-A patch configuration. The results suggest that hydrolysis of ATP to ADP by endogenous ATPases leads to the development of gradients of [ATP] and [ADP] between the bath and the 'inside' of the open cell. By re-phosphorylating ADP, CrP is able to dissipate these gradients, revealing the 'true' ATP dependence of channel activity, which is the same as that in the 1-0 patch configuration. 6. In order to estimate the contribution of KATP channel activity to the rat cardiac action potential at different [ATP] we have made the following measurements. Using electrodes of resistance 2-8 MCI the density of KATP channels was 10-3 + 0-1 channels per patch (n = 162). The single KATP channel current-voltage relationship in 4 mMexternal K+ is approximately linear (between -80 and + 20 mV) with a conductance of 23 pS. In the presence of 200 /ZM-ADP, 50 ptm-GDP and 1 mM-free Mg2+, the ATP MS 7648
C. G. NICHOLS AND W. J. LEDERER dependence of channel activity was well fitted with ki,ATP = 1OOUM and Hill coefficient = 2. 7. We have incorporated these relationships into a rat ventricular cell action potential model. The model predicts that the action potential duration will shorten as KATP channel activity increases and that significant shortening of the action potential may result from changes of [ATP] in the millimolar range. 92
INTRODUCTION
K+-selective channels that are inhibited by intracellular ATP (KATP channels) have been implicated in the shortening of the cardiac action potential under conditions of metabolic blockade (Noma, 1983; Trube & Hescheler, 1984; Allen & Orchard, 1987; Belles, Hescheler & Trube, 1987; Fosset, De Weille, Green, SchmidAntomarchi & Lazdunski, 1988; Elliott, Smith & Allen, 1989). Evidence has recently been provided to show that, by shortening the action potential, KATP currents are largely responsible for early failure of contraction in rat ventricular myocytes exposed to complete metabolic blockade (Lederer, Nichols & Smith, 1989) or substrate-free hypoxia (Stern, Silverman, Houser, Josephson, Capogrossi, Nichols, Lederer & Lakatta, 1988), and in ferret hearts exposed to metabolic blockade (Elliott et al. 1989). The mechanism by which the current is activated in these situations remains a matter of controversy. In isolated patches, KATP channels are blocked by micromolar levels of ATP (Noma, 1983; Findlay, 1988 a, b). Biochemical and nuclear magnetic resonance measurements of [ATP] in the whole heart suggest that in metabolic blockade, mechanical failure and action potential shortening occur at millimolar levels of ATP (Williamson, 1966; Hearse, 1979; Matthews, Radda & Taylor, 1981; Allen, Morris, Orchard & Pirolo, 1985; Elliott et al. 1989) and it has therefore been argued that this [ATP] is too high for decreased [ATP] to be the mechanism underlying action potential shortening and twitch failure (Trube & Hescheler, 1984; Allen et al. 1985; Elliott et al. 1989). A possible solution to this controversy is that the ki, ATP ([ATP] causing halfmaximal inhibition of channel activity) in vivo is actually much higher than that measured in isolated patches, i.e. some modulator of ATP sensitivity is lost on isolation of the patch. Kakei, Noma & Shibasaki (1985) and Noma & Shibasaki (1985) reported that the ATP sensitivity in the open-cell attached (0-C-A) patch, and whole-cell configurations (where ki, ATP = 500 pM) is greater than in inside-out patches (ki, ATP = 20-100 ,M; Noma, 1983; Findlay, 1988a; Lederer & Nichols, 1989). This has been taken as suggestive evidence that the ATP sensitivity of KATP channels in vivo may be altered by some cytosolic, or cyto-skeletal, element that is lost on patch excision (Noma & Shibasaki, 1985; Elliott et al. 1989). The difference between results obtained in open-cell attached and inside-out patches needs explanation if we are to understand the regulation of KATP channels in vivo. In this study, we have investigated the ATP sensitivity of KATP channels in permeabilized rat heart cells using the O-C-A patch configuration (Kakei et al. 1985). Our results suggest that measurements of ATP sensitivity in this preparation are confounded by [ATP] gradients, and that the presence of [ATP] gradients may be shifting the true ATP dependence (Lederer & Nichols, 1989). The true ATP
KATP CHANNEL ACTIVITY IN RAT HEART 93 dependence of channel activity in the O-C-A patch configuration appears to be the same as that found in isolated inside-out membrane patches, with channel blockade occurring at micromolar [ATP]. However, channel density is sufficiently high that even given this sensitivity to ATP, we predict that channel activity will affect membrane potential when ATP falls below, or ADP rises above, normal levels. Preliminary reports of some of the findings of this study has been given to the Physiological Society (Lederer & Nichols, 1988) and to the Biophysical Society (Nichols & Lederer, 1989 a). METHODS
Preparation of single rat ventricular myocytes Single ventricular myocytes were obtained from adult rat hearts by established enzymatic dissociation techniques (see Lederer & Nichols, 1989). Experimental and analysis Experiments were performed at room temperature, in one of two experimental chambers that were mounted on the stage of an inverted microscope (Nikon, Inc., Garden City, NY, USA). The first chamber (Cannell & Lederer, 1986), used in whole-cell, cell attached and open-cell attached patch experiments, allowed rapid changes of the solution bathing the cells. In some of these experiments, cell images were recorded throughout the experiment with a CCD video camera (Pulnix Inc., Sunnyvale, CA, USA) mounted on the microscope. Cell length could be measured from the video signal by a video dimension analyser (Steadman, Moore, Spitzer & Bridge, 1988). Microelectrodes (2-8 MO) were pulled from thin-walled filamented borosilicate glass (1-5 mm o.d., TW150F-6, WPI Inc., New Haven, CT, USA) on a horizontal puller (BB-CH Mechanex, Geneva, Switzerland). The open-cell attached patch configuration was obtained as follows. After formation of an on-cell patch, a second (large-bore) microelectrode filled with high-K+, zero-Na+ solution containing 1 % saponin (w/v) was brought up to the downstream end of the cell. Positive pressure was applied to the rear of the saponin-containing pipette to eject solution onto the surface of the cell. Permeabilization was assessed by the development of a slight swelling of the cell within 1-5 s of exposure to saponin (Kakei et al. 1985). The saponin-containing pipette was then withdrawn. A second type of chamber (Qin & Noma, 1988) was used in the isolated patch experiments. The experimental details of the fabrication, and use, of this 'oil-gate' chamber are described in Lederer & Nichols (1989). Patch-clamp currents were recorded using a Dagan Model 8900 Patch Clamp (Dagan Corp., Minneapolis, MN, USA) with a 10 GOQ headstage, and filtered at 1 or 10 kHz. Signals were digitized at 22 kHz (Neurocorder, Neurodata, New York, NY, USA) and stored on videotape. Experiments were replayed through an 8-pole Bessel filter (at 500 or 1000 Hz) and digitized at 20 kHz into a microcomputer (Compaq Computer Corp., Houston, TX, USA; Data Translation, Marlborough, MA, USA) using Vacuum Mk II software (M.B.C. Systems, Baltimore, MD, USA) for subsequent analysis using Lotus 1-2-3 (Lotus Development Corp., Cambridge, MA, USA).
Pipette solutions Pipettes were filled with either high-K+ (extracellular) solution containing (mM): 140 KCl; 1 KEGTA, 10 K-HEPES,; or with low-K+ (extracellular) solution containing (mM): 140 NaCl, 4 KCl, 1 CaCl2, 5 Na-HEPES, each buffered to pH 7-25.
Bathing solutions Intact cells. During experiments the cells were bathed in a solution of the following composition (mM): 135 NaCl, 4 KCl, 1 MgCl2, 0 or 2 CaCl2, and 10 glucose. The solution pH was 7-4. Glucose was replaced by 10 mM-2-deoxyglucose at least 20 min before exposure to cyanide. Sodium cyanide (2 mM) was added from a concentrated solution (200 mM-NaCN, 670 mM-HEPES) immediately before use. Deoxyglucose and cyanide were added to inhibit glycolysis and oxidative phosphorylation respectively (cf. Allen et al. 1985). Permeabilized cells. Before permeabilization, cells were perfused with an extracellular solution
C. G. NICHOLS AND W. J. LEDERER containing (mm): 135 NaCi, 4 KCl, 1 MgCl2, 10 HEPES, and 10 glucose. The pH was 7-3 and the solution was nominally Ca2+ free. This prevented the development of [Ca2+], overload when perfusion was switched to the following (intracellular) solution (mm): 140 KCl, 10 K-HEPES, 0-5 MgCl2, 4 or 2 K-ATP, 1 K-EGTA. Solution pH was 7-25. inside-out patches, the bath (intracellular) InIide-out isolated patches. In the experiments using Mg2+, 1 K-EGTA. Solution pH was 7-25. solution contained (mm): 140 KCl, 10 HEPES, 10-5 free 2 Patches were isolated in solution containing an additional 2mM-K-ATP andmM-MgCl2.
94
94
RESULTS
KATP channel activation in rat ventricular myocytes
high-K+
In normal (low-K+) extracellular solutions, with a pipette solution at the outer surface of the patch membrane, KATP channel activity was very rarely to bathing solutions observed in on-cell patches. After addition of 2 of K+ channels a increase in the activity 10 containing mm-2-deoxyglucose, large of after the addition cyanide (Fig. could eventually be recorded, typically 1-15 The onset of channel activity preceded the onset of contracture development by 1A). 1-2 min. Assuming that the number of functional channels in the patch was approximated by the maximum observed number of overlaps of channel openings (in this case seven), then the maximum observed open-state probability at 0 mV (given byI/[ni], where I = mean patch current, n = number of channels observed in patch, i = single-channel current) varied between 0 4 and 0-9 (n = 5 experiments). In the experiment shown in Fig. 1B, field stimulation was used to initiate twitch contractions. This caused capacitative current surges that are recorded as regular spikes on the patch current record. It is apparent that the activation of KATP channels begins as the twitch amplitude begins to decline. The disappearance of the before the onset of contracture. twitch (lower panel) begins approximately This result is consistent with the results of Lederer et al. (1989) who showed that decline of the twitch under these conditions was due principally to a decrease in action potential duration caused by the activation of a large K+ conductance. The increase in K+ conductance is attributable to activation of KATP channels.
mM-cyanide
min
1 min
ATP dependence of KATP channels in open-cell attached patches Figure 2A shows typical records of patch membrane current and cell length from an experiment using the open-cell attached patch technique. At the start of the and a cell-attached patch was formed experiment, the cell was bathed in 2 approximately one-third of the way from one end of the cell. Immediately preceding the onset of the record, approximately half of the cell (away from the patch) was permeabilized by exposure to saponin (Kakei et al. 1985). At the start of the record, the bathing solution was switched to one containing 0 1 Approximately twenty-five KATP channels were activated. Lowering the bath concentration of ATP ([ATP]) to less than mm generally caused no change or increased, channel activity but always increased the rate of run-down of channel activity. In order to minimize the rate of channel run-down, exposure to was always used to obtain a control measure of channel activity (Kakei et al. 1985). The experiment of Fig. 2A illustrates the protocol used to measure the ATP dependence of open-cell attached channel activity. The cell was exposed to 01 again. in Fig. 2A) and then to ATP, then to a test solution (0 4
mM-ATP
mM-ATP.
0.1
mM-ATP
in, 0-1 mM-ATP
041 mM-ATP
mM-
95 KATP CHANNEL ACTIVITY IN RAT HEART Between exposures to low-ATP solutions, the preparation was exposed to high (2 or 4 mM) ATP for at least 2 min in a further attempt to minimize channel run-down (Ohno-Shosaku, Zunkler & Trube, 1987; Findlay, 1988 b). The patch current was averaged over 1-2 min after attaining a steady level, and the average current in the A
CN-
Cal
0
E
_0
'D
135e 1 min
CN-
B
-E 100 r /
_
120L 1 min
Fig. 1. Activation of KATP channels in rat ventricular myocytes exposed to complete metabolic blockade. A and B, slow time-base records of on-cell patch current (above), and cell length (below). 2-Deoxyglucose (10 mM) was present throughout. Cyanide (2 mM) was applied for the time indicated by the bar. In B, note that externally applied stimuli trigger twitch contractions, and induce a large artifact on the current record. The onset of KATP channel activity precedes the onset of contracture (A) and accompanies the decline of the twitch (B). Channel activity reversed on removal of cyanide (A).
test solutions was expressed as a fraction of that measured in the steady state at 041 mM-ATP. Where more than 50% run-down occurred between initial and final exposure to 0-1 mM-ATP, the data were rejected from analysis. For test solution = zero ATP, channel run-down was rapid and these acceptance criteria were not employed. In these experiments (n = 8), post-control channel activity was 23-6 + 5-5 % of precontrol activity. Thus, the data obtained in zero ATP may be less reliable than the data obtained with other test solutions.
C. G. NICHOLS AND W. J. LEDERER
96 A
