Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 1998 Mar 1;507(Pt 2):315–324. doi: 10.1111/j.1469-7793.1998.315bt.x

Modulation of reconstituted ATP-sensitive K+ channels by GTP-binding proteins in a mammalian cell line

Jorge A Sánchez *, Tohru Gonoi *, Nobuya Inagaki †, Toshiaki Katada ‡, Susumu Seino †
PMCID: PMC2230792  PMID: 9518695

Abstract

  1. The action of GTP-binding proteins on ATP-sensitive potassium (KATP) channels was investigated. KATP channels were expressed in a mammalian cell line (COS-1 cells) by cotransfecting vectors carrying the sulphonylurea receptor (SUR1) and BIR (Kir6.2), a member of the inward rectifier K+ channel family. G proteins were also tested on KATP channels composed of an isoform of SUR1, SUR2A, in combination with Kir6.2.

  2. The α and βγ subunits of the GTP binding protein Gi were tested separately in inside-out patches under continuous recording. Gα-i1 increases the activity of SUR1-Kir6.2 and SUR2A-Kir6.2 channels by 200 and by 30 %, respectively.

  3. Gα-i2 does not increase the activity of SUR1-Kir6.2 channels, but increases the activity of SUR2A-Kir6.2 channels by 30 %.

  4. Control experiments showed that GTPγS, a specific activator of G proteins, and heat- inactivated Gα-i1 do not increase the single channel activity.

  5. No effects of the other subunits (βγ) from either Gi1 or Gi2 on the single channel activity were observed.

  6. The protein kinase C inhibitors H7 and an inhibitory peptide (FARKGALRQKNV) had no effect on the modulatory action of Gα-i1 on SUR1-Kir6.2 channels.

  7. We conclude that both types of reconstituted KATP channels are modulated by G proteins.

In recent years, modulation of ion channel activity by membrane-delimited complexes has been shown. In this type of modulation, guanosine nucleotide-binding proteins (GTP-binding proteins) couple to ion channels without the involvement of cytosolic second messengers. A well documented example is the inward rectifier potassium channel in heart muscle. This potassium channel opens by activation of muscarinic receptors via a GTP-binding protein. In addition, several K+ and Ca2+ channels have been shown to alter their activity by membrane-delimited modulation (for reviews see Hille, 1992; Brown, 1993). Insulin secretion is modulated by ligands that act via GTP- binding proteins. Cellular targets for G proteins include ATP-sensitive K+ channels (KATP) (Ribalet, Eddelstone & Ciani, 1988b; De Weille, Schmid-Antomarchi, Fosset & Lazdunski, 1989). These channels play a key role as a link between metabolism and membrane excitability in pancreatic β-cells (Cook & Hales, 1984) as well as in cardiac myocytes (Noma, 1983). KATP channels from pancreatic β-cells and cardiac myocytes have been recently cloned and functionally expressed. They are composed of two subunits: a sulphonylurea receptor (SUR) and Kir6.2, a member of the inward rectifier K+ channel family (Sakura, Ämmälä, Smith, Gribble & Ashcroft, 1995; Inagaki et al. 1995, 1996). SUR1 and Kir6.2 reconstitute the β-cell KATP channel, while SUR2 (now referred to as SUR2A) and Kir6.2 reconstitute cardiac and skeletal-muscle KATP channel. Recently, variants of SUR2 (SUR2B) have also been identified (Isomoto et al. 1996; Chutkow, Simon, Le Beau & Burant, 1996). While SUR probably confers the ATP and sulphonylurea sensitivity, Kir6.2 probably forms the K+ ion permeable domain (Ämmälä, Moorhouse & Ashcroft, 1996; Inagaki et al. 1996). However, a recent study has suggested that Kir6.2 itself could also confer the ATP sensitivity (Tukker, Gribble, Zhao, Trapp & Ashcroft, 1997). The expression of KATP channels in a heterologous expression system provides a unique opportunity to study the interaction between KATP channels and G proteins without the involvement of second messengers, G proteins, or receptors present in native cells.

The present experiments examine the effects of G proteins on KATP channels expressed in COS-1 cells. We found dramatic effects in the open probability of K+ currents. Some of these effects have not been described in native cells before.

METHODS

Purification of G proteins

Heterotrimeric forms of G proteins, Gi1 and Gi2 were purified from bovine brain membranes and separated into GTPγS-bound α (Gα-i1 and Gα-i2) and βγ (Gβγ-i1 and Gβγ-i2) subunits, respectively, as described previously (Kobayashi et al. 1990; Kontani, Takahashi Inanobe Ui & Katada, 1992). The βγ subunits were stored at a concentration of 25 μm in a solution containing: 50 mm Na-Hepes (pH 7.4) and 0.7 % Chaps at -80°C. Biological activity of our βγ subunit preparation was assayed and reported previously (Yamada et al. 1994). Thc GTPγS-bound α subunits were stored at a concentration of 2 μm in a similar solution that also contained 0.1 mm MgCl2.

Cell culture and transfection

COS-1 cells were plated at a density of 3 × 105 per dish (35 mm in diameter) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplement with 10 % fetal calf serum. The mammalian expression vectors: pCMV6bSUR1 (1.5 μg) carrying SUR1, or pCMV6bSUR2A (1.5 μg) carrying SUR2A, and pCMVmBIR (1.5 μg) carrying Kir6.2, as well as the expression plasmid vector for the green fluorescent protein (0.05 μg) as reporter gene (Marshall, Molloy, Moss, Howe & Hughes, 1995), were cotransfected into COS-1 cells with pAdVantage (Promega, WI, USA), Lipofectamine and Opti-MEM reagents (Life Technologies, Inc., Gautherburg, MD, USA). Electrophysiological experiments were performed in transfected cells that were selected by their green fluorescence. Recordings were made 1–5 days after transfection.

Solutions

The pipette and bath solution contained (mm): 140 KCl; 2 MgCl2 and 5 EGTA. The solutions were buffered with Hepes (10 mm) at pH 7.3. The estimated free Mg2+ concentration in this solution is 0.4 mm according to calculations described by Fabiato (1988). Aliquots from the solutions containing the G protein subunits were taken just before each test to obtain the desired final concentration of G proteins. The bath solution facing the intracellular side of the membrane was continuously superfused with the control and ATP-containing solutions at a rate of 5 ml min−1. When G proteins were tested, a static bath was used and the test solutions were applied by hand. The protein kinase inhibitors: H7 (Funakoshi, Japan) and the synthetic peptide FARKGALRQKNV, corresponding to the pseudosubstrate region of protein kinase C (Seikagaku Corporation, Japan) were dissolved in water and tested at the indicated concentrations.

Electrophysiological methods

The patch clamp technique was used in the inside-out configuration (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Pipettes were double-pulled from soft glass and the tips were coated with Sylgard and fire-polished to resistances of about 8–10 MΩ. Currents were recorded with an EPC-7 amplifier (List Electronics, Germany).

Data collection

Single channel currents were continuously stored on VCR tape with a digital data recorder for later analysis. The holding potential was -60 mV. Patches were first recorded in a control solution containing 1 μm ATP. Thereafter, the patch was exposed to solutions containing increasing nucleotide concentrations: 10, 50, 100, 1000 μm and returned to 1 μm. A final control record in a preselected ATP concentration was taken before G proteins were tested. Uninterrupted recordings of at least 4 min were obtained in each solution. Data were analysed by pCLAMP (ver. 6, Axon Instruments, CA, USA). The amplitudes, closed durations, and open durations were measured individually and visually checked for accuracy. Times of transitions were defined as crossing of the 50 % threshold between adjacent levels. Scatter plots of event amplitudes versus duration were displayed for each analysis.

The open state probability (Po) was calculated according to eqn (1),

graphic file with name tjp0507-0315-m1.jpg (1)

where N is the number of channels active in the patch, tj is the time spent at each current level j, and T is the duration of the recording (Fenwick, Marty & Neher, 1982; Spruce, Standen & Stanfield, 1987). Since the number of channels in each individual patch is unknown, we expressed our data as NPo. The relative open probability was calculated as the ratio between NPo in the test solution and the corresponding value in control conditions. We assumed that N remains constant throughout the course of the experiment. To characterize the single channel activity of a given patch in a particular solution, we calculated NPo in 5 s intervals during the whole time of recording and these values were averaged. Experiments were performed at room temperature, 20–22°C.

The fitting of numerical formulae to experimental data employed a non-linear least squares algorithm. Parameter values given in the table are expressed as means ± standard error of the mean. When calculating statistical significance, Student's paired t test was used with significance at the level P < 0.05.

RESULTS

The actions of G proteins on SUR1-Kir6.2 channels

Inagaki et al. (1995) indicated that KATP channels can be expressed in COS-1 cells by cotransfection of SUR1 plus Kir6.2. Our experiments confirm these observations. Figure 1 shows unitary currents recorded from ATP-sensitive channels. In this experiment, channel activity was first seen in the presence of 1 μm ATP (Fig. 1A). When the cytoplasmic face of the membrane was exposed to higher ATP concentrations, the open-state probability was greatly reduced (Fig. 1B-D). This reduction is concentration dependent and channel activity becomes negligible when ATP concentration is 100 μm or greater. Figure 1E shows the relationship between single channel activity (measured as NPo, see Methods) and the ATP concentration. Open symbols correspond to data from the experiment shown in panels A-D. The points were fitted to the Hill equation:

graphic file with name tjp0507-0315-m2.jpg (2)

where A is an amplitude factor that corresponds to the single channel activity in the absence of ATP, Ki is the mid-point of inhibition by ATP, and nH is the Hill coefficient. Half-maximal inhibition was obtained at 14 μm with a Hill coefficient of 2.

Figure 1. The ATP sensitivity of reconstituted K+ channels (SUR1 and Kir6.2).

Figure 1

Open in a new tab

Panels A-D show single channel recordings in the inside-out configuration at several ATP concentrations: 1 μm in A, 10 μm in B, 50 μm in C, and 100 μm in D. Panel E, ○, single channel activity from panels A-D. The abscissa represents the ATP concentration (μm) in the bath solution and the ordinate the single channel activity measured as the open probability times the number of active channels (see Methods). The smooth curve is the best fit of eqn (2) with A = 0.9, Ki = 14.5 μm, and nH = 2.0.

Next, the effect of G proteins on ATP-sensitive channels was investigated. In most experiments, we tested the G protein subunits in the presence of 10 or 100 μm ATP. We performed the experiments in the presence of ATP because previous reports have shown that ATP is required in native cells for G protein actions (Ito, Vereecke & Carmeliet, 1994; Terzic, Tung, Inanobe, Katada & Kurachi, 1994). The primary action of Gα-i1 on SUR1-Kir6.2 channels is to increase their open probability. This is shown in Fig. 2. Panel 2A shows control records obtained in the presence of 100 μm ATP from the experiment illustrated in Fig. 1. In panel 2B, GTPγS-bound Gα-i1 at a concentration of 0.01 nM was added to the intracellular face of the membrane. The amplitude of the currents remained unchanged but NPo increased from 0.04 to 0.08. When we applied Gα-i1 at higher concentrations, there was a further increase in the open probability of the channels, as shown in panels C and D: NPo increased to 0.16 and to 0.39 in the presence of 0.1 nM and 1 nM Gα-i1, respectively. A further increase to 10 nM did not bring about any additional increase in the open probability of the channels. This was also observed in a separate experiment. The mean ratio between NPo values in the presence of 10 nM and the corresponding values obtained in 1 nM was 1.10, suggesting that saturation of the effect is reached at 1 nM. Therefore, this concentration was used to characterize the action of α and βγ subunits of Gi in the experiments summarized in Table 1. At least three single channel levels are obvious in Fig. 2C-D, indicating that more than one channel was active at any given time. This fact complicates the analysis of distribution of open and closed times and the evaluation of Gα-i1 on kinetic constants. For this reason, we constructed open time histograms from idealized transitions without fitting a sum of exponentials to the distributions. Figure 3 shows open time histograms from the experiment shown in Figs 1 and 2. Panel 3A shows the control histogram and panel B the histogram obtained in the presence of 1 nM Gα-i1. It is clear that the number of events increases quite significantly in the presence of Gα-i1. These observations indicate that Gα-i1 increases the open probability of SUR1-Kir6.2 channels. A quantitative analysis of the effect of Gα-i1 on the open probability requires an estimate on the number of channels present in a given patch. This number cannot be calculated in a straightforward manner and the maximum number of levels provides only a lower limit. Therefore, we compared data from different experiments as the relative open probability (see Methods). In six experiments, this value averaged 2.87 ± 0.83 in the presence of 100 μm ATP. Since this concentration is fairly high, we also performed similar experiments but in the presence of 10 μm ATP. The relative open probability was 2.70 ± 0.63 (n = 5) in this case. These numbers are not significantly different. Therefore, we collected all the experiments performed with this subunit at these concentrations to produce the mean value shown in Table 1. Gα-i1 increases the open probability of SUR1-Kir6.2 channels by more than twofold. This effect is completely reversible after washout. In contrast to these results, Gα-i1 does not increase the single channel activity in the absence of MgATP. In two experiments, the open probability remained within 10 % of the corresponding value in the control solution (data not shown), in agreement with the findings of Ito et al. (1994) and Terzic et al. (1994), performed in native cells.

Figure 2. The stimulatory effect of Gα-i1 on KATP channels (SUR1-Kir6.2).

Figure 2

Open in a new tab

Panel A shows control records, panel B in the presence of 0.01 nM Gα-i1, panel C in 0.1 nM Gα-i1, and panel D in 1 nM Gα-i1. 100 μm ATP was present throughout. Same experiment as in Fig. 1.

Table 1.

The effects of G proteins on the open probability of KATP channels

SUR1-Kir6.2 channel
Po,test/Po,control (n) Po,wash/Po,control (n)
Gα-i1 2.78 ± 0.75 (11) 1.06 ± 0.19 (11)
Gα-i2 0.77 ± 0.11 (8) 0.92 ± 0.15 (7)
Gβγ1 0.98 ± 0.13 (5) 1.00 ± 0.13 (5)
Gβγ2 0.95 ± 0.09 (4) 0.93 ± 0.11 (2)
SUR2A-Kir6.2 channel
Po,test/Po,control (n) Po,wash/Po,control (n)
Gα-i1 1.27 ± 0.12 (7) 1.01 ± 0.07 (6)
Gα-i2 1.34 ± 0.13 (5) 0.98 ± 0.03 (3)
Open in a new tab

Concentration of α and βγ subunits is 1 nM.

Figure 3. The effect of Gα-i1 on the open time of KATP channels (SUR1-Kir6.2).

Figure 3

Open in a new tab

Panel A shows the open time histogram in control solution and panel B in 1 nM Gα-i1. Same experiment as in Figs 1 and 2. Note that the vertical scale in A differs from that in B. 100 μm ATP was present throughout.

In contrast to the enhancement of single channel activity of SUR1-Kir6.2 channels produced by Gα-i1, Gα-i2 does not increase the open probability of SUR1-Kir6.2 channels. Figure 4A shows a continuous recording of SUR1-Kir6.2 channels in the presence of 10 μm ATP. In panel B, Gα-i2 (1 nM) was added to the bath solution which produced a moderate decline in single channel activity. This effect was reversible as shown in panel C. The effect of Gα-i2 on the protracted activity of the channels is illustrated in Fig. 5A. The relative time that channels remain open over the whole recording time is illustrated. Open symbols represent NPo calculated from records obtained in the control solution. Filled symbols represent the activity in the presence of Gα-i2. Although there is some scattering in the single channel activity under each experimental condition, the channels become less active in the presence of Gα-i2 in a reversible manner. Figure 5B and C show open time histograms from records obtained in the control solution and in the presence of Gα-i2, respectively, from the same experiment. Clearly, Gα-i2 decreases the number of transitions of SUR1-Kir6.2 channels between the closed and open states. In similar experiments, Gα-i2 decreased the relative open probability to 0.80 ± 0.18 in the presence of 10 μm ATP and it decreased it to 0.75 ± 0.16 (n = 5) when 100 μm ATP was added to the bath solution. The difference was not significant and all individual values were gathered to produce the results summarized in Table 1.

Figure 4. The inhibitory effect of Gα-i2 on KATP channels (SUR1-Kir6.2).

Figure 4

Open in a new tab

Panel A shows control records, panel B in 1 nM Gα-i2 and C shows records after wash. 10 μm ATP was present throughout.

Figure 5. The inhibitory effect of Gα-i2 on KATP channels (SUR1-Kir6.2).

Figure 5

Open in a new tab

Panel A shows the single channel activity over time. ○, open probability in the control solution; •, in the presence of 1 nM Gα-i2. Each point represents the NPo value computed during 5 s. Panels B and C represent open time histograms in the control solution and in the presence of Gα-i2, respectively. Each histogram represents data obtained during 140 s of recording. Same experiment as in Fig. 4.

Because G proteins used in the present experiments are activated with GTPγS, a non-hydrolysable GTP analogue, it may be argued that GTPγS, released from the G proteins, increases the channel activity by activating endogenous G proteins of COS-1 cells. To test this possibility, we examined the action of GTPγS on the channel activity in the presence of several ATP concentrations (1, 10, and 100 μm). In two to three experiments performed at each ATP concentration, we found that GTPγS added to the bath solution at a concentration of 100 μm reversibly decreased the channel activity by 30–60 %. This indicates that the stimulatory actions of G proteins on reconstituted KATP channels cannot be attributed to GTPγS. In addition, we also tested heat-inactivated Gα-i1 to examine the specificity of the G protein effects further. In two experiments, we observed a non-significant decline in the open probability of SUR1-Kir6.2 channels of 4 %. The fact that GTPγS does not increase the single channel activity of reconstituted KATP channels as it does in pancreatic β-cells, suggests that whatever G proteins may be present in COS-1 cells, they lack the regulatory capabilities that the G proteins of β-cells have on KATP channels.

SUR1-Kir6.2 channels are sensitive to modulation by the α subunit of G proteins as shown in Figs 1-5. On the other hand, βγ subunits do not have any influence on these channels. Figure 6A shows a continuous recording lasting several minutes of SUR1-Kir6.2 channels, in the presence of 100 μm ATP. At the time indicated (arrow), Gβγ-i2 (1 nM) was added to the bath solution. No apparent changes were observed even after five minutes of recording. The fact that these channels were responsive to ATP is illustrated in Fig. 6B-E. Panel 6B shows records taken at the beginning of the experiment in the presence of 1 μm ATP. In panel C, the patch was exposed to an ATP concentration of 100 μm and a significant reduction in single channel activity was produced. In panel D the channels were exposed to Gβγ-i2 without major effects. In Figure 6E the patch was washed with a Gβγ-i2-free solution containing 1 μm ATP and the single channel activity returned to similar levels to those shown in panel 6B. Table 1 summarizes the action of several subunits on SUR1-Kir6.2 channels. Neither Gβγ-i1 or Gβγ-i2 had any effect on the open probability.

Figure 6. The effect of Gβγ-i2 on KATP channels (SUR1-Kir6.2).

Figure 6

Open in a new tab

Panel A shows a continuous recording of single channel activity in the presence of 100 μm ATP. The arrow shows the time of application of Gβγ-i2 (1 nM). Panel B shows records in the presence of 1 μm ATP taken before the records shown in panel A. Panels C and D show records of single channel activity in the presence of 100 μm ATP taken from panel A. Panel C shows records obtained before, and panel D after, the application of Gβγ-i2. The records were taken at the times indicated by the bars in panel A and are plotted in an expanded time scale. Panel E shows the increase in single channel activity after returning to 1 μm ATP. Note that the time scale in A differs from that in B-E. Same experiment throughout.

The actions of α subunits are not mediated by protein kinase C

Ribalet & Eddlestone (1995) suggested that inhibitors of protein kinase C reduce the activity of KATP channels of mammalian cell lines. Furthermore, they proposed that protein kinase C inhibitors prevent the stimulatory action of G proteins on channel activity. In our experiments, SUR1-Kir6.2 channels are insensitive to protein kinase C inhibitors. Figure 7A shows single channel activity of SUR1-Kir6.2 channels in the control solution. At least, three channels were active in the patch. In panel B, the protein kinase C inhibitor, H7 (100 μm) was added to the bath solution and no apparent changes occurred. Figure 7C-D illustrates the influence of H7 on the stimulatory action of Gα-i1 in a separate experiment. Panel 7C shows single channel activity in the presence of 10 μm ATP, where only one opening was apparent. In panel D, Gα-i1 (1 nM) was added in combination with H7 (100 μm). The relative open probability increased to 2.17 and up to three channels opened simultaneously. In three experiments, the relative open probability was 2.34 ± 0.10 when H7 and Gα-i1 were added simultaneously, suggesting that this protein kinase C inhibitor does not affect the stimulatory action of Gα-i1. Similarly, in two experiments performed in the presence of the inhibitory peptide, the mean increase in the relative open probability was 2.70.

Figure 7. The effect of H7 on SUR1-Kir6.2 channels.

Figure 7

Open in a new tab

Two separate experiments. Panel A shows the single channel currents in 1 μm ATP and panel B after the addition of 100 μm H7. Panel C shows, from a separate experiment, a continuous recording in the presence of 10 μm ATP and panel D after the simultaneous addition of 100 μm H7 plus 1 nM Gα-i1.

The actions of G proteins on SUR2A-Kir6.2 channels

Co-expression of SUR2A with Kir6.2 results in functional KATP channels that are about 10 times less sensitive to ATP than SUR1-Kir6.2 channels (Inagaki et al. 1996). The possibility that G proteins modulate the activity of SUR2A-Kir6.2 channels was investigated. We found that both Gα-i1 and Gα-i2 have a stimulatory effect on single channel activity. Figure 8A shows a continuous recording of SUR2A-Kir6.2 channels exposed to 100 μm ATP. The single channel activity was quite high even in the presence of a high ATP concentration confirming the low ATP sensitivity of these channels. When Gα-i1 was added to the perfusate (arrow) there was a significant increase in the channel activity that lasted as long as Gα-i1 was present in the bath solution. When the test solution was washed out (arrow), channel activity recovered to control levels. Figure 8B-D shows fragments of the record shown in panel A in an expanded time scale. The dashed lines mark the levels of channel activity observed during the application of Gα-i1 (panel C). At least seven channels were active in the patch. Panels B and D show that multiple openings were less frequent in the control solution and seven simultaneous openings were never observed. On average, Gα-i1 increases the open probability of SUR2A-Kir6.2 channels by 30 % (Table 1).

Figure 8. The stimulatory effect of Gα-i1 on KATP channels (SUR2A-Kir6.2).

Figure 8

Open in a new tab

Panel A shows a continuous recording of single channel activity. Arrows bracket time of application of 1 nM Gα-i1. Panels B-D show records taken from panel A at the times indicated by the bars. Note that the time scale in A differs from that in B-D. B and D were taken in control solution and C in the presence of Gα-i1. The horizontal dashed lines indicate different levels of single channel activity. Note the larger number of simultaneous openings in the presence of Gα-i1. 100 μm ATP was present throughout.

In contrast to the inhibitory actions of Gα-i2 on SUR1-Kir6.2 channels, this subunit increases the open probability of SUR2A-Kir6.2 channels. Figure 9A shows a continuous recording of SUR2A-Kir6.2 channels in the presence of 100 μm ATP. In panel B, Gα-i2 (1 nM) was applied. The dashed lines indicate the levels of channel activity recorded in the presence of Gα-i2. At least four channels were active in this patch and the open probability was relatively high since the current level corresponded, for the most part, to that of two or three openings. On the other hand, in the control record illustrated in panel A, only one channel was active most of the time. Panel 9C shows the single channel activity expressed as NPo as a function of time. Open symbols represent the channel activity in the control solution and filled symbols in the presence of Gα-i2. The horizontal line indicates the mean NPo value when Gα-i2 was present in the bath solution. It clearly stands above the mean channel activity level in the control solution indicating the stimulatory effect of Gα-i2 on SUR2A-Kir6.2 channels. As summarized in Table 1, Gα-i2 increased the open probability by 34 %.

Figure 9. The stimulatory effect of Gα-i2 on KATP channels (SUR2A-Kir6.2).

Figure 9

Open in a new tab

Panel A shows the control records and panel B in the presence of 1 nM Gα-i2. The horizontal dashed lines indicate different levels of single channel activity. Panel C shows the single channel activity over time. ○, activity in control solution; •, in the presence of 1 nM Gα-i2. The horizontal line indicates the mean activity in the presence of Gα-i2. 100 μm ATP was present throughout.

DISCUSSION

ATP-sensitive K+ channels and G proteins

Our major finding is that the activity of reconstituted KATP channels is modulated by the application of exogenous G proteins to the intracellular surface of membrane patches. This modulation is specific to the α subunits and it is not shared by the βγ subunits. Our results can be interpreted by a direct action of the α subunits on the KATP channels in a membrane-delimited way without the intervention of diffusible second messengers. Our experiments indicate that co-expression of sulphonylurea receptors (SUR1 or SUR2A) in combination with Kir6.2 results in functional KATP channels that are prone to modulatory influences by G proteins.

Previous studies have shown that the activity of KATP channels of cardiac cells and insulin-secreting cell lines is enhanced by activation of G proteins. Findlay (1987) and Kirsch, Codina, Birnbaumer & Brown (1990) showed that the addition of the non-hydrolyzable GTP analogue guanosine 5′-O-(3-thiotriphosphate) (GTPγS), enhances KATP channel activity. Ito et al. (1994) and Terzic et al. (1994) confirmed the stimulatory action of GTPγS on KATP channels in heart muscle and found similar effects in the presence of AlF4−. It is well known that millimolar concentrations of fluoride activate heterotrimeric G proteins. The action of fluoride depends on the formation of multifluorinated complexes with traces of aluminum and requires the presence of magnesium (Sternweiss & Gilman, 1982). Although these observations clearly indicate that G proteins play a role in channel modulation, they do not provide direct evidence of which G protein and subunits are actually involved. This problem was approached by Kirsch, Codina, Birnbaumer & Brown (1990) in heart muscle and by Ribalet & Eddlestone (1995) in insulin-secreting cells. Kirsch et al. (1990) found that the effects of GTPγS are mimicked by the subunits of Gi: Gα-i1, Gα-i2 and Gα-i3. In agreement with these observations, we found that Gα-i1 and Gα-i2 enhance the channel activity of SUR2A-Kir6.2 channels. SUR2A is a recently cloned isoform of the sulphonylurea receptor SUR1 (Inagaki et al. 1996). When SUR2A is co-expressed with Kir6.2, ATP-sensitive channels, whose properties greatly resemble those of KATP channels found in heart muscle, are reconstituted (Inagaki et al. 1996). On the other hand, co-expression of SUR1 with Kir6.2 results in KATP channels that are very similar to those found in pancreatic islets and other insulin-secreting cells (Inagaki et al. 1995). Ribalet & Eddlestone (1995) described findings that Gα-i3 stimulates the KATP channel of HIT and RIN insulin-secreting cell lines but did not test the effects of Gα-i1 or Gα-i2. We found that Gα-i1 stimulates SUR1-Kir6.2 channels but we observed that Gα-i2 decreases their open probability. At this point, it is unclear whether Gα-i2 causes inhibition of SUR1-Kir6.2 channels directly or whether this action is related to the inhibitory effect of GTPγS on KATP channels that we observed in the present experiments. On the other hand, since we found that Gα-i2 significantly increases the activity of SUR2A-Kir6.2 channels, these results clearly indicate that the sensitivity of SUR1-Kir6.2 channels to Gα-i2 is different from that of SUR2A-Kir6.2 channels.

The role of protein kinase C in G protein actions

Somatostatin is a peptide hormone with hyperglycaemic properties that increases the activity of KATP channels (Fosset, Schmid-Antomarchi, De Weille & Lazdunski, 1984; Ribalet, Eddlestone & Ciani, 1988a; De Weille et al. 1989). The involvement of protein kinase C in the somatostatin response has been proposed because the kinase is an important route that stimulates opening of KATP channels. It has further been shown that the sulphonylurea glibenclamide suppresses the somatostatin response (De Weille et al. 1989) and that G proteins play a role in the stimulation of KATP channels by somatostatin (Ribalet & Eddlestone, 1995). It is conceivable that G proteins stimulate KATP channels directly. However, similar to other examples of membrane-delimited modulation of ion channels, the possibility that G proteins interact with KATP channels indirectly, via a non-diffusible membrane component, has not been ruled out. In fact, Ribalet & Eddlestone (1995) proposed that protein kinase C mediates the G protein actions on KATP channels. According to their view, Gα-i enhances the activity of phospholipase C. This in turn would stimulate protein kinase C, leading to phosphorylation of KATP channels. An alternative possibility also proposed by Ribalet & Eddlestone (1995) assumes that Gα-i interacts with KATP channels directly and that this interaction is facilitated by protein kinase C. Our experiments do not support the former scheme since we observed that Gα-i still promotes channel activity in the presence of protein kinase C inhibitors. Our reconstitution experiments favour the view that either Gα-i directly interacts with KATP channels, or that the signal that links the interaction between Gα-i and KATP channels is a membrane-delimited component different from protein kinase C, that is present in the surface membrane of COS-1 cells.

Acknowledgments

This work was supported by Scientific Research Grants and a Grant-in-Aid for Scientific Research on Priority Areas of ‘Channel-Transporter Correlation’ from the Ministry of Education, Science and Culture, Japan, by grants from the Yamanouchi Foundation for Research on Metabolic Disorders, the Uehara Memorial Foundation and the Naito Foundation, and by a grant for studies on the pathophysiology and complications of diabetes from Tsumura Pharma Ltd. J. S. and T. G. were supported by Scientific Research Grant (Centre of Excellence) from the Ministry of Education, Science and Culture, Japan.

References

  1. Ämmälä C, Moorhouse A, Ashcroft FM. The sulphonylurea receptor confers diazoxide sensitivity on the inwardly-rectifying K-channel Kir6.1. The Journal of Physiology. 1996;494:709–714. doi: 10.1113/jphysiol.1996.sp021526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brown AM. Membrane-delimited cell signaling complexes: direct ion channel regulation by G proteins. Journal of Membrane Biology. 1993;131:93–104. doi: 10.1007/BF02791318. [DOI] [PubMed] [Google Scholar]
  3. Chutkow WA, Simon C, Le Beau MM, Burant CF. Cloning, tissue expression, and chromosomal localization of SUR2, the putative drug-binding subunit of cardiac, skeletal muscle, and vascular KATP channels. Diabetes. 1996;45:1439–1445. doi: 10.2337/diab.45.10.1439. [DOI] [PubMed] [Google Scholar]
  4. Cook DL, Hales CH. Intracellular ATP directly blocks K+ channels in pancreatic β cells. Nature. 1984;311:271–273. doi: 10.1038/311271a0. [DOI] [PubMed] [Google Scholar]
  5. De Weille J R, Schmid-Antomarchi H, Fosset M, Lazdunski M. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP. Proceedings of the National Academy of Sciences of the USA. 1989;86:2971–2975. doi: 10.1073/pnas.86.8.2971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods in Enzymology. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  7. Fenwick EM, Marty A, Neher E. Sodium and calcium channels in bovine chromaffin cells. The Journal of Physiology. 1982;331:599–635. doi: 10.1113/jphysiol.1982.sp014394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Findlay I. The effects of magnesium upon adenosine triphosphate-sensitive potassium channels in a rat insulin-secreting cell line. The Journal of Physiology. 1987;391:611–629. doi: 10.1113/jphysiol.1987.sp016759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fosset M, Schmid Antomarchi H, De Weille JR, Lazdunski M. Somatostatin activates glibenclamide-sensitive and ATP-regulated K+ channels in insulinoma cells via a G-protein. FEBS Letters. 1984;242:94–96. doi: 10.1016/0014-5793(88)80992-x. [DOI] [PubMed] [Google Scholar]
  10. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv. 1981;391:85–100. doi: 10.1007/BF00656997. [DOI] [PubMed] [Google Scholar]
  11. Hille B. G protein-coupled mechanisms and nervous signaling. Neuron. 1992;9:187–195. doi: 10.1016/0896-6273(92)90158-a. [DOI] [PubMed] [Google Scholar]
  12. Inagaki N, Gonoi T, Clement JP, IV, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J. Reconstitution of IKATP: An inward rectifier subunit plus the sulphonylurea receptor. Science. 1995;270:1166–1170. doi: 10.1126/science.270.5239.1166. [DOI] [PubMed] [Google Scholar]
  13. Inagaki N, Gonoi T, Clement JP, IV, Wang C Z, Aguilar-Bryan L, Bryan J, Seino S. A family of sulphonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron. 1996;16:1011–1017. doi: 10.1016/s0896-6273(00)80124-5. [DOI] [PubMed] [Google Scholar]
  14. Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, Matsuzawa Y, Kurachi Y. A novel sulfonylurea receptor forms with BIR(Kir6.2) a smooth mucle type ATP-sensitive K+ channel. Journal of Biological Chemistry. 1996;271:24321–24324. doi: 10.1074/jbc.271.40.24321. [DOI] [PubMed] [Google Scholar]
  15. Ito H, Vereecke J, Carmeliet E. Mode of regulation of G protein of the ATP sensitive K+ channel in guinea-pig ventricular cell membrane. The Journal of Physiology. 1994;478:101–107. doi: 10.1113/jphysiol.1994.sp020233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kirsch GE, Codina J, Birnbaumer L, Brown AM. Coupling of ATP sensitive K+-channels to A1 receptors by G proteins in rat ventricular myocytes. American Journal of Physiology. 1990;259:H820–826. doi: 10.1152/ajpheart.1990.259.3.H820. [DOI] [PubMed] [Google Scholar]
  17. Kobayashi I, Shibasaki H, Takahashi K, Tohyama K, Kurachi Y, Ito H, Ui M, Katada T. Purification and characterization of five different α subunits of guanine-nucleotide-binding proteins in bovine brain membranes: Their physiological properties concerning the activities of adenylate cyclase and atrial muscarinic K+ channels. European Journal of Biochemistry. 1990;191:499–506. doi: 10.1111/j.1432-1033.1990.tb19149.x. [DOI] [PubMed] [Google Scholar]
  18. Kontani K, Takahashi K, Inanobe A, Ui M, Katada T. Molecular heterogeneity of the βγ-subunits of GTP-binding proteins in bovine brain membranes. Archives of Biochemistry and Biophysics. 1992;294:527–533. doi: 10.1016/0003-9861(92)90720-h. [DOI] [PubMed] [Google Scholar]
  19. Marshall J, Molloy R, Moss GWJ, Howe JR, Hughes TE. The jellyfish green fluorescent protein: a new tool for studying ion channel expression and function. Neuron. 1995;14:211–215. doi: 10.1016/0896-6273(95)90279-1. [DOI] [PubMed] [Google Scholar]
  20. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983;305:147–148. doi: 10.1038/305147a0. [DOI] [PubMed] [Google Scholar]
  21. Ribalet B, Eddelstone GT. Characterization of the G protein coupling of a somatostatin receptor to the KATP channel in insulin-secreting mammalian HIT and RIN cell lines. Journal of Physiology. 1995;485:73–86. doi: 10.1113/jphysiol.1995.sp020713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ribalet B, Eddelstone GT, Ciani S. Metabolic regulation of the K(ATP) and a Maxi-K(V) channel in the insulin secreting RINm5F cell. Journal of General Physiolgy. 1988;92:219–237. doi: 10.1085/jgp.92.2.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ribalet B, Eddelstone GT, Ciani S. Role of G proteins in K(ATP) channel modulation by the neuropeptide somatostatin. Biomedical Research. 1988;12:37–40. [Google Scholar]
  24. Sakura H, Ämmälä C, Smith PA, Gribble FM, Ashcroft FM. Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel expressed in pancreatic β-cells, brain, heart, and skeletal muscle. FEBS Letters. 1995;377:338–344. doi: 10.1016/0014-5793(95)01369-5. [DOI] [PubMed] [Google Scholar]
  25. Spruce AE, Standen NB, Stanfield PR. Studies of the unitary properties of adenosine-5′-triphosphate-regulated potassium channels of frog skeletal muscle. The Journal of Physiology. 1987;382:213–236. doi: 10.1113/jphysiol.1987.sp016364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sternweiss PC, Gilman AG. Aluminum: A requirement for activation of the regulatory component of adenylate cyclase by fluoride. Proceedings of the National Academy of Sciences of the USA. 1982;79:4888–4891. doi: 10.1073/pnas.79.16.4888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Terzic A, Tung RT, Inanobe A, Katada T, Kurachi Y. G proteins activate ATP-sensitive K+ channels by antagonizing ATP-dependent gating. Neuron. 1994;12:885–893. doi: 10.1016/0896-6273(94)90340-9. [DOI] [PubMed] [Google Scholar]
  28. Tukker SJ, Gribble FM, Zhao C, Trapp S, Ashcroft FM. Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature. 1997;387:179–183. doi: 10.1038/387179a0. [DOI] [PubMed] [Google Scholar]
  29. Yamada M, Ho YK, Lee RH, Kontani K, Takahashi K, Katada T, Kurachi Y. Muscarinic K+ channels are activated by βγ subunits and inhibited by the GDP-bound form of α subunit of transducin. Biochemical and Biophysical Research Communications. 1994;200:1484–1490. doi: 10.1006/bbrc.1994.1618. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES