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MS 1577 Received 16 August 2000; accepted 31 August 2000.
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ABSTRACT |
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C35.
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INTRODUCTION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The ATP-sensitive K+ (KATP) channel represents a unique group of potassium channels that are inhibited by intracellular ATP in many tissues, such as islets of the pancreas, cardiac muscle, skeletal muscle, smooth muscle and brain tissue (Ashcroft, 1988; Ashcroft & Ashcroft, 1990). The inhibitory effect of ATP on the KATP channel current links the metabolic state of the cell to the cross-membrane K+ flux and the electrical activity of the membrane. Therefore, KATP channels can act as a sensor, or transducer, of the cellular metabolic state (Trapp & Ashcroft, 1997). A key question about this scenario, however, remains unanswered: how does the KATP channel open in the intact cell at cytoplasmic ATP concentrations that would be sufficient to inhibit the channel almost completely in an isolated membrane patch (Ashcroft, 1998)? Although ADP is a key regulator of KATP channel activity (Findlay, 1988; Nichols et al. 1996), it has become clear from many studies that other cellular factors can also regulate ATP sensitivity and KATP channel opening (Babenko et al. 1998).
Recently, a direct association between anionic membrane phospholipids, such as phosphatidylinositols, and KATP channel activity has been found (Hilgemann & Ball, 1996; Fan & Makielski, 1997). The finding that these lipids reduce the sensitivity of KATP channels to ATP (Shyng & Nichols, 1998; Baukrowitz et al. 1998; Fan & Makielski, 1999) provides a possible mechanism whereby cells regulate the ATP sensitivity of KATP channels in vivo. Therefore, it has been speculated that the sensitivity of KATP channels to the metabolic level might be modified in intact cells in response to some extracellular or intracellular stimuli by a change in membrane phosphoinositide levels. Unfortunately, few pathophysiological stimuli have been clearly identified.
Metabolic level changes are often associated with cell stress. Cardiac ischaemia, for example, impairs the cellular metabolism and induces cell stress at the same time. Under ischaemic conditions, cardiac cells are exposed to numerous chemical and physical stresses, including increased production of reactive oxygen species, mechanical strain, pacing, haemodynamic load, osmotic stress and metabolic deprivation (reviewed by Jennings et al. 1995). Treating eukaryotic and prokaryotic cells with cell stress-inducing agents results in modulation of the levels of polyphosphoinositides (Einspahr et al. 1988), two isomers of which were later identified as phosphotidylinositol 4,5-bisphosphate (PI(4,5)P2; Pical et al. 1999) and phosphotidylinositol 3,5-bisphosphate (PI(3,5)P2), via rapid activation of phosphatidylinositol kinases (Dove et al. 1997). Wortmannin, an inhibitor of phosphatidylinositol kinases (reviewed by Fruman et al. 1998), reduces KATP channel activity, a finding that is consistent with the suggestion that phosphoinositides play a role in regulating this channel (Xie et al. 1999a,b). Some stress stimuli, such as oxidant stress and hyposmotic shock, reportedly modify KATP channels, although the mechanism of such modification is not yet known (Tokube et al. 1996, 1998; Ichinari et al. 1996). Based on all of this information, it seems reasonable to test the hypothesis that cell stress affects KATP channel activity by regulating membrane phosphoinositide levels.
In the present study, we show an association between cell stress and KATP channel activity, and we provide evidence that this relationship is mediated by phosphoinositide-induced KATP channel modification. We focused our study on the 'traditional' KATP channel that is found in pancreatic 
-cells, cardiac muscle and skeletal muscle and that is composed of two subunits: a sulfonylurea receptor (SUR1 or SUR2) and an inwardly rectifying K+ channel (Kir6.2) (Aguilar-Bryan et al. 1995; Inagaki et al. 1996). The first series of experiments was designed to examine the effects of stress stimulation on KATP channels. Various stress stimuli, such as osmotic stress, photoirradiation, heat shock and hydroxyl radicals, reportedly stimulate the synthesis of phosphatidylinositol phosphates (Dove et al. 1997; Low et al. 1997; Lin et al. 1997; Toker & Cantley, 1997; Kabuyama et al. 1998; Jones et al. 1999; Pical et al. 1999). In our study, we induced cell stress by exposing the cells to a UV-rich irradiation source. The major advantage of this stimulus over other stress stimuli is the quick delivery of a controlled amount of energy to the selected cells without the introduction of any physical or chemical disturbance. We found that photoirradiation conditionally activated KATP channels, and we present evidence that such activation is caused by (1) an increase in the maximal open probability, mainly resulting from prolonged burst openings, and (2) a decrease in ATP inhibition. Next, we studied the possible role of phosphoinositides relative to the effects of irradiation on KATP channels. We compared the effects of irradiation and phosphoinositides on KATP channels and determined similarities in the changes in channel properties in response to the two treatments. The effect of irradiation on phosphoinositides was confirmed by measuring phosphoinositide levels.
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METHODS |
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Native cardiac KATP channels
Native KATP channel currents were measured in cardiac ventricular myocytes isolated from rat ventricles using methods described previously (Fan & Makielski, 1999). Briefly, in accordance with national guidelines, adult female Wistar rats (150-250 g) were anaesthetized with 75 mg kg-1 ketamine and 10 mg kg-1 xylazine. Heparin (500 i.u.) was given at the same time to prevent coagulation. After adequate anaesthesia was achieved, sternotomy was performed and the heart exposed. Artificial perfusion of the heart was established by cannulation of the aorta. The heart was then removed and placed in a Langendorf perfusion apparatus and an enzymatic method was used for isolation of single ventricular cells for electrophysiological experiments. Alternatively, the hearts were used for the measurement of phosphoinositides (described later).
Expression of recombinant cDNAs and truncation of Kir6.2 cDNA
Mouse SUR2 and mouse Kir6.2 cDNA clones or rat SUR1 and mouse Kir6.2 cDNA clones were coexpressed in the African Green monkey kidney COS-1 cell line (ATCC, Rockville, MD, USA) using the LipofectAMINE transfection kit (Gibco BRL, Gaithersburg, MD, USA). The SUR2 cDNA used in these studies was the cardiac form of SUR2A, with terminal usage of exon 38A (Isomoto et al. 1996) in the full-length variant (Chutkow et al. 1999) including exons 14 and 17. SUR1 cDNA was cloned from a RINm5F insulinoma cDNA library (Tokuyama et al. 1996) and modified by insertion of an exon 17 segment (Fan & Makielski, 1997). A polymerase chain reaction (PCR)-based site-directed mutagenesis kit ExSite (Stratagene, Inc., La Jolla, CA, USA), was used to generate the carboxyl-terminal truncation (Kir6.2C35) at amino acid residue 35 of the carboxyl-terminus of mouse Kir6.2. The resulting PCR product was subsequently subcloned into a PCR3.1 vector (T/A cloning kit, Clontech Laboratories, Inc., Palo Alto, CA, USA) and verified by sequencing.
Chemicals
Inside-out single- or multiple-channel currents were recorded with an intracellular solution containing (mM): 140 KCl, 2 EGTA, 0·5 MgCl2, 5·5 glucose, 5 Hepes (pH 7·4) and ATP (1 or 10 µM, as indicated in the text) and an extracellular solution containing (mM): 10 KCl, 120 NaCl, 1·8 CaCl2, 0·48 MgCl2, 5·5 glucose and 5 Hepes (pH 7·2). On-cell patch-clamp recording was made when the cells were perfused with the above intracellular solution exclusive of ATP. The small amount of ATP included in the intracellular solution induced very little (< 10 %), if any, inhibition, whereas it was sufficient to promote phosphorylation or a polymerization reaction. Phosphoinositides (from bovine brain; Sigma Chemical Co.) containing phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol bisphosphate (PIP2) and phosphatidylserine (PS) were dispersed in solutions by a 10 min ultrasonication on ice. The lipid-containing solutions were used in experiments shortly after the dispersal procedure and were applied to the inner side of the patch membrane. Wortmannin and LY-294002 (Alexis Biochemicals, USA) were first dissolved in DMSO and then dispersed into the solution with a final concentration of DMSO of < 0·1 %. Hydrogen peroxide (H2O2) was from Fisher Scientific Co. All other chemicals used in this study, if not specified, were from Sigma.
Light source and irradiation
Photoirradiation was conducted in a recording chamber with a 100 W mercury light (HBO 100 W, manufactured by Osram, Germany, mounted in a lamp house made by Chuo Technical Co., USA). The light was delivered to the sample chamber through a reflect-cube (custom-made by Nikon Co., Japan) mounted on a Nikon inverted microscope with a ×10 objective. The irradiation was controlled by changing the amount of light allowed to pass through a shutter placed between the light source and the recording chamber and was monitored with a phototransducer (Tracor Northern, USA) connected to an oscilloscope. The phototransducer was calibrated by comparison with a CDR-2 All-Wave ultraviolet intensity meter (Ultra LUM, USA). The lamp delivered light to the preparation chamber with a spectrum of 10 mW cm-2 at 365 nm (UVC), 1·8 mW cm-2 at 300 nm (UVB) and 2 mW cm-2 at 254 nM (UVA) (the errors were ± 10 %). The light intensity at other wavelengths was not measured, but could be deduced from the spectrum provided by the manufacturer.
During the initial phase of this study, we examined ATP hydrolysis and photolysis to evaluate the influence of irradiation-induced ATP degradation, which would possibly decrease the ATP concentration and cause channel activation. ATP was dissolved in the intracellular solution. The inorganic phosphorus concentration was measured using a kit from Sigma. Photoirradiation for 2 min did not produce a significant amount of phosphorus in a solution containing 1 mM ATP. Photolysis was monitored by measuring the UV spectra (200-300 nm) of ATP using a UV1200-S spectrophotometer (Shimadzu Co., Japan). No detectable difference was seen after 2 min of irradiation. In inside-out patch-clamp experiments, flow of the perfusion solution was maintained to minimize light-induced dissociation.
Electrophysiological recordings
The patch-clamp and data acquisition systems used were Axopatch 200B with a 1200 DMA interface and pCLAMP6.0 software (Axon Instruments, USA) running on a PC. Solution changes in the bath (membrane side of channel in excised patches) were achieved within 100 ms by means of a rapid solution exchange system (DAD-12, ALA Scientific Instruments, USA). All current recordings were filtered at 0·5-2 kHz and digitized at 2-20 kHz. Outward currents are shown as upward deviations from the closed level. The current traces were plotted after being manually corrected for baseline shift and filtered with a low-pass digital filter at a cut-off frequency of 500 Hz. Unless specified, currents were recorded at a membrane potential of 0 mV. Experiments were performed at room temperature.
Analysis of single-channel data
For patches containing five or fewer active channels (a software limitation), open activity was assessed by the open probability (Po), which was measured using a conventional event analysis method (Spruce et al. 1987). In macropatch recordings containing more than five active channels, the apparent open probability (NPo) was used, where N is the number of channels, an unknown parameter in a macropatch. NPo was calculated as the average current in a 5 s time window divided by the single-channel current amplitude determined under the same recording conditions. To avoid confusion in terminology, 'open probability' is used to generally describe the open activity of the channel, which can be Po or NPo depending upon how it was measured. For a set of pooled data, if all data in the set were obtained by measuring Po, then this set of data will be noted as Po. NPo was used otherwise.
In event analysis, a 50 % threshold criterion was used to detect events, and all events were confirmed visually. Single-channel kinetics were analysed with an established method used in our previous work (Fan & Makielski, 1999). Briefly, after detecting events, open and closed time distributions were constructed against a logarithmic time scale with event duration log-binned at a resolution of 25 bins per log unit and a minimum resolution of 25 bins per log unit. A minimum resolution of 150 µs was set for the event analysis to match the filtration frequency of 2 kHz. This value was used in correcting mean closed times. Mean open times were not corrected for reasons given previously (Fan & Makielski, 1999). A burst duration was defined as the duration between two closed events whose durations were both longer than a critical time (according to a calculation given previously (Fan & Makielski, 1999); typically 3-5 ms).
Measurement of phosphoinositides
Phosphoinositides were analysed by thin-layer chromatography (TLC) (Hajra et al. 1988). Rat hearts (minced with scissors) or cultured cells were washed with ice-cold buffer containing: 110 mM KCl, 25 mM Hepes, 2 mM EGTA, 50 µM ATP, 1 mM MgCl2; pH 7·4 adjusted with KOH. The buffer was supplemented with a cocktail of protease inhibitors including 17 µg ml-1 4-(2-aminoethyl)-benzenesulfonyl fluoride), 10 µg ml-1 aprotinin, 5 µg ml-1 leupeptin, 2 µg ml-1 bestatin, 2 µg ml-1 pepstatin and 14 µg ml-1 calpain inhibitor 1 (Roche Molecular Biochemicals, USA). The tissues or cells were then homogenized in 5 ml of the same buffer. The homogenate was centrifuged at 1000 g for 10 min at 4°C, and the supernatant was then centrifuged at 31 000 g for 30 min at 4°C. The resultant pellet together with 500 µl ice-cold buffer were collected and mixed. The liposome suspension, or freshly isolated cells, was then divided into equal aliquots (100 µl each). The samples were incubated with appropriate inhibitors according to the experimental design, followed by exposure to the mercury light in the recording chamber for 0, 30, 60 and 120 s. After irradiation, liposomes were prepared from the cell samples as described above. Phospholipids were extracted from the liposomes in an acidified buffer (1·2 ml buffer plus 0·4 ml 6 n HCl) with 2·4 ml of chloroform:methanol (1:1, v/v). The mixture was centrifuged at 1000 g for 5 min. The organic phase was collected and washed once with 5 ml of the acidified aqueous phase of chloroform-methanol-water (1:12:12, v/v). The solvents were evaporated with nitrogen at 40°C. Lipid samples dissolved in chloroform-methanol-water (75:25:2, v/v) were loaded onto a K2C2O4-pretreated silica gel 60 plate (Fisher Scientific, USA) and developed in chloroform-methanol- acetone-acetic acid-water (40:15:13:12:7, v/v). The lipids were visualized with copper acetate (Fewster et al. 1969), and the amount was evaluated by densitometry and Scion image processing software (Scion Co., USA). A 0·1 mg phosphoinositide standard (Sigma) was also subjected to the lipid extraction procedure and run alongside the samples on TLC.
Statistical analysis
Data were analysed statistically using either Student's t test when two treatment groups were compared, or one-way analysis of variance (ANOVA) followed by a post hoc Student-Newman-Keuls test when all pairwise comparisons among the different treatment groups were conducted. Tests were considered significant when P < 0·05. All values are reported as means ± S.E.M.
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RESULTS |
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Activation of KATP channels by photoirradiation in intact cells
The effect of photoirradiation on KATP channels was examined in intact cardiac myocytes and COS-1 cells expressing SUR2-Kir6.2 channels. Figure 1 illustrates an experimental recording of a patch containing multiple channels initially recorded in the cell-attached configuration on a rat ventricular myocyte. After establishment of the cell-attached patch-clamp configuration, the cell was exposed to mercury light radiation via the microscope; the exposure lasted 45-60 s. While KATP channel activity was not observed before irradiation, obvious openings of KATP channels were recorded during and after irradiation. In 12 cells tested, seven exhibited clear openings of KATP channels after the irradiation was started. The KATP channel currents were confirmed using the inside-out configuration that was formed subsequently by excision of the patch after irradiation. As shown in the later part of the recording trace in Fig. 1A, the patch membrane was excised from the cell, and the sensitivity of the channels to inhibition by ATP was measured. Upon excision of the patch membrane into a solution with an ATP concentration ([ATP]) of 1 µM, outward KATP channel currents were recorded in all of the successful patch excisions. The ATP sensitivity was obtained by varying [ATP] in the intracellular solution. Changes in [ATP] were made by a series of both increasing and decreasing concentration steps of 10-20 s. This protocol has been shown to be effective at minimizing possible errors caused by run-down of the channels (Fan & Makielski, 1999). Compared with the channels that did not undergo irradiation, the ATP sensitivity of the irradiated channels was reduced considerably (without irradiation, [ATP] producing half-maximal inhibition (Ki) = 41·8 ± 8·6 µM, the Hill coefficient (S)= 1·2 ± 0·2, the number of experiments (n) = 19; with irradiation, Ki = 398 ± 188 µM, S = 1·3 ± 0·3, n = 6; P < 0·05 for Ki, but P > 0·05 for S).
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A, multiple-channel current recording. The current was initially recorded in the cell-attached configuration. The patch membrane was excised at the time marked Inside-out. The cell was bathed in a high K+ solution (140 mM), and the patch membrane was held at 0 mV throughout the recording period. ATP was applied at the concentrations indicated by the bars above the current trace. Irradiation indicates the period during which the patched cell was exposed to photoirradiation (see text for details of photoirradiation treatment). In this and subsequent figures, the dotted line through the current recording indicates the closed channel level. B, relationships between concentration and the ATP inhibition of KATP channels. | ||
Analysis of irradiation-induced activation in excised patches
Photoirradiation also affected KATP channels in patches shortly after their excision. The effects in excised patches allowed direct comparison of channel properties before and after irradiation, in the same patch and also probably the same channels. In these experiments, photoirradiation was applied to excised patch membranes while KATP channel currents were recorded in the inside-out patch-clamp configuration, during perfusion with intracellular solution containing modulators of channel activity such as ATP (see Methods).
As shown in Fig. 2, multiple KATP channel currents were recorded from either rat ventricular myocytes (Fig. 2A) or from COS-1 cells transfected with SUR2 and Kir6.2. Again, no KATP channel activity was observed in the cell-attached patch-clamp configuration. After patch excision, control ATP sensitivity was measured as described above. Thereafter, the intracellular side of the membrane was perfused with solution containing 1 µM ATP for 30 s to record the maximal open probability (defined as the open probability that is measured either by Po or NPo in the absence of inhibitory [ATP]; Fan & Makielski, 1999). During this period some patches exhibited an appreciable decrease in channel activity as shown by the increased number of bursts and shortening of the burst durations, probably caused by run-down. After this measurement, the mercury light was introduced onto the tip of the electrode via the microscope; exposure lasted for 30 s. Channel activity was monitored during irradiation. The maximal open probability and ATP sensitivity were measured again after the irradiation was stopped. Irradiation induced a significant increase in the maximal open probability of the active channels. The increase in maximal open probability was quantified as the ratio of maximal open probability after irradiation to maximal open probability before irradiation.
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A, activation of KATP channels by photoirradiation in a patch membrane from a rat ventricular myocyte. ATP was applied at the concentrations indicated by the bars above the current trace. During all other periods not marked with a specific [ATP], ATP was present at 1 µM. Irradiation indicates the period during which the patch was exposed to photoirradiation. B, effect of irradiation on the concentration-dependent inhibition of KATP channels by ATP. | ||
It is worth mentioning that, in two patches (out of 8), we observed a decrease in maximal open probability. We attributed the decrease in these two patches to the loss of some active channels. Loss of active channels became particularly evident in patches exposed to irradiation for longer periods (> 100 s, not included in the following statistical summary). Statistically, however, the ratio was significantly greater than 1 (Table 1), confirming a net increase in maximal open probability after irradiation. Under control conditions, no open channel event in 1 mM ATP was observed. In contrast, open channel events could be identified clearly after irradiation. This result indicates that irradiation reduces the ATP sensitivity.
Table 1. Statistical summary of photoirradiation-induced activation of KATP channels
| Parameters | Control | Irradiation | Low intensity |
No MgATP | 10 µM WMN | 100 µM WMN | Potassium ascorbate |
Tocopherol |
| n | 19 | 10 | 3 | 6 | 6 | 6 | 6 | 5 |
| Change of Po,max | 1 | 1·47 ± 0·15 * a,b,c,d,e,f |
1·21 ± 0·15 | 0·88 ± 0·09 b |
1·13 ± 0·07 c |
0·81 ± 0·05 d |
0·82 ± 0·06 e |
0·84 ± 0·04 f |
| Ki (µM) | 41·8 ± 8·6 | 807 ± 153 * a,b,c,d,e |
637 ± 134 * f,g,h,i |
287 ± 69 a |
375 ± 97 * b,f |
115 ± 24 c,g |
147 ± 29 d,h |
136 ± 36 e,i |
| S | 1·2 ± 0·2 | 1·3 ± 0·2 | 1·1 ± 0·1 | 1·4 ± 0·2 | 1·2 ± 0·1 | 1·2 ± 0·1 | 1·3 ± 0·2 | 1·2 ± 0·2 |
ATP at 1 µM and MgCl2 at 0·5 mM were present in the intracellular solution during irradiation except for the parameter 'No MgATP'. WMN, wortmannin. Low intensity, irradiation at 1/10 of the original level. Comparisons by one-way ANOVA were made among all groups for each parameter. *P < 0·05, for any treated group vs. the control group. P < 0·05, for any two of the other groups, indicated by letters.
A quantitative measure of the ATP sensitivity change is presented as the half-inhibitory [ATP] (Ki) and slope factor (S) (Table 1) from the fit of a Hill saturation function to the relationship between ATP inhibition and [ATP] (Fig. 2B). As summarized in Table 1, the maximal open probability and Ki were both significantly altered by 30 s irradiation. The changes in these parameters might have been involved in the increased channel activity observed in both cell-attached and excised patches. Similar irradiation-induced activation was also seen in SUR1-Kir6.2 channels expressed in COS-1 cells (data not shown). We also examined the relationship between the intensity of the light and channel activation by reducing the overall intensity to approximately 1/10 of its original level. Under this lower intensity irradiation, the effect on maximal open probability became less clear, probably the result of run-down that might have masked the activation effect. The reduction of ATP inhibition, however, remained distinct (Table 1), but was less than that observed at higher intensity irradiation.
The effect of irradiation, particularly on the ATP sensitivity which is less subject to 'run-down', was spontaneously reversible, albeit partially, over a time period of 10-20 min after irradiation. Figure 3A shows a recording during one experiment in which the patch was irradiated for 20 s at low intensity (1/10, as described earlier). This treatment decreased the Ki for ATP inhibition from 33 to 464 µM (measured 1 min after irradiation). The Ki returned to a lower value of 101 µM 10 min thereafter, without any additional treatment (Fig. 3C). Similar results were obtained in two other experiments. These results suggest that direct photochemical modification of the channel protein, which in most cases should not be readily reversible, is less likely to account for the activation. Longer periods and/or stronger irradiation caused changes that were less reversible over the same time period of recovery (Fig. 3D). Interestingly, we found that Ca2+ could facilitate such reversibility. The patch shown in Fig. 3B was exposed to high intensity irradiation for 40 s, which apparently had a stronger effect, but addition of Ca2+ (20 µM, 2 min) to the bath solution reversed this change. Statistical data summarized in Fig. 3D show a comparison of Ki measured with and without Ca2+ and spontaneous recovery after the same dose of irradiation.
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A, spontaneous recovery. The patch from a rat cardiac myocyte was irradiated for 20 s at low intensity (1/10, see text). The break in the current recording signifies a period of about 10 min. B, effect of irradiation and Ca2+ on ATP inhibition. The patch was irradiated for 40 s. Ca2+ at 20 µM was applied for 2 min as indicated, during the break in the recording. C, concentration dependence of ATP inhibition obtained from the data in A, before, and 1 and 11 min after, irradiation. The lines are fits of the experimental data with the expression described in the legend to Table 1. The half-maximal inhibitory concentration (Ki) values were 33, 464 and 101 µM, with Hill coefficient (S) values of 1·1, 1 and 1·3, respectively, before ( | ||
The effects of irradiation on KATP channels were further analysed kinetically in two patches that contained only a single active channel. In the example given in Fig. 4A, a SUR2-Kir6.2 channel was active from the beginning of the experiment when the excised patch was perfused with 1 µM ATP. Histograms of open and closed time distributions resulting from the single-channel current of another patch excised from a rat ventricular myocyte are given in Fig. 4B. In accordance with data obtained previously (Fan & Makielski, 1999), the open time histogram of the control could be fitted with a three-exponential distribution function, and the closed time histogram could be fitted with a four-exponential distribution function. The parameters of the fits are given in Table 2. Irradiation did not alter the number of components in the closed time distribution. Rather, it reduced the components corresponding to the gaps between bursts relative to the closures within the bursts. The number of components in the open time distribution were also not altered. However, the irradiation did increase the two slower components of the open time distribution. Table 2 also gives the mean burst duration measured before and after irradiation. In fact, the most obvious change was a considerable prolongation of the mean burst duration.
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A, photoirradiation-induced activation of single-channel current recorded from a COS-1 cell transfected with SUR2-Kir6.2. The membrane was held at 0 mV. B, effects of photoirradiation on open and closed time distributions of single-channel currents recorded at 0 mV from a patch excised from a rat ventricular myocyte. The parameters of the probability density functions are given in Table 2. | ||
Table 2. Effects of irradiation on closed and open time distributions
| Closed times | Open times | Burst | |||||||||||||
 c,1 |
sc,1 | c,2 |
sc,2 | c,3 |
sc,3 | c,4 |
sc,4 | o,1 |
so,1 | o,2 |
so,2 | o,3 |
so,3 | b |
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| Control | 0·15 | 690 | 1·1 | 51 | 14 | 20 | 171 | 6·1 | 0·5 | 1·6 | 4·5 | 2·5 | 55 | 19 | 210 |
| Irr. | 0·16 | 636 | 1·3 | 50 | 14·5 | 5·5 | 92 | 1·6 | 0·4 | 2 | 3·5 | 5·6 | 131 | 38 | 709 |
c,i and o,i (in ms) are closed and open time constants, respectively, and sc,i and so,i (in relative units) are scale factors of various exponential components in the functions fitted to the closed and open time histograms, respectively, in Fig. 4B. b (in ms) is the mean burst duration.
Phosphoinositides and irradiation-induced activation
As mentioned earlier, in this study we contemplated using photoirradiation as an experimental protocol to elucidate the connection between cell stress stimuli and KATP channel activity. Initially, we did not specify the involvement of phosphoinositides as an exclusive target. Rather, we were looking for any significant regulatory mediators that might possibly connect the irradiation and the effect on the channel. A number of intracellular regulatory factors can activate KATP channels (Babenko et al. 1998), e.g. MgATP, which activates KATP channels even in inside-out patches (Findlay & Dunne, 1986). By eliminating MgCl2 and ATP from the cytoplasmic solution, we examined whether MgATP was required for irradiation-induced activation. The ratio of maximal open probability of KATP channels obtained in the absence of MgATP was < 1, which was significantly different from that in the presence of MgATP (Table 1). However, in spite of a reduction in maximal open probability, prolonged open channel events were still sometimes observed in the absence of MgATP. Under these conditions, irradiation also produced a small decrease in ATP sensitivity that was less than that seen in the presence of MgATP (Table 1). These results indicate that MgATP, at least in part, interferes with the irradiation-induced alteration of KATP channel activity. The requirement of MgATP led us to consider the involvement of a phosphorylation event, of which phospholipids could be substrates. Indeed, Xie et al. (1999b) proposed that MgATP is bridged to KATP channel activity via PI kinase-catalysed phosphorylation of phosphatidylinositols. Therefore we compared the effects of irradiation (Figs 2 and 5) with those of phosphoinositides extracted from bovine brain (Fig. 5; see also our previously published data, Fan & Makielski, 1999). We found that the effects resembled each other qualitatively, as did their slow reversibility. In fact, many studies have indicated that cell stress produces phosphoinositides which lead to downstream responses. Two approaches have commonly been used to study such a causal connection. The first is the use of inhibitors of enzymes needed for phosphoinositide metabolism to block the response. The second approach is to correlate the phosphoinositide levels with the downstream response. A number of phosphoinositide signalling pathways are mediated by phosphoinositide kinases, the inhibition of which stops the corresponding signal transduction process. We tested two available inhibitors of PI kinases, wortmannin and LY-294002. After pretreatment of the patch with 10 µM wortmannin for 5 min, the ratio of maximal open probability of KATP channels before and after irradiation (2 out of 6 patches having a ratio > 1) differed considerably from the data obtained without wortmannin treatment (where 6 out of 8 patches had a ratio > 1). In another four patches, the maximal open probability decreased (Fig. 6A). At a concentration of 10 µM, although wortmannin significantly retarded the irradiation-induced decrease in ATP inhibition, the ATP sensitivity was still significantly lower than that of the control (Table 1), implying that the effect of irradiation was not completely prevented. We therefore used 100 µM wortmannin and observed a significant reduction of the effect of irradiation on ATP sensitivity (Fig. 6B). In this group of six experiments, none of the patches had a maximal open probability ratio > 1, and the ATP sensitivity was statistically higher than that in the absence of wortmannin, but was not different from that of the control (Table 1). There was no perceivable effect on the activity of KATP channels, either before or after irradiation, with 10 µM LY-294002 (with 
5 min preincubation). These results agree with data of two recent studies (Xie et al. 1999a,b), in which it was reported that a concentration of wortmannin of 100 µM is critically needed for prevention of phosphoinositide-mediated KATP channel modification. Although wortmannin-sensitive responses are often found relative to PI 3-kinases or PI 4-kinases, at a concentration of 100 µM, wortmannin can also affect other enzymes, making an exclusive conclusion based solely on these experiments almost impossible. In this respect, however, it should be noted that some technical restraint could contribute to the effects observed as the sensitivity of the drugs, especially wortmannin, to light could reduce their effectiveness during photoirradiation.
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A, KATP channel current was recorded from a COS-1 cell cotransfected with SUR2 and Kir6.2. Phosphoinositides (PPIs, extracted from bovine brain, 0·5 mg ml-1) were added to the perfusate of the patch membrane for 5 min. ATP was applied at the times and concentrations indicated by the bars above the trace. B, concentration dependence of ATP inhibition measured in A, before and after treatment of the patch with PPIs. The lines are fits of the experimental data with the expression described in the legend to Table 1. Ki was 40 µM and 5 mM, and S was 1·2 and 1·3, before ( | ||
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A, wortmannin (WMN, 10 µM) partially prevented the activation of KATP channels by irradiation in an inside-out patch from a rat ventricular myocyte. B, wortmannin (100 µM) abolished the irradiation-induced activation of KATP channels. C, potassium ascorbate (15 mM) also prevented the activation of KATP channels by irradiation in a rat ventricular myocyte. ATP (1 µM) was included in the perfusion solution throughout. Irradiation indicates the period during which the patches were exposed to photoirradiation. | ||
Effect of photoirradiation on phosphoinositide levels
The distribution of phosphoinositide species, including PI, PIP isomers and PIP2 isomers, in cells or membrane liposome vesicles extracted from rat heart and COS-1 cells was analysed by TLC after various irradiation exposure times (0, 30, 60 and 120 s) under conditions similar to those of the patch-clamp experiments. The phosphoinositides used to compare effects on channel activity (Fig. 5) were also analysed at the same time. The phosphoinositide preparation used contained a similar number of lipid spots on TLC as those extracted from rat heart (Fig. 7). We found that irradiation noticeably elevated the amount of PIP2. Individual isomers of PIP2, namely PI(3,5)P2, PI(4,5)P2, and 3,4-bisphosphate (PI(3,4)P2), migrated close together on TLC and could not be identified separately. In the study reported here, further isolation of the individual isomers was not pursued. Changes in other phosphoinositide species, if any, were not immediately as noticeable as those of PIP2. The total amount of PIP2 measured after 60 s irradiation was increased to 270 ± 25 % (P < 0·05, n = 5) of the control level from samples before irradiation. The irradiation-induced rise in the PIP2 level was abolished by 100 µM wortmannin (Fig. 7B). Similar results were obtained in four other independent samples. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) was detectable, albeit at lower resolution. The change in the PIP3 level after irradiation was 'subtle' compared with that of PIP2 (Fig. 7B). Because of technical limitations, quantitative analysis of PIP3 levels was not performed. It should be noted that although we tried to keep the experimental conditions as close to those of the patch-clamp experiments as possible, differences did occur. One uncontrollable factor that we noticed was the high density of vesicles or cells, which might absorb the light in the chamber and cause an unknown decrease in the irradiation dose.
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A: left panel, images of a TLC plate stained using copper acetate. The phosphotidylinositols were extracted from liposomes prepared from rat heart. The liposomes were exposed to similar amounts of photoirradiation in the recording chamber before lipid extraction. The image was digitized and enhanced. Lanes 1-4, exposure of the samples to irradiation for 0, 30, 60 and 120 s, respectively. Lane 5, phosphoinositides (Sigma) used for experiments shown in Fig. 5. Right panel: plots of density functions of the phosphotidylinositol levels measured from images in A using Scion image processing software. The plots from top to bottom correspond to lanes 1-5, respectively. PE, phosphatidylethanolamine; PS, phosphatidylserine; PC, phosphatidylcholine; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate; PIP2, phosphatidylinositol bisphosphate; Ori, origin. B, effect of wortmannin. Images (left) and density functions (right) of phosphatidylinositols extracted from vesicles incubated with wortmannin (100 µM, lane 2) before and during exposure to irradiation for 100 s. Lanes 1 and 3, control samples from the same heart, without and with irradiation, respectively. PIP3, phosphatidylinositol trisphosphate. | ||
Effect of free radical scavengers
Among the early and immediate consequences of exposure of proteins and lipids to irradiation is the formation of reactive oxygen intermediates, which themselves are cell stress agents. Several reports have demonstrated that some species of free radicals can activate KATP channels (Tokube et al. 1996, 1998; Ichinari et al. 1996). In guinea-pig ventricular myocytes, the effects of free radicals include an increase in the maximal open probability and a decrease in ATP inhibition. Using a radical-induction agent, H2O2 (4 mM), we confirmed these results in rat ventricular myocytes and also in SUR2-Kir6.2 recombinant channels expressed in COS-1 cells. Wortmannin at a concentration of 100 µM reduced the effects of H2O2 (Z. Fan, unpublished observation), a result that appeared to correlate well with the irradiation effects. Such information led us to suggest that free radicals participate in the irradiation-induced activation of KATP channels. The free radical scavengers ascorbic acid and tocopherol (Gotoh & Niki, 1992) were tested to determine whether they could attenuate the effects of irradiation. Before and during irradiation, 15 mM potassium ascorbate was added to the intracellular perfusate. Irradiation treatment of the patch for 30 s did not increase the channel activity, and very little change in ATP sensitivity was observed (Fig. 6C). In the same patch, we later repeated the irradiation for another 30 s in the absence of potassium ascorbate and observed noticeably changed opening kinetics of the channel and long bursts of openings (data not shown). Data from five experiments were summarized statistically and are compared with data in the absence of ascorbate in Table 1. Pretreatment of the cells with (+)-
-tocopherol (15 i.u.) for 90 min also attenuated the effects of irradiation (Table 1). We further examined the effects of potassium ascorbate and tocopherol on the amount of total phosphoinositides. When potassium ascorbate (10 mM) and tocopherol (10 i.u.) were included in the buffer solution during the preparation of liposomes, the PIP2 level after photoirradiation (60 s) was not different from control, but was significantly different from the level in the absence of drugs (120 ± 30 %, n = 3 vs. 270 ± 25%, n = 5, P < 0·05).
Effects of photoirradiation on Kir6.2C35
In the experiments described so far, KATP channels composed of two subunits, SUR2 or SUR1 and Kir6.2, were tested. A characteristic feature of the effect of phosphoinositides on KATP channels is that their primary target is the Kir6.2 subunit. Therefore we investigated whether the effects of photoirradiation had the same profile of subunit dependence. If phosphoinositides are involved, irradiation-induced activation will predictably also activate the Kir6.2 current. Kir6.2 can be expressed without the SUR subunit (Tucker et al. 1997; John et al. 1998). Single-channel currents of Kir6.2C35 expressed in COS-1 cells were recorded for this series of experiments. The ATP sensitivity was tested after establishment of an inside-out patch clamp. ATP inhibited multiple-channel currents with a higher Ki than that needed to inhibit KATP channels with SUR. It is also notable that the current events of Kir6.2C35 were gated faster than those of KATP. These characteristics agree well with our previous observations (Fan & Makielski, 1999) and those of others (Tucker et al. 1997; John et al. 1998). As shown in Fig. 8A, after an ATP-sensitivity test, the patches were exposed to irradiation for 30 s. Channel activity decreased, rather than increased, in response to the irradiation. An increase in maximal open probability was never observed in the experiments using Kir6.2C35. However, the channels (6 out of > 30 channels) that remained active after the irradiation was stopped exhibited reduced ATP sensitivity. The channels remaining active had a Ki of 638 ± 139 µM (n = 4) compared with 278 ± 52 µM (n = 9, P < 0·05) obtained before irradiation. Figure 8B shows the relationship between ATP concentration and inhibition measured after irradiation (superimposed on the control data).
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C35
A, effects of irradiation on multiple channel currents of Kir6.2 | ||
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DISCUSSION |
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In this study, we report that a cell stress stimulus, photoirradiation, activates KATP channels of rat heart and recombinant KATP channels composed of SUR2 and Kir6.2 in intact cells and in excised patches. Based on our results, we propose that the modification favouring channel opening triggered by cell stress stimuli is mediated, at least in part, by changes in membrane phosphoinositide levels.
Multiple components of irradiation-induced activation
A prominent increase in KATP channel activity was perceived within tens of seconds after the start of irradiation. However, KATP channel openings in intact cells are difficult to analyse. For example, photoirradiation might indirectly induce channel activity by changing ATP hydrolysis or the ATP/ADP ratio, which is a well-known regulatory factor of KATP channels. The reduced ATP sensitivity in patches excised from cells exposed to irradiation (Fig. 1), however, clearly indicates that a change in the intrinsic KATP channel sensitivity is at least a partial cause of channel activation. In excised patches, we dissected the activation into two major components: (1) an increase in maximal open probability as a result of the prolongation of bursts and the shortening of closures between the bursts, and (2) a decrease in ATP sensitivity. Both occur at approximately the same time and remain after irradiation. The increase in maximal open probability was less consistent, probably due to 'contamination' caused by irradiation-induced channel inactivation. This is indicated by a significantly higher maximal open probability ratio being measured from only those channels that survived irradiation. A number of well-studied KATP channel activators also increase channel openings in multiple ways. Among these activators, polyphosphatidylinositols, judged by their effects on single channels (Fan & Makielski, 1997, 1999; Shyng & Nichols, 1998; Baukrowitz et al. 1998), are qualitatively the closest to irradiation. Interestingly, the effect of irradiation is slowly and partially reversible, and has subunit preference for Kir6.2, as do the phosphoinositides (Fan & Makielski, 1997; Shyng & Nichols, 1998; Baukrowitz et al. 1998). Quantitatively, the effects of phosphoinositides are greater than those of irradiation (a 125-fold vs. 20-fold increase in the Ki of ATP inhibition). In addition, the ATP sensitivity changes almost concurrently with the maximal open probability following irradiation, whereas it is delayed when exogenous phosphoinositides are applied (Shyng & Nichols, 1998; Fan & Makielski, 1999). The discrepancies may reflect differences in the dynamic distribution and localization of exogenous and endogenous lipids, although at present a definite explanation is not available.
Mechanism of the effects
Besides the similarity of action, other evidence also correlates the irradiation with phosphoinositides. First, observation of the irradiation effect in patches and isolated vesicles suggests a membrane-delimited process whereby phosphorylation of phosphoinositides can still take place, whereas most stress-activated protein kinase pathways are in the cytoplasm (Tibbles & Woodgett, 1999). In this regard, MgATP regulation of a Na+-Ca2+ exchanger was also observed in vesicles in which the PIP2 level was raised in seconds (Berberian et al. 1998). Second, Ca2+ facilitation of the recovery from the effect of irradiation can be explained by 'lost contact' with PIP2 due to neutralization of the charges (Fan & Makielski, 1997) and/or activation of phospholipase C (Xie et al. 1999b). Third, activation is attenuated in the absence of MgATP, implying that a phosphorylation process may be involved. Fourth, an elevated PIP2 level during the time course of the irradiation-induced activation directly supports our hypothesis. The identity of the PIP2 isomer(s) primarily responsible does not seem to be critical, because all three PIP2 isomers can activate KATP channels (Z. Fan, unpublished observation). PIP3 also activates KATP channels (Shyng & Nichols, 1998; Harvey et al. 2000). Compared with PIP2, the change in PIP3 level (Fig. 7B) is subtle and thus is unlikely to play a dominant role. We note from the literature that PIP2 and PIP3 levels are not necessarily correlated. Free radical-induced PI(3,4)P2, for example, does not correlate with PIP3 (Van der Kaay et al. 1999). Finally, 100 µM wortmannin reduces irradiation-induced activation of KATP channels as well as the rise in PIP2. Wortmannin at this concentration inhibits all types of PI 3-kinases and some PI 4-kinaeses (Fruman et al. 1998). LY-294002, which does not block type III PI 3-kinase and PI 4-kinases (Fruman et al. 1998), failed to intervene in the effect of the irradiation. This result is not surprising, because a similar pharmacological profile has been reported for MgATP-dependent activation and receptor-induced inhibition of cardiac KATP channels. The wortmannin sensitivity was cited as evidence of the involvement of PI kinase, hypothetically some PI 4-kinase(s) (Xie et al. 1999a,b). Without any evidence to disprove it, we adopt this rationale to interpret our results.
What is the upstream step that transforms irradiation energy into stimulation of enzymatic production of phosphoinositides? One plausible step is the production of free radicals (Kabuyama et al. 1998). In our study, free radical scavengers indeed retarded irradiation-induced activation, thereby suggesting such a possibility. Coincidentally, free radicals have been shown to increase the opening of cardiac KATP channels by increasing the open probability and reducing the ATP sensitivity (Tokube et al. 1996, 1998; Ichinari et al. 1996). It will be intriguing to test whether agents that induce free radicals change PIP2 levels. Nevertheless, the sensitivity of irradiation-induced activation and raised PIP2 levels to both wortmannin and free radical scavengers has accordingly reflected a possible causal relationship. Irradiation also induces inactivation of KATP channels, which was seen more persistently in Kir6.2C35. The current study was not designed to investigate this inactivation, so the reason for it is unknown. However, free radicals can inhibit some K+ channels including IRK3, another inwardly rectifying K+ channel, probably through a direct interaction (Duprat et al. 1995). To summarize, we have proposed, and our data support, a hypothetical scheme for how photoirradiation, and probably other cell stress stimuli, modifies KATP channels. This process contains the minimal steps shown in scheme 1 (where hv is the energy of a photon). We remain cautious about suggesting phosphoinositides as the sole mediator, considering that irradiation can produce broad biological effects. Our data do not totally exclude other mechanisms such as direct structural modification of the channels. Elucidation of this hypothetical scheme and its alternatives is currently underway.
Cell stress and KATP: possible physiological implications
Physical and chemical stimuli such as UV irradiation, photochemical treatment, osmotic stress, heat shock and oxygen free radicals are often used to study the susceptibility of various subcellular systems to cell stress in various types of cells. Many of these stimuli share an array of common or closely related signal cascades to evoke a cellular response. For instance, osmotic stress (Dove et al. 1997), UV irradiation and interleukin-2 (Jones et al. 1999) all elevate PI(3,5)P2 levels that trigger similar downstream events. The results of our study are therefore likely to have physiological implications in terms of the response of KATP channels to other physical stimuli of cell stress. Indeed, as we mentioned earlier, oxygen free radicals also activate KATP channels, and so does physical strain on the membrane (VanWagoner, 1993). Even if a common response to all stress stimuli is still questionable, activation of KATP channels as a possible mediator in the cellular response to stress conditions is implicated. Vice versa, cell stress is implicated as a factor that modifies the sensitivity of KATP channels to ATP inhibition. This modification may be part of the solution to the mysterious openings at high [ATP] in vivo, considering that cells are subject to an incredible variety of stress stimuli under physiological, and even more so under pathological, conditions. In general, the recent dogma that phosphoinositides regulate most, if not all, inwardly rectifying K+ channels, makes the extrapolation of our findings for these channels testable.
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We thank Drs Xi He and Talent I. Shevchenko for help in some of the experiments and Dr P. Hoffman for kindly providing us with rat ventricular myocytes. We also thank Dr David Armbruster for critical reading of this manuscript. We are grateful to the support provided by the Department of Physiology, The University of Tennessee Health Science Center. This work is supported in part by National Institutes of Health grants HL-58133 and GM61943 to Z. Fan.
Corresponding author
Z. Fan: The Department of Physiology, University of Tennessee, College of Medicine, Memphis, TN 38163, USA.
Email: zfan{at}physio1.utmem.edu
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, data collected from patches excised from cells exposed to irradiation. The lines are fits of the experimental data with the expression given in the legend to Table 1. The dashed line is derived from data from cells not exposed to irradiation (actual data points are shown in Fig. 2B).
and 
) after irradiation. D, statistical evaluation of the Ki of ATP inhibition measured in control (C), < 3 min after irradiation (Irr/s), > 10 min after irradiation (Irr/l) and after irradiation and exposure to Ca2+ (Irr/Ca). The number of measurements is indicated in parentheses. One-way ANOVA was performed for all data groups. *P < 0·05, significant difference between the data groups indicated.