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Am J Physiol Heart Circ Physiol 275: H751-H759, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 3, H751-H759, September 1998

Inhibition of ATP-induced increase in muscarinic K+ current by trypsin, alkaline pH, and anions

Apisate Pleumsamran, Michael L. Wolak, and Donghee Kim

Department of Physiology and Biophysics, Finch University of Health Sciences, The Chicago Medical School, North Chicago, Illinois 60064

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

In atrial cells, the open probability of G protein-activated ACh-sensitive K+ (KACh) channels can be increased approximately fivefold by intracellular ATP (ATPi). Using inside-out patches, we examined how proteases, changes in intracellular pH, and different anions affect G protein-mediated activation and ATP-induced stimulation of the KACh channel. Treatment with trypsin (0.5 mg/ml) removed the GTP dependence of the KACh channel and abolished the ATP-induced stimulation. Intracellular GTP activated KACh channels at all intracellular pH values tested (6.0-8.0), with the concentration at which half-maximal activation (K1/2) occurred ranging from 0.3 (pH 8.0) to 6.7 (pH 6.0) µM. However, the ATPi-induced increase in KACh channel activity was inhibited at pH 8.0 (K1/2 = pH 7.4). All anions tested except sulfate, phosphate, fluoride, and iodide supported GTP-induced activation. Of the anions that supported GTP-induced activation, only citrate blocked the ATP-induced stimulation of the KACh channel. These results indicate that the GTP- and ATP-mediated effects on the KACh channel use separate signaling pathways. The ATP-mediated effect involves a trypsin- and pH-sensitive mechanism.

atrial cells; G protein; acetylcholine; proteases

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ACETYLCHOLINE (ACh) activates the G protein-coupled K+ (KACh) current in pacemaker and atrial cells, thus regulating heart rate and rhythm (29). Recent studies have shown that on ACh binding to the muscarinic receptor, the beta gamma -subunits of the pertussis toxin-sensitive G protein (Go/Gi family) activate the KACh channel by directly interacting with the cytoplasmic domains of the channel protein (8, 17, 25). At present, the G protein-induced opening of the KACh channel is believed to be the only physiologically relevant mechanism by which ACh elicits the KACh current.

Our recent studies (6, 13) have shown that whether the KACh channel is activated via endogenous G protein by ACh or via exogenously applied beta gamma -subunits, adding ATP (1-4 mM) to the cytoplasmic side of inside-out patches increases the channel open probability approximately fivefold. The stimulatory effect of ATP on the G protein-activated KACh channel has also been reported by other investigators (4). These findings indicate that the ATP-dependent mechanism may have an important modulatory effect on KACh channel function. Because adenylimidodiphosphate, a nonhydrolyzable analog of ATP, does not produce the same effect as ATP (14), hydrolysis of ATP may be necessary to increase the channel activity. The precise mechanism of the ATP effect on the KACh channel, however, is not known.

Several earlier studies reported that in the absence of ACh and intracellular GTP (GTPi), application of ATP to the cytoplasmic side of the atrial membrane activates the KACh channel (3, 10). This effect of ATP has been attributed to the generation of active Galpha -GTP from inactive Galpha -GDP via a transphosphorylation reaction catalyzed by membrane-bound nucleoside diphosphate kinase (NDPK) using ATP as the substrate (3, 10, 23). However, such a mechanism for KACh channel activation may not occur under physiological conditions when a sufficient amount of GTP (~100 µM) is already present in the cell for G protein function. Furthermore, it is clear that intracellular ATP (ATPi) itself, whatever the source might be, does not activate the KACh channel under physiological conditions, because cell-attached patches show only a very low, basal level of activity (mainly due to GTP in the cell) and cells contain 4-5 mM ATP. In a recent study (31), it was shown that in the presence of an agonist, NDPK does not participate in the formation of GTP by phosphate transfer from ATP. Therefore, we speculate that the role of the ATP-dependent mechanism may be to modulate the degree of activation produced by a given level of G protein stimulation rather than to activate the channel.

To further understand the behavior of the KACh channel with respect to G protein-induced activation and ATP-induced stimulation, we used experimental conditions that might alter the function of cytoplasmic domains of KACh channel protein. The cytoplasmic domains of the KACh channel have been shown to be important in the activation process because they contain the interaction sites for beta gamma -subunits (9, 26). We considered three maneuvers that might affect the behavior of the cytoplasmic domains of the KACh channel: 1) treatment with trypsin and other proteases that would be expected to cleave parts of the intracellular domains, 2) changes in intracellular pH (pHi) that would alter the net charge of the channel molecule, 3) and use of different anions, some of which might affect protein conformation and function (20, 27).

The results of our study show that the ATP-induced stimulation of the KACh channel could be selectively inhibited by various experimental conditions used in this study, without disruption of the G protein coupling to the KACh channel. This provides evidence that G protein is not involved in ATP-induced changes in KACh channel activity. Therefore, the two processes (activation by G protein and stimulation by ATP) are independent and separate mechanisms that control KACh channel activity. Studies with anions show that Cl- plays an important, permissive role in G protein- and ATP-mediated effects on KACh channel function.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell preparation. Hearts from 1- to 2-day-old rats (Sprague-Dawley) were dissociated with collagenase and trypsin (13). Animals were used in accordance with the Guide for the Care and Use of Laboratory Animals [DHHS Publication No. (NIH) 85-23]. Rats were rapidly decapitated, and right and left atrial tissues from whole hearts were excised and placed in Ca2+-free Hanks' medium (Sigma Chemical, St. Louis, MO). The tissues were then cut into small pieces (<1 mm3) with a sharp blade and placed in Hanks' balanced salt medium containing 0.05% collagenase (type II; Worthington) and 0.06% trypsin (from bovine pancreas; Sigma Chemical). Tissues were incubated at 37°C and agitated for 7 min. Suspended cells were then removed and added to an equal volume of 50% fetal calf serum to inhibit the activity of the enzymes. Remaining tissues were incubated in a fresh enzyme solution and allowed to dissociate for another 7 min. This procedure was repeated five times. Dissociated cells were collected, centrifuged, and placed in the growth medium consisting of culture medium (Dulbecco's modified Eagle's medium; Sigma Chemical), 10% fetal calf serum, and 0.1% penicillin-streptomycin. Cells were plated on glass coverslips and placed in a 37°C incubator gassed with 5% CO2-95% air for 18-24 h.

Electrophysiology. Gigaseals were formed using Sylgard-coated thin-walled borosilicate pipettes (Kimax) with 3- to 4- MOmega resistances. Channel currents were recorded with an Axopatch 200 patch-clamp amplifier, digitized with a PCM adapter (VR10, Instrutech, Elmont, NY) and stored on videotape using a videotape recorder (JVC). The recorded signal was filtered at 2 kHz using an eight-pole Bessel filter (Frequency Devices, Haverhill, MA) and transferred to a computer (Dell) using the Digidata 1200 interface (Axon Instruments, Foster City, CA). Continuous single-channel currents were then analyzed with the pClamp program without further filtering (version 6.0.3). Data were analyzed to obtain duration histogram, amplitude histogram, single-channel conductance in picosiemens, and channel activity [averaged NPo, where N is number of channels and Po is probability of a channel being open]. NPo was determined from 1-2 min of channel openings at the beginning and end of each treatment. Current tracings shown in Figs. 1, 2, 4, and 7 were filtered at 100 Hz. Data are represented as means ± SE. Student's t-test was used to test for significance at the level of 0.05.

Solutions and materials. The pipette and bath solutions contained (in mM) 140 KCl, 2 MgCl2, 10 HEPES, and 5 EGTA (pH 7.2). To change solutions perfusing the cytosolic surface of the inside-out patches, we brought the pipette with the attached membrane to the mouth of the polypropylene tubing through which flowed the desired solution at a rate of ~1 ml/min. For studies with changes in pH and ATP, amounts of MgCl2 and ATP were determined to produce desired concentrations of free Mg2+ and MgATP using EQCAL software (Biosoft, Milltown, NJ). Free Mg2+ concentration ([Mg2+]) in solutions was always kept constant at 1.0 mM. All experiments were performed at 24-26°C.

ACh, GTP, GTPgamma S, and ATP were purchased from Boehringer Mannheim Chemicals (Indianapolis, IN). Trypsin (porcine pancreas, type 2), trypsin inhibitor (soybean, type 1), and all K salts were purchased from Sigma Chemical.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Inhibition of ATP-induced increase in KACh channel activity by trypsin. Trypsin has been reported to cause activation of the KACh channel in atrial cells when applied to the cytoplasmic side of inside-out patches, even in the absence of an agonist and GTPi (16). Thus trypsin removes the G protein dependence of the KACh channel. Therefore, we examined whether trypsin also affected the ATP-dependent stimulation of the KACh channel. First, we examined whether trypsin had an effect on the KACh channel already activated with GTP. Inside-out patches were formed with ACh in the pipette (10 µM), and GTP (100 µM) was applied to the bath solution to activate the KACh channels. Addition of trypsin (0.5 mg/ml) to the bath solution did not significantly affect the KACh channel activity for at least 8 min (Fig. 1A). Trypsin (0.5 mg/ml) applied alone in the absence of GTP slowly activated the KACh channel and reached a steady-state level in ~3 min. Addition of GTP to the bath solution caused a slight increase in KACh channel activity in some patches, but had no significant effect in other patches (Fig. 1B). Single-channel conductance (36 ± 2 pS) and mean open time (0.9 ± 0.1 ms) determined after trypsin treatment were not significantly different from the values obtained during activation by GTP (35 ± 2 pS, 1.0 ± 0.1 ms; n = 3 patches). These results are in agreement with earlier findings that a short period of exposure to trypsin removes the requirement for G protein for KACh channel activation and does not affect the channel gates that govern the channel conductance and open time duration.


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  		Fig. 1.   Effect of trypsin on ACh-sensitive K+ (KACh) channel in presence and absence of GTP. A: an inside-out patch was formed with ACh (10 µM) in pipette. Cell membrane potential was held at -60 mV. GTP (100 µM) was applied to bath, and trypsin (0.5 mg/ml) was applied ~3 min later. B: trypsin was applied first, and GTP (100 µM) was added subsequently. Bar graph on right summarizes effect of GTP and trypsin on KACh channel activity. A and B on bottom of bar graph refer to experiments shown in A and B on left. KACh channel activity (averaged NPo, where N is number of channels and Po is probability of a channel being open) was determined from ~2 min of channel openings before and after each treatment. Each bar represents mean and SE of 5 determinations. No significant difference was present between values within groups A or B.

When KACh channels were first activated with 100 µM GTP, subsequent application of 1 mM ATP produced a marked increase in channel activity, as shown in Fig. 2A. This stimulation by ATP was observed in every patch we tested (n > 50). In other patches, trypsin (0.5 mg/ml) was applied after activation of the KACh channel with GTP and 1 mM ATP was added ~3 min later (Fig. 2B). In all 12 patches tested, trypsin treatment completely inhibited the ATP-induced stimulation of the KACh channel. To be sure that the trypsin molecule itself did not interfere with ATP binding to its site, trypsin was washed off after 3 min of perfusion and ATP was added 1-2 min later. In these patches, no stimulatory effect of ATP was present. When KACh channels were activated by trypsin in the absence of GTP, the KACh channel also failed to respond to ATP (Fig. 2C). Trypsin solution boiled for 5 min failed to inhibit the ATP-induced stimulation of the KACh channel (Fig. 2D). In addition, the stimulatory effect of ATP was preserved when trypsin inactivated by incubation with trypsin inhibitor (1 mg/ml) was used. Figure 2F summarizes the results of these experiments and shows that trypsin produces a selective and irreversible modification of the KACh channel (or an associated ATP-sensitive regulatory protein) either in the presence or absence of GTP such that the channel is no longer able to be stimulated by ATP. This effect of trypsin is probably caused by proteolytic cleavage of a certain intracellular domain(s). Interestingly, when trypsin was applied after the KACh channels were modified by GTP and ATP, trypsin did not reverse the ATP-induced increase in channel activity (Fig. 2E). This suggests that the cytoplasmic cleavage site is no longer accessible to the enzyme, possibly because of a conformational change induced by ATP. If it is assumed that G protein and ATP act directly on the KACh channel, these results support the view that two gates, the G protein- and the ATP-sensitive gates, regulate KACh channel activity and that each gating mechanism works independently.


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  		Fig. 2.   Inhibition of ATP-induced stimulation of KACh channel activity by trypsin. Cell-attached patches were formed, and membrane potential was held at -60 mV, as shown by ~30 s of initial activity (A-C). A: an inside-out patch was formed with 10 µM ACh in pipette, and GTP (100 µM) was applied to activate channel. ATP (1 mM) was then added to bath solution. B: after activation of channel with GTP (100 µM), 0.5 mg/ml trypsin was added. ATP (1 mM) was then added. C: an inside-out patch was formed with ACh in pipette. Trypsin was applied and washed off when a steady-state level of channel activity was present (~3 min). ATP (1 mM) was applied 3 min later. D: same experiment as B except that trypsin solution was boiled for 5 min. E: trypsin was applied to patch after GTP and ATP had increased channel activity. F: results of these experiments shown as a bar graph. Each bar represents mean and SE of 4-6 determinations. Significant difference between values within each group before and after addition of ATP was found in groups A, D, and E (P < 0.05).

Inhibition of ATP-induced increase in KACh channel activity by alkaline pH. Protons can alter ion channel function by several mechanisms such as entering and blocking the pore, changing electrostatic interactions between different regions, and altering channel conformation. We examined whether the KACh channel is sensitive to changes in pHi with respect to GTP- and ATP-induced effects in channel activity. Inside-out patches were formed with ACh in the pipette, and GTP was applied to the bath solution kept at pH 7.0. After ~2 min, the pH of the bath solution was changed from 7.0 to 6.0 or 8.0 in increments of 0.5 pH unit. In other experiments, we started with a bath solution at pH 6.0 and increased the pH to 8.0 in increments of 0.5 pH unit. In all experiments, free Mg2+ was kept constant at 1 mM by adjusting the amount of MgCl2. KACh channel activity was not significantly affected by changes in pHi except at pH 6.0. At pH 6.0, the channel activity was 72 ± 6% of that observed at pH 7.0 (Fig. 3A). The effect of changing pHi on single-channel amplitude occurred quickly, within 10 s. In general, the single-channel conductance was decreased by acidic pH and increased by alkaline pH, although a statistically significant decrease was observed only at pH 6.0 when compared with that at pH 7.0 (Fig. 3B). A similar effect of pHi on conductance was present in the presence of GTP and ATP.


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  		Fig. 3.   Effect of intracellular pH (pHi) on channel activity and single-channel conductance. Cytoplasmic side of inside-out patches was perfused with internal solution containing GTP (100 µM) or GTP + ATP (1 mM) at desired pH (6.0-8.0). Channel activity (A) and conductance (B) were determined at each pH value. Each value is mean and SE of 5 determinations. * Significantly different from value determined at pH 7.0 (P < 0.05).

To determine whether pHi alters the concentration at which half-maximal activation occurred (K1/2) for GTP, we examined the effect of pHi on the GTP concentration ([GTP])-NPo relationship. GTP was applied to the cytoplasmic side of inside-out patches starting at 0.1 µM and progressively increased to 100 µM, allowing a steady-state level of channel activity (NPo) to be achieved at each [GTP]. NPo was determined for each [GTP] and plotted as a function of [GTP] as shown in Fig. 4. Compared with the relationship obtained at pH 7.0, those at pH 6.0 and 6.5 were shifted to the right and those at pH 7.5 and 8.0 were shifted to the left. K1/2 values were (in µM) 6.7, 1.4, 1.1, 0.5, and 0.3 at pH 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. The Hill coefficients ranged from 3 to 4, indicating that cooperativity of G protein binding to the channel was not greatly affected by changes in pHi from 6.0 to 8.0. Thus, within the pH range of 6.0-8.0, GTP was able to activate the KACh channel, although some changes in conductance and sensitivity to GTP were present. Also, the mean open times, determined from patches showing mainly one level of channel opening, remained unchanged; they were 1.0 ± 0.1, 0.9 ± 0.1, and 0.8 ± 0.1 ms at pH 6.0, 7.0, and 8.0, respectively.


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  		Fig. 4.   Intracellular GTP concentration ([GTP])-channel activity relationships at different pHi. Inside-out patches were perfused with increasing [GTP], and channel activity was plotted as a function of [GTP] at various pHi. Data points (n = 4-6 patches for each point) were fitted with a Hill equation (f = 1/[1 + (k/[GTP])nH], where k is [GTP] at which half-maximal activation occurs and nH is Hill coefficient). Values of k were (in µM) 6.7, 1.4, 1.1, 0.5, and 0.3, and nH values were 3.0, 3.1, 4.3, 3.7, and 3.0 at pH 6.0, 6.5, 7.0, 7.5, and 8.0, respectively. Vertical lines on certain data points indicate SE.

We then examined the effect of pHi on the ATP-induced stimulation of the KACh channel activity in the presence of 100 µM GTP, a concentration that is sufficient to produce the maximal G protein-mediated activation of the channel at all pHi levels (pH 6.0-8.0). The pH of the pipette solution (extracellular) was kept at 7.2, and the bath solution with a desired pH was used. Free [Mg2+] was kept at 1 mM, and MgATP was adjusted to 1 mM when using solutions at different pH. GTP was applied first to activate the channel, and ATP was added after ~2 min. When the pH of the bath solution was 6.0, 6.5, and 7.0, the ATP-induced increase in channel activity was consistent and clearly observed in every patch tested (Fig. 5A). The top tracing in Fig. 5A shows a current recording from an inside-out patch perfused with a bath solution kept at pH 6.0. To clearly illustrate the effect of ATP, a KACh current record from a patch with a low activity is shown. As we described earlier, the ATP-induced prolongation of open time duration was evident from such patches with a low level of activity (see ATP increases open time duration but not opening frequency). However, at pHi 7.5, the degree of channel stimulation by ATP was reduced, and at pHi 8.0, a nearly complete reduction in the ATP effect was present. A plot of the ATP-induced increase in channel activity as a function of pHi shows clearly that alkaline pH (pH 8.0) selectively inhibits the ATP-induced stimulation. The lack of effect of ATP on the open time duration at pH 8.0 can also be clearly observed. The half-maximal effect of ATP occurred at pH 7.4. Changing extracellular pH from 7.2 to 6.0 or 8.0 at constant pHi (bath solution) did not produce significant effects on the ATP-induced channel activity. For example, at extracellular pH of 6.0, 7.0, and 8.0, the ATP-induced increases were 4.6 ± 0.9-, 4.3 ± 0.8-, and 4.4 ± 1.1-fold, respectively (n = 4 patches each). Thus these results show that changes in pHi between 6.0 and 8.0 affect both the G protein-induced activation of the channel and the ATP-induced stimulation of the channel activity. The effects of pHi on the two nucleotide-induced changes in KACh channel function are qualitatively opposite, supporting the view that GTP- and ATP-induced effects on the KACh channel involve separate gating mechanisms.


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  		Fig. 5.   ATP-induced stimulation of KACh channel activity at different pHi. Inside-out patches were formed with ACh in pipette solution (pH 7.2), and cytoplasmic sides were perfused with a solution at a desired pH (6.0-8.0). GTP (100 µM) was applied to activate channel, and then ATP (1 mM) was added to stimulate channel further without altering pH of bath solution. Three current tracings obtained at pH 6.0 (A), 7.5 (B), and 8.0 (C) are shown. Expanded current tracings are shown on right. D: plot of ATP-induced increase vs. pH is fitted with a Hill equation (f = 1/[1 + ([pH/k])nH]), where k (7.4) is pH at which ATP-induced increase in channel activity is half maximal and nH (1.1) is Hill coefficient. Each point is mean of 5 determinations.

ATP increases open time duration but not opening frequency. Previously, ATP has been reported to increase the mean open time of the KACh channel from ~1.0 to 3-5 ms (6, 13). However, the measurement of mean open times in patches containing multiple channels is clearly not accurate and is subject to large errors. Therefore, it is not clear how much of the ATP-induced increase in channel activity is related to changes in open time duration. Because ATP activates the channel when applied in the absence of GTP and ACh (3, 10), it is possible that ATP also augments channel activity by increasing the frequency of opening when GTP and ACh are present. To determine whether ATP affects only the open time duration or, in addition, has an effect on frequency of opening, we measured the number of transitions from the closed to the open state during 60-s periods. Despite large differences in the response to ATP at different pH values, ATP did not produce a significant change in the number of closed-to-open transitions at pH 6.0, 7.0, or 8.0 (Fig. 6). These results provide evidence that the increase in KACh channel activity produced by ATP is primarily caused by changes in the open time duration. Therefore, trypsin and alkaline pH probably impair the function of the gate that governs the open time duration, which can be separated from the G protein-sensitive gate, which has no effect on the open time duration.


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  		Fig. 6.   Effect of ATP on number of closed-to-open transitions of KACh channel during a 60-s period. Inside-out patches with ACh in pipette were formed, and GTP was applied to activate channel. After ~3 min, 1 mM ATP was added to bath solution to stimulate channel activity. This procedure was performed using bath solution maintained at pH 6.0, 7.0, and 8.0. Each bar represents number of closed-to-open transitions during a 60-s period of channel activity observed in presence of GTP alone or GTP and ATP. ATP did not produce a significant change in number of closed-to-open transitions at each pH.

Dependence of ATP-induced stimulation of KACh current on anions. It has been reported that the sensitivity of the KACh channel to G protein is dependent on the type of anion used (21). Therefore, it was of interest to know whether anions also affect ATP-induced stimulation of the KACh current. We studied the effect of GTP and ATP on the KACh channel using bath solutions containing K-acetate, K-gluconate, K-aspartate, K-glutamate, K-sulfate, K-citrate, K-phosphate, K-iodide, or K-bromide in place of K-chloride. K+ concentration was kept constant at 140 mM for each salt solution. In inside-out patches with ACh in the pipette, GTP was applied to determine whether KACh channels could be activated in a solution containing a particular anion and, if activation occurred, whether subsequent addition of 1 mM ATP stimulated the KACh channel activity.

With ACh in the pipette, GTPi was clearly able to activate the KACh channels in inside-out patches bathed in solution containing all but sulfate, phosphate, iodide, and fluoride (Table 1). Within the group of anions that allowed the activation of the KACh channels by GTP, all but citrate supported ATP-induced stimulation of the KACh channel (Fig. 7A). Interestingly, when 3 mM KCl was present in the bath solution together with K3-citrate (45.7 mM), ATP was able to produce an increase in KACh channel activity (3.8 ± 1.1-fold; n = 4 patches), indicating that Cl- plays an important role in the ATP-mediated effect (Fig. 7B).

                              
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  		Table 1.   GTP- and ATP-dependent effects on muscarinic KACh channel in solution containing various anions


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  		Fig. 7.   Effect of GTP, ATP, and Cl- on KACh channel activity in solution containing K-citrate and K-phosphate (140 mM K+). A: an inside-out patch with ACh in pipette was formed, and GTP was applied to bath solution containing citrate as anion. Subsequent application of 1 mM ATP failed to increase channel activity. B: same experiment as in A except that 3 mM KCl was present in bath solution along with K-citrate. C: same experiment as in A except that phosphate was used as anion. After GTP + ATP was found to have no effect, phosphate was replaced with Cl-. D: same experiment as in A except that 135 mM K-phosphate and 5 mM KCl were used in bath solution. Under this condition, GTP activated channels and ATP further increased channel activity. Changing bath solution to that containing 140 mM KCl did not significantly change channel activity.

In patches in which the anions (sulfate, phosphate, iodide, and fluoride) did not support GTP-induced activation, subsequent application of ATP also failed to activate the KACh channels (n = 4 each). An example is shown in Fig. 7C, in which GTP and ATP were unable to activate the channels in phosphate solution but clearly activated the channels in Cl- solution. Interestingly, the presence of only 5 mM KCl in the phosphate-containing solution permitted GTP to activate and ATP to stimulate the KACh channel (Fig. 7D). Additional experiments indicated that only a minimal amount of Cl- (0.5 mM) was necessary to permit activation of the KACh channel by GTP, although a higher Cl- concentration ([Cl-]; 1 mM or more) was required to produce an increase in channel activity by ATP. These results indicate that Cl- plays an important role in both G protein- and ATP-dependent signaling pathways involved in KACh channel activation.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The G protein-mediated mechanism is generally believed to be the pathway by which ACh activates the KACh channel. However, studies during the last several years indicate that ATPi might also be an important modulator of KACh channel function by increasing the open probability of the G protein-activated channels. The present study was therefore done to further examine the behavior of the KACh channel with respect to G protein-mediated activation and ATP-mediated stimulation under various experimental conditions. We studied the effect of proteases, pH, and different anions to determine how each experimental manipulation modifies the two nucleotide-dependent processes. Our study shows that trypsin, which can activate the KACh channel in the absence of GTP and ATP, blocks the ATP-induced increase in channel activity. Alkaline pH and certain anions can selectively block the ATP-dependent process without uncoupling of signaling between the muscarinic receptor, G protein, and the KACh channel. These results suggest that the G protein-mediated activation involves a mechanism distinct from the ATP-induced stimulation of the KACh channel.

Trypsin inhibits ATP-dependent stimulation of KACh channel activity. Application of trypsin to the cytoplasmic side of atrial membrane has previously been shown to cause spontaneous activation of the KACh channel even in the absence of GTP and ACh (16). Thus trypsin produces the same net effect as the beta gamma -subunits of the pertussis toxin-sensitive G protein, which activates the KACh channel by binding directly to the cytoplasmic domains of the channel protein (1, 8, 18). These results indicated to us that trypsin must have caused a substantial functional change in the cytoplasmic domain(s) of the channel protein. Therefore, it was of interest to determine whether trypsin could affect the ATP-dependent stimulation of the channel activity. The results show that trypsin treatment abolishes the ATP-induced increase in channel activity, suggesting that a cytoplasmic gating particle sensitive to ATP has probably been removed. It is unlikely that trypsin affected the amino acid residues near or at the pore regions, because single-channel kinetics, conductance, and K+ selectivity remained unchanged. It is possible that trypsin blocked the interaction between the KACh channel and an associated regulatory molecule that confers the ATP sensitivity to the channel. G protein-coupled muscarinic K+ channel proteins GIRK1 and CIR, two cloned subunits that form nativelike KACh channels when coexpressed in oocytes or mammalian cells, have also been reported to respond to ATP (15, 17). Therefore, if such a regulatory molecule is the transducer of the ATP effect, then it is also present in oocytes and mammalian cells used for transfection. Although many studies have been done using GIRK1/CIR expressions, no specific domains of the KACh channel subunits that confer ATP sensitivity have yet been identified. The KACh channel subunit that possesses the ATP-sensitive gate may be more closely associated with CIR, because it is activated by ATP when expressed alone (17). Recently, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] has been reported to activate the inwardly rectifying K+ channels such as ATP-sensitive and muscarinic-gated K+ channels after these channels have desensitized or run down (7, 28). Whether the effect of ATP observed in this study is related to the effect of PtdIns(4,5)P2 is not known, but this seems unlikely because channel desensitization or rundown was minimal in atrial cell patches and ATP itself only minimally activates the KACh channel under our experimental conditions. Our results show that trypsin affects both GTP- and ATP-mediated mechanisms: removal of GTP dependence in channel activation and removal of the ATP effect. The ability of the KACh channel to be in the GTP-induced open state can therefore be completely uncoupled from its ability to respond to ATPi, indicating that the two processes very likely involve separate sites on the channel protein.

Sensitivity of KACh channel to pHi. Protons have multiple effects on ion channel function such as binding to the ion permeation pathway and blocking the channel, altering the surface charge near the ion pore and influencing channel conduction by an electrostatic mechanism, and modifying the channel conformation (2, 24, 30). Therefore, changes in pHi are expected to affect certain aspects of KACh channel function. Our results show that alkaline pH increases the sensitivity of the KACh channel to GTP, whereas it decreases the sensitivity to ATP. This opposite relationship supports the view that the GTP- and ATP-induced effects on the KACh channel activity occur via different processes. If the ATP-induced stimulation were via NDPK and G protein as suggested previously (10), such an opposite relationship would not be observed.

The half-maximal effect of ATP is observed at pH 7.4. Because pHi in healthy cells is ~7.1, the ATP-dependent effect on the KACh channel would be expected to occur whenever the KACh channels are activated by ACh or any other agonists whose receptors are coupled to the KACh channel (11, 12, 19). If pHi decreases, for example, by lactate overproduction during ischemia, the G protein-mediated activation of the KACh current would be decreased but the ATP-dependent effect would be unaffected. We have shown previously (22) that neuronal G protein-coupled K+ channels have sensitivity to ATP similar to that of atrial KACh channels. Therefore, both atrial and neuronal K+ channels are expected to be regulated by pHi in a similar way with respect to GTP- and ATP-induced changes in channel activity.

Dependence of KACh channel function on anions. The intracellular solution used in all experiments contained 140 mM KCl. Because normal intracellular [Cl-] is ~4-10 mM, it was of interest to know whether the ATP-induced stimulation can still occur in a solution containing low [Cl-] and whether other anions can support the ATP effect on the KACh channel. Our results show that anions typically used in electrophysiological experiments all support both GTP- and ATP-dependent effects on the KACh channel. Even for citrate ions, which selectively inhibited the ATP-dependent stimulation without blocking the GTP-induced activation of the KACh channel, only a few millimolar amounts of Cl- were sufficient to overcome the inhibitory effect. Therefore, the ATP-dependent effect on the KACh channel should occur in a physiological medium that contains Cl-, phosphate, bicarbonate, negatively charged groups on proteins, and organic ions such as ATP and phosphocreatine.

The permissive action of Cl- on the ATP effect is particularly significant. Cl- has been shown to inhibit the catalytic rate of GTPase of G proteins by increasing the affinity of Galpha to GTP (5). Cl- also destabilizes the normal structure of myosin (27) and enhances rundown of ATP-sensitive K+ channel in muscle (20). Thus one can speculate that Cl- allows ATP to work on the channel by affecting the stability of the cytoplasmic domains of the channel protein. If a change in intracellular Cl- occurs, for example, this could influence the KACh current by modulating the amount of stimulation produced by ATP.

In summary, we examined the effects of proteases, changes in pH, and different anions on the functional properties of the KACh channel. We show that trypsin can remove the GTP dependence of the KACh channel while inhibiting the ATP-dependent stimulation. Alkaline intracellular solution (pH 7.5-8.0) increases the sensitivity of the KACh channel to GTP, whereas it blocks the ATP-dependent stimulation of the KACh channel activity. Most anions can support both GTP- and ATP-dependent effects on the KACh channel; however, citrate selectively blocks the ATP-induced stimulation of the KACh channel without inhibiting the G protein-induced activation. Presence of a small amount of Cl- (3 mM) was sufficient to permit ATP to stimulate the K+ channel. These findings support the view that GTP- and ATP-induced effects are independent events, presumably involving different cytoplasmic domains of the KACh channel protein.

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: D. Kim, Dept. of Physiology and Biophysics, Finch Univ. of Health Sciences/The Chicago Medical School, 3333 Green Bay Rd., North Chicago, IL 60064.

Received 13 January 1998; accepted in final form 24 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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Am J Physiol Heart Circ Physiol 275(3):H751-H759
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society



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