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Originally published In Press as doi:10.1074/jbc.M707695200 on December 27, 2007

J. Biol. Chem., Vol. 283, Issue 9, 5306-5316, February 29, 2008
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Both Gi and Go Heterotrimeric G Proteins Are Required to Exert the Full Effect of Norepinephrine on the β-Cell KATP Channel*

Ying Zhao{ddagger}, Qinghua Fang§, Susanne G. Straub{ddagger}, and Geoffrey W. G. Sharp{ddagger}1

From the {ddagger}Department of Molecular Medicine, College of Veterinary Medicine and §Department of Applied Engineering and Physics, Cornell University, Ithaca, New York 14853-6401

Received for publication, September 13, 2007 , and in revised form, December 7, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of norepinephrine (NE), an inhibitor of insulin secretion, were examined on membrane potential and the ATP-sensitive K+ channel (KATP) in INS 832/13 cells. Membrane potential was monitored under the whole cell current clamp mode. NE hyperpolarized the cell membrane, an effect that was abolished by tolbutamide. The effect of NE on KATP channels was investigated in parallel using outside-out single channel recording. This revealed that NE enhanced the open activities of the KATP channels ~2-fold without changing the single channel conductance, demonstrating that NE-induced hyperpolarization was mediated by activation of the KATP channels. The NE effect was abolished in cells preincubated with pertussis toxin, indicating coupling to heterotrimeric Gi/Go proteins. To identify the G proteins involved, antisera raised against {alpha} and β subunits (anti-G{alpha}common, anti-Gβ, anti-G{alpha}i1/2/3, and anti-G{alpha}o) were used. Anti-G{alpha}common totally blocked the effects of NE on membrane potential and KATP channels. Individually, anti-G{alpha}i1/2/3 and anti-G{alpha}o only partially inhibited the action of NE on KATP channels. However, the combination of both completely eliminated the action. Antibodies against Gβ had no effects. To confirm these results and to further identify the G protein subunits involved, the blocking effects of peptides containing the sequence of 11 amino acids at the C termini of the {alpha} subunits were used. The data obtained were similar to those derived from the antibody work with the additional information that G{alpha}i3 and G{alpha}o1 were not involved. In conclusion, both Gi and Go proteins are required for the full effect of norepinephrine to activate the KATP channel.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Insulin secretion is inhibited by {alpha}2-adrenergic receptor activation (1, 2). The {alpha}2-adrenergic receptors at the plasma membrane are linked to pertussis toxin (PTX)2-sensitive Gi and Go proteins. Upon interacting with the {alpha}2-adrenergic receptors, catecholamines (i.e. epinephrine and norepinephrine (NE)), two important physiological inhibitors of insulin secretion, activate Gi and/or Go proteins and inhibit the exocytosis of insulin-containing granules (2). Four mechanisms have been suggested to account for their inhibitory effect: 1) activation of ATP-sensitive K+ channels (KATP) and repolarization of the β-cells; 2) inhibition of L-type Ca2+ channels; 3) decreased activity of adenylyl cyclase; and 4) inhibition of exocytosis at a "distal" site in stimulus-secretion coupling, which is beyond the elevation of intracellular Ca2+ and beyond the potentiating actions of cAMP and diacyglycerol (2). Although it is generally accepted that the "distal" inhibitory effect is the most dominant, the relative contribution of each of these four mechanisms to the overall inhibition of insulin release is not clear.

Considering the effects of catecholamines on the membrane potential in the insulin-secreting cells, it is clear that catecholamines can repolarize the cell membrane in a PTX-sensitive manner. However, the explanations for this phenomenon are mixed. Abel et al. (3) reported that adrenaline-induced hyperpolarization was due to activation of KATP channels but that the effect was insufficient to cause the sustained inhibition of insulin secretion in INS-1 cells associated with it (i.e. the distal effect is dominant). Schermerhorn and Sharp (4) also found that NE acted on the KATP channel and produced different effects on [Ca2+]i in oscillating and nonoscillating HIT-T15 cells. However, Rorsman et al. (5) proposed that epinephrine suppressed mouse β-cell electrical activity by a G protein-dependent mechanism, which culminated in the activation of a sulfonylurea-insensitive low conductance K+ channel distinct from the KATP channel. Further evidence for the existence of this G protein-regulated low conductance K+ channel comes from studies with SUR1 knock-out mice that do not have functional KATP channels. Islet cells from these mice are hyperpolarized by epinephrine in a PTX-sensitive manner, and the hyperpolarization is due to activation of a low conductance K+ channel (6). The expression and catecholamine control of the KATP channel and the low conductance channel in β-cells of different origins and conditions (e.g. knockouts) remain to be defined. However, although it is established that the action of catecholamines is coupled to the activation of Gi and Go proteins, information about the exact identities of these G proteins and the corresponding subunits is not available. Therefore, in this study, we investigated the effects of NE on the membrane potential and on the KATP channels in INS 832/13 cells, and we identified the G proteins involved. For this, we used a combination of whole cell configuration and outside-out single channel recording to monitor the membrane potential and single KATP currents in parallel. In order to clarify which G proteins and subunits are responsible for the actions of NE, we used antisera raised against various G protein {alpha} and β subunits, anti-G{alpha}common, anti-Gβ, anti-G{alpha}i1/2/3, and anti-G{alpha}o. We also used small peptides specific for the C termini of the {alpha} subunits that specifically block G protein interactions with receptors. The antisera or peptides were diffused into the cells separately and in combination, and their effects on the INS 832/13 cells were determined. NE hyperpolarized the cell membrane via enhancing the open activities of the KATP channels in an ATP-dependent manner. Although the action of NE was mediated by G{alpha}i and G{alpha}o subunits, the effects of the two types of subunits were not redundant. Applying one of their corresponding antibodies or peptides only partially inhibited the action of NE. The effect of NE was fully blocked only by the combination of antibodies against G{alpha}i1/2/3 and G{alpha}o and by the blocking peptides for G{alpha}i1/2 and G{alpha}o2 but not the peptides for Gi3 and G{alpha}o1. The data show that activation of KATP channels by NE contributes to the NE-induced inhibition of insulin release in the INS 832/13 cells and that the action requires both G{alpha}i1/2 and G{alpha}o2 proteins.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—INS 832/13 cells (a kind gift by Dr. C. B. Newgard) were cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 µg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 95% air, 5% CO2 atmosphere. The cells (passage numbers 60–66) were divided once a week by treatment with trypsin, and the medium was changed twice between divisions. The measurements were performed 1–2 days after cell division.

Electrophysiology and Solutions—Both whole cell current clamp mode and outside-out single channel recording were applied. Whole cell recordings were performed using fire-polished electrodes, pulled from borosilicate glass and showing an open resistance of 2–5 megaohms. Signals were amplified and acquired by a PULSE 8.75-controlled EPC-10 amplifier (HEKA Electronics). Data were filtered through a four-pole low pass Bessel filter at 2 kHz, and the sampling rate was 10 kHz. For single channel recording, data were further digitally filtered at 500 Hz. Capacitative transients and series resistance were compensated, using the circuitry incorporated in the amplifier. The cells were continuously superfused with standard extracellular solution (solution A) containing 140 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 2.6 mM CaCl2, and 10 mM HEPES-NaOH (pH 7.4) and glucose with adjusted concentrations. For the perforated whole cell configuration, the pipette solution (solution B) was composed of 10 mM KCl, 76 mM K2SO4, 10 mM NaCl, 1 mM MgCl2, 10 mM HEPES-KOH (pH 7.3) and amphotericin B (200 µg/ml). For the standard whole cell configuration, the patch pipette was filled with solution C containing 145 mM potassium glutamate, 8 mM NaCl, 1.0 mM MgCl2, 2 mM ATP-Mg, 0.5 mM GTP, 0.3 mM cAMP, and 10 mM HEPES-KOH (pH 7.3). The membrane potential was measured under perforated or standard whole cell configurations, and during the recording periods the cells were bathed in solution A supplemented with 16.7 mM glucose. The outside-out patch was established after the formation of standard whole cell configuration and was continuously bathed in solution A supplemented with 5 mM tetraethylammonium and 2.8 mM glucose. The pipette solution specific for the single channel recording (solution D) was composed of 140 mM KCl, 8 mM NaCl, 1.0 mM MgCl2, 0.1 mM ATP-Mg, 0.5 mM GTP, 0.3 mM cAMP, 5 mM EGTA, and 10 mM HEPES-KOH (pH 7.3). NE and tolbutamide were freshly prepared in stock solutions before experiments and were added into solution A at final concentrations of 5 and 300 µM, respectively. All experiments were performed at room temperature (20–23 °C). The antibodies used in this study were all purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies were added to the pipette solution with adjusted concentrations and were applied intracellularly by dialysis. In order to reach equilibrium of the antibodies between the pipette tip and the cytosol, monitoring the membrane potential as well as establishing the outside-out membrane patch were performed 2–5 min after the cell membrane was ruptured. Experiments using the peptides analogous to the C termini of G{alpha}i1/2, G{alpha}i3, G{alpha}o1, G{alpha}o2, and a randomized peptide were performed exactly as those using the antibodies.

The analysis program of single channel recording was written in our laboratory based on the software of Igor Pro version 5.03 (WaveMetrics, Inc.). The maximum number of functional channels (N) in the patch was estimated by observing the number of peaks detected on the histogram distribution of amplitude. As an index of channel activity, NPo (the maximum number of channels in the patch times the open probability) was calculated as follows,

Formula(Eq. 1)
where T represents the total recording time, n is the number of channels open, and tn is the recording time during which n channels are open. Therefore, NPo can be calculated without making assumptions about the total number of channels in a patch or the open probability of a single channel. In order to control the variations in NPo among patches, the NPo values were generally expressed in normalized form relative to the NPo under pretest conditions. The NPo was evaluated at 0 mV.

Statistics—Data are presented as means ± S.E. Significance tests were performed using Student's test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of NE on the Membrane Potential of INS 832/13 Cells—The perforated whole cell configuration was applied to investigate the effect of NE on the membrane potential in intact INS 832/13 cells. The cells were continuously superfused with solution A containing 16.7 mM glucose, and the voltages were monitored by using pipette solution B. In Fig. 1A, the membrane potential was depolarized by high glucose to ~–40 mV with continuous bursts of action potentials to ~20 mV, which were triggered by rapid Ca2+ influx. When challenged with NE (5 µM) in contact with the extracellular membrane surface, the cell was markedly hyperpolarized to ~–60 mV and was redepolarized to ~–40 mV upon the removal of NE, showing that the effect of NE had a rapid onset and was rapidly reversible. The apparent delay in onset of the action of NE (Fig. 1A) and tolbutamide (Fig. 1B) was caused by the perifusion system and a lag period before NE and tolbutamide reach the cells. The sensitivity of NE-induced hyperpolarization to the classic KATP channel inhibitor, tolbutamide, was tested. Tolbutamide (300 µM) reversed the NE-induced fall in membrane potential (Fig. 1B). Similarly, when tolbutamide was added first, NE had no effect on membrane potential (not shown). NE was not capable of hyperpolarizing the cells that were precultured with PTX (150 ng/ml) for 24 h (Fig. 1C), indicating the involvement of Gi and/or Go proteins in the regulation of the membrane potential by NE. Data presented in Fig. 1, A–C, respectively, are typical of three sets of experiments. As summarized in Fig. 1D, on average, 5 µM NE hyperpolarized the membrane potential from –36 ± 1.5 mV to –55 ± 2.1 mV (n = 14, p < 0.01), and tolbutamide counteracted this hyperpolarization by restoring the membrane potential from –57 ± 3.2 to –31 ± 1.8 mV (n = 10, p < 0.01). In the cells pretreated with PTX, the membrane potentials before and after the challenge with NE were –43 ± 4.0 and –45 ± 3.7 mV (n = 5, p > 0.05). Similarly, PTX treatment blocked the effect of NE to decrease the frequency of Ca2+-triggered action potentials. Analysis of the data in Fig. 1 showed that the frequency of the action potentials was 20.6 ± 5.5/min prior to NE treatment, 17.7 ± 4.2/min during NE treatment (p > 0.05, n = 5) and 16.8 ± 4.3/min after the washout of NE. These data indicate that NE hyperpolarized the cell membrane via its effect on the KATP channels. To this point, the observations were obtained in the relatively intact cell and could not elucidate whether the change of membrane potential was made by the direct interaction between the KATP channels and the Gi/o proteins or by indirect interaction with other cytosolic messengers. To address this, the same experimental procedures were repeated, except that the standard whole cell configuration was employed and the patch pipette was filled with solution C containing 2 mM ATP, 0.5 mM GTP, and 0.3 mM cAMP. As shown in Fig. 2A, the cell membrane was again reversibly hyperpolarized by 5 µM NE. When ATP was omitted from the pipette solution, the cell was hyperpolarized, and, as expected, NE was not capable of inducing any further fall of membrane potential (Fig. 2B). The effects of NE on the membrane potential recorded under the standard whole cell configuration in the presence and absence of ATPi are summarized in Fig. 2C. In the presence of 2 mM ATP in the pipette solution C, the membrane potentials were –41 ± 3.5 mV (control), –58 ± 3.1 mV (with NE), and –42 ± 4.7 mV (wash out). These values are similar to those obtained with the perforated whole cell configuration. The effect of NE on membrane potential was significantly different from both the pre-NE and post-NE values (n = 5, p < 0.01). When ATPi was omitted, the membrane potentials were not significantly changed by NE (n = 5, p > 0.05), remaining at ~–60 mV before, during, and after NE treatment.


Figure 1
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  		FIGURE 1.
Effect of NE on the membrane potential under control and PTX-treated conditions. The representative traces shown in A–C were obtained using the perforated whole cell current clamp configuration. In A, NE (5 µM) reversibly hyperpolarized the membrane. In B, the NE-induced hyperpolarization was blocked by 300 µM tolbutamide, indicating that the effect of NE was on the KATP channel. In C, NE failed to induce hyperpolarization in PTX-pretreated cells. D summarizes the effects of NE on membrane potential under the different experimental conditions imposed. The lines indicate the perfusion periods of NE and tolbutamide. **, p < 0.01; #, p > 0.05 relative to control values.

 
Effects of NE on ATP-sensitive K+ Channels under Single Channel Recording Conditions—The effects of NE on single KATP channels were examined in detail. All single channel recordings presented in this paper were derived exclusively from currents passing through KATP channels in outside-out patches and with the membrane potential clamped at 0 mV. The Ca2+-activated nonselective cation channels and the Ca2+-activated K+ channels were eliminated by keeping the [Ca2+]i low, <10–9 M, and by applying 5 mM tetraethylammonium extracellularly (7). Because the number of operational KATP channels per membrane patch is often unknown, NPo was introduced to estimate the channel activity (see "Experimental Procedures").


Figure 2
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  		FIGURE 2.
Effect of NE on the membrane potential in the absence and presence of ATP. The representative traces in A and B were obtained under the standard whole cell current clamp configuration. In Fig. 2A, the cell membrane was reversibly hyperpolarized by NE (5 µM) from ~–40 to ~–60 mV with 2 mM Mg-ATP in the pipette solution. In Fig. 2B, NE lacked the ability to further hyperpolarize the cell when ATP was omitted from the pipette solution. C, summary of the effects of NE on the membrane potential in the presence and absence of ATP in the pipette solution. The lines indicate the perfusion periods of NE. *, p < 0.05; #, p > 0.05, relative to control values.

 
Fig. 3A shows the effect of NE (5 µM) on single KATP channels in an outside-out membrane patch. The pipette solution D was in direct contact with the cytosolic side of the membrane patch, and the outside of the patch was continuously superfused with solution A. The single channel currents were monitored before, during, and after NE treatment. In Fig. 3A, NE-induced enhancement of the open activity of KATP channels was clearly visualized, and this effect was completely reversible, since the channel activity decreased to the control level upon removal of NE (Fig. 3E). The values of NPo determined under test conditions were normalized to the control value, which was displayed as 100%. As summarized in Fig. 3E, on average, 5 µM NE increased the value of NPo from 100% in the control situation to 209 ± 17% and returned back to 106 ± 11% of the pretest value upon removal of NE (0.1 mM ATPi was present, n = 12, p < 0.01). When ATP was excluded from the pipette solution, the channels exhibited robust open activity, and the channel activity could not be further increased by the application of NE (Fig. 3B), which is similar to the situation shown in Fig. 2B. The results shown in Fig. 3, A and B, are typical of two sets of experiments under different conditions of ATPi. On average, when ATPi was omitted, the mean value of NPo was 103 ± 3.4% in the presence of NE (n = 7, p > 0.05). When tolbutamide was present, the subsequent addition of NE had no effect on K+ channel activity (not shown).

These data are consistent with the observations shown in Fig. 2 that the NE-induced modification of electrical properties in INS 832/13 cells cannot be detected in the absence of ATPi. The current frequency distribution for the channels studied under control and NE-treated conditions in the presence of 0.1 mM ATP is shown in Fig. 3C and in the absence of ATP in the pipette in Fig. 3D. There is no evidence for the presence of more than one channel in the cell under either of these conditions. In order to investigate whether NE also influences the conductance of single KATP channels, single KATP currents were recorded in the presence and absence of 5 µM NE over the range of –40 to +60 mV. The mean values of single current amplitudes were plotted versus the corresponding clamped voltages (Fig. 3F). The current-voltage relationship was nearly linear at negative potentials but showed a slight inward rectification at membrane potentials higher than 10 mV, as previously described in RINm5F cells by Findlay (8). The two I-V curves obtained under control and test conditions were overlapping. The slope conductance fitted between –40 and 20 mV was ~20 picosiemens in control and 19.2 picosiemens in the presence of NE, indicating that application of NE did not induce any significant modification of single channel current amplitude. The single channel conductance of 20 picosiemens is similar to that of other reports for the KATP channel in clonal cells (8–10).

Effects of Antibodies against Common G{alpha} Subunits on the NE-induced Electrical Changes—To identify the G protein subunits involved in NE-mediated hyperpolarization and activation of the KATP channels, antibodies raised against various G protein subunits were tested. Anti-G{alpha}common (2 µg/ml) was added to the pipette solutions C and D, for the voltage measurement and outside-out single channel recordings, respectively. Once the standard whole cell configuration was established, 2–5 min was allowed for the antibodies to reach equilibrium between the pipette tip and the cytosol. The results in Fig. 4A clearly show that upon application of anti-G{alpha}common, the membrane voltage remained at ~–40 mV during the whole perfusion process, without exhibiting hyperpolarization in the presence of NE. As summarized in Fig. 4C, anti-G{alpha}common significantly counteracted the effect of NE on the membrane potential. The membrane potential was –42 ± 4.3 mV in control, –41 ± 3.7 mV in the presence of NE, and –42 ± 4.3 mV upon removal of NE (n = 15, p < 0.01). In order to exclude possible nonspecific effects of anti-G{alpha}common, the experiments were controlled by replacing anti-G{alpha}common with IgG and by measuring the response of the membrane potential to NE. As shown in Fig. 4C, NE induced its anticipated effect on membrane potential, and the results were analogous to those shown in Fig. 2C. The effect of anti-G{alpha}common on the NE-induced activation of KATP channels was investigated by adding anti-G{alpha}common (2 µg/ml) to pipette solution D. 5 µM NE failed to induce a visible change of channel activity (Fig. 4B). As summarized in Fig. 4D, with anti-G{alpha}common in the pipette solution D, the normalized NPo was almost unchanged from 100% in control to 103 ± 3.3% in the presence of 5 µM NE and 99 ± 4.8% with the removal of NE (n = 18, p > 0.05). However, the NE effects remained in a series of control experiments using IgG. NPo was increased by NE from 100% to 221 ± 12% and back to the precontrol level of 98 ± 2.7% when NE was washed out (n = 6, p < 0.01).


Figure 3
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  		FIGURE 3.
NE enhances the KATP channel activity without changing the single channel conductance. A, a representative KATP current trace recorded under the outside-out configuration. The cytosolic side of the patch was in direct contact with the pipette solution containing 0.1 mM ATP. The outside of the membrane patch was superfused with the standard extracellular solution and exposed to 5 µM NE during the period indicated. In Fig. 3B, the membrane patch was treated in the same way as in Fig. 3A, except that ATP was omitted from the pipette solution. Shown is the current frequency distribution for the channel under control and NE-treated conditions in the presence of 0.1 mM ATP (C) and in the absence of ATP in the pipette (D). E, summary of NE effects on the open activities of single KATP channels, in terms of NPo values, in the presence and absence of ATP in the pipette solution. *, p < 0.05; #, p > 0.05 relative to control values. F, current-voltage relationship (I-V curves) of single channel KATP currents plotted in the presence or absence of 5 µM NE.

 


Figure 4
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  		FIGURE 4.
Polyclonal antibodies against common {alpha} subunits of G proteins (anti-G{alpha}common) abolished NE-induced electrical modification of the cell membranes. In A and B, anti-G{alpha}common (2 µg/ml) was present in the pipette solutions. A, a representative trace of the membrane potential recorded in the absence and presence of 5 µM NE. B, a representative trace of the single KATP channel recording in an outside-out patch recorded in the absence and presence of 5 µM NE. C and D summarize the effects of anti-G{alpha}common on membrane potential and NPo in the absence and presence of NE. For control experiments, anti-{alpha}common was replaced by IgG. **, p < 0.01; #, p > 0.05, relative to control values.

 
Effects of Antibodies against Specific Subunits of G{alpha}i and G{alpha}o on the NE-induced Electrical Changes—From the results presented above, we predicted that at least one of these two types of subunits, G{alpha}i and G{alpha}o, should be able to mimic, to some extent, the effects of anti-G{alpha}common on the electrical properties in INS 832/13 cells. To test this, monoclonal antibodies raised specifically against G{alpha}i1/2/3 and G{alpha}o were applied. Adding anti-G{alpha}i1/2/3 or anti-G{alpha}o (2 µg/ml for each) to the cytosolic side of the patches did not fully block NE (5 µM) to increase KATP channel activity. As can be seen in the summary in Fig. 5A, in the presence of individual anti-G{alpha}i1/2/3 and anti-G{alpha}o, NE still increased the normalized NPo values from 100% to 156 ± 7.8% and to 148 ± 5.1%, respectively for anti-G{alpha}i1/2/3 (n = 17, p < 0.05) and anti-G{alpha}o (n = 19, p < 0.01), respectively. After removal of NE, both NPo values determined in the presence of G{alpha}i1/2/3 and anti-G{alpha}o individually returned to the precontrol levels.

Because anti-G{alpha}i1/2/3 and anti-G{alpha}o only partially attenuated the effects of NE (by ~44 and ~52%, respectively), the effect of these antisera in combination was examined. In the patches treated with G{alpha}i1/2/3 and anti-G{alpha}o simultaneously, the normalized NPo, in the presence of NE, was 101 ± 6.1% (n = 15, p < 0.01). This represents a complete inhibition of the effects of NE on KATP channel activity by anti-G{alpha}i1/2/3 and anti-G{alpha}o when applied together. In considering why individual anti-G{alpha}1/2/3 and anti-G{alpha}o only partially block the action of NE, several possibilities exist. First, G{alpha}i1/2/3 and G{alpha}o are redundant, and both have the same targeting sites on the KATP channel. In this case, the partial inhibition observed could be due to an insufficient amount of antibodies applied. Second, the effects of G{alpha}i1/2/3 and G{alpha}o on the KATP channels might not be redundant, so that both independently impair the closure of KATP channels by targeting different sites. In this case, each alone would not fully block the effect of NE. Third, the inhibitory effect observed from one of the two types of antibodies, either anti-G{alpha}i1/2/3 or anti-G{alpha}o, could be artifactual, due to cross-reaction between the two antibodies (unlikely, since both antibodies were affinity-purified). To answer the questions of redundancy and cross-reactivity, the experiments were repeated, except that the concentrations of the individual anti-G{alpha}i1/2/3 and anti-G{alpha}o were modified either by increasing 2-fold (4 µg/ml) or by lowering 10-fold (0.2 µg/ml). Comparing the summary data in Fig. 5B with that present in Fig. 5A, increasing the amount of antibody did not change the outcome from that seen with the initial concentration of 2 µg/ml. At higher concentrations of anti-G{alpha}i1/2/3 or anti-G{alpha}o (4 µg/ml for each) the NPo values in the presence of NE were 151 ± 4% (n = 13, p < 0.01) and 152 ± 6% (n = 14, p < 0.01), respectively (similar to the results in Fig. 5A). Full inhibition was only achieved by combining both antibodies (NPo = 108 ± 4%, n = 15, p < 0.01). Thus, the effects of individual G{alpha}i1/2/3 and G{alpha}o are not redundant. The possibility of cross-reaction was minimized by decreasing the concentrations of anti-G{alpha}i1/2/3 and anti-G{alpha}o to 0.2 µg/ml for each (one-tenth of the original amount of antibody). Under these conditions, when any possible cross-reaction should be much reduced or eliminated, the effects of anti-G{alpha}i1/2/3 and anti-G{alpha}o were still clearly observed (see Fig. 5C). Anti-G{alpha}i1/2/3 and anti-G{alpha}o attenuated the effects of NE on NPo by ~34% (NPo = 164 ± 4%, n = 11, p < 0.01) and ~28% (NPo = 172 ± 7%, n = 9, p < 0.01), respectively. When applied together, the effect of the two antibodies was greater than either one alone and resulted in 51% inhibition (NPo = 149 ± 5%, n = 11, p < 0.01). Next, both anti-G{alpha}i1/2/3 (2 µg/ml) and anti-G{alpha}o (2 µg/ml) were applied simultaneously to counteract NE-induced hyperpolarization under the standard whole cell configuration. As summarized in Fig. 5D, the mean values of the membrane potentials were –43 ± 3.7 mV, –45 ± 4.2 mV, and –43 ± 5.1 mV, before, during, and after NE treatment, respectively (n = 15, p > 0.05), data that are consistent with the observations from the single channel experiments presented earlier. Control experiments (shown in Fig. 5, A–D) were performed with mouse and goat IgG, separately or combined as appropriate.


Figure 5
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  		FIGURE 5.
Individual monoclonal antibodies raised against G{alpha}i or G{alpha}o subunits only partially inhibit the effects of NE on KATP channels, whereas the combination of the two inhibits completely. The effects of individual anti-G{alpha}i/anti-G{alpha}o, the combination of these two, and IgG on the activity of KATP channels (NPo) in the absence and presence of 5 µM NE are summarized in A–C, respectively. The summaries are for three different concentrations of individual anti-G{alpha}i/anti-G{alpha}o, IgG, and the combinations applied. The effects of the combination of anti-G{alpha}i/anti-G{alpha}o and of IgG on the membrane potential are summarized in D.*, p < 0.05; **, p < 0.01; #, p > 0.05 relative to control values.

 
Effects of Gβ on NE-induced Electrical Activities—To evaluate the contribution of Gβ subunits to the effects of NE, polyclonal antibodies raised against Gβ subunits (anti-Gβ) were added to the pipette solution C or D, with respect to voltage measurement and outside-out single channel recording. In the presence of anti-Gβ, the example traces corresponding to the effects of NE on the membrane potentials and on the KATP channels are presented in Fig. 6, A and B, respectively. In contrast to the effects of anti-G{alpha}common, anti-G{alpha}i1/2/3, and anti-G{alpha}o, NE-mediated regulation of the membrane potential and the KATP channels was not changed by anti-Gβ. As summarized, in the presence of anti-Gβ, NE reduced membrane potential from –45 ± 3.2 to –58 ± 4.7 mV (n = 9, p < 0.01) (Fig. 6C) and increased NPo from 100% to 212 ± 12% (n = 14, p < 0.01) (Fig. 6D), similar to the control experiments. In addition to concluding that the β{gamma} subunit has no role in the activation of the KATP channels, these results also exclude an effect of NE on K+ channels of the Kir3 family in these cells, which are activated by β{gamma} (11–14).


Figure 6
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  		FIGURE 6.
Lack of effect of polyclonal antibodies raised against β subunits of G proteins (anti-Gβ) on NE-induced modification of the membrane potential and of KATP channel activity. A and B, anti-Gβ (2 µg/ml) was present in the pipette solutions. A and B are representative traces of membrane voltage and outside-out single channel recordings. C and D summarize the lack of effect anti-Gβ on the membrane potential and NPo of single KATP channels in the absence or presence of NE. For the control experiments, anti-Gβ was replaced by IgG in the pipette solution. **, p < 0.01, relative to control values.

 
In order to check the results obtained with antibodies against G{alpha}i and G{alpha}o and in order to obtain further information on the specificity of the Gi and Go proteins involved, we used a second independent approach to block the effect of the Gi and Go proteins. This was to prevent the activation of the G proteins at the {alpha}2-adrenergic receptor with peptides containing the same sequences as the last 11 amino acids of the C termini of G{alpha}i1/2, G{alpha}i3, G{alpha}o1, and G{alpha}o2 (15, 16), using a randomly synthesized peptide (GiR) as control. These specific peptides have been shown to effectively block G protein interaction with the receptors for thrombin and adenosine (15, 16) and to block the action of adenosine on a potassium channel using a whole cell technique similar to that used here (16). The results of these experiments are shown in Fig. 7. NE increased NPo from 100% to 226 ± 6% (n = 6, p < 0.01). Using concentrations of 20 µM, peptides for G{alpha}i1/2 reduced the effect of NE to 144 ± 3% (n = 6, p < 0.01). Peptides for G{alpha}o2 reduced the NE effect to 143 ± 6% (n = 4, p < 0.05), essentially to the same extent as those for G{alpha}i1/2. Importantly, peptides for G{alpha}i3 and G{alpha}o1 had no significant effects. Using concentrations of 60 µM, the effects of the peptides were almost identical to those of peptides used at 20 µM. Thus, a peptide concentration as low as 20 µM is sufficient to produce maximal effects. Finally, different combinations of peptides were used. The combination of peptides for G{alpha}i3 and G{alpha}o1 had no significant effect. The combination of peptides for G{alpha}i3 and G{alpha}o2 reduced the effect of NE to 142 ± 9% (n = 8, p < 0.01), similar to the effect of G{alpha}o2 alone. The combination of peptides for G{alpha}i1/2 plus G{alpha}o1 reduced the NE effect to 144 ± 9% (n = 6, p < 0.05), similar to those for G{alpha}i1/2 alone. The last combination to be tested, that for the G proteins that appear to be the mediators for NE (i.e. G{alpha}i1/2 plus G{alpha}o2), reduced the effect of NE to 105 ± 1% (n = 7, p < 0.01), effectively eliminating the response to NE. These results confirm those obtained with antibodies against the {alpha} subunits and the conclusion that both Gi and Go proteins are required to fully activate the channel. Furthermore, they demonstrate the specificity of the G protein involvement in that G{alpha}i3 and G{alpha}o1 are without effect.


Figure 7
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  		FIGURE 7.
The effects of peptides and combinations of peptides corresponding to the C-terminals of G{alpha} subunits on the activity of KATP channels (NPo) in the absence and presence of 5 µM NE. The data are for two different concentrations of the peptides and the combinations applied. The individual peptides corresponding to the C termini of G{alpha}i1/2 or G{alpha}o2 subunits only partially inhibit the effects of NE on KATP channels, whereas the combination of the two inhibit completely. Peptides corresponding to G{alpha}i3 or G{alpha}o1, a combination of the two, and a randomized control peptide had no effect. Combinations of peptides for active (G{alpha}i1/2 or G{alpha}o2) and inactive (G{alpha}i3 or G{alpha}o1) {alpha} subunits inhibited similarly to the effect of the active peptides alone.

 
Given these data and the conclusion that both Gi and Go proteins appear to be required for full activation of the KATP channel, an alternative explanation could be that two distinct channels are responsible for the effects of NE and that G{alpha}i2 works on one and G{alpha}o2 works on the other. Reasons for thinking this are the reports that islets from SUR1–/– mice are still responsive to epinephrine-induced hyperpolarization. This would argue that the KATP channel is not the inward rectifier responsible for the hyperpolarization in these mouse β-cells (17). Also, Kir3.1 is present in at least some β-cells; its current is pertussis toxin- and therefore Gi/Go protein-sensitive and causes hyperpolarization in response to norepinephrine (18). Rorsmann (5) also proposed that epinephrine causes hyperpolarization via a G protein-sensitive, sulfonylurea-insensitive low conductance potassium channel distinct from KATP. An obvious candidate for an additional channel would be Kir3.1, which is G protein-sensitive and has been reported present in mouse β-cells and clonal cell line(s) derived from mice (18). Finally, somatostatin activates two inwardly rectifying K+ channels in MIN-6 cells (also mouse-derived), the KATP and a G protein-gated inwardly-rectifying K+ channel (19). Despite these reports, the possibility of two distinct channels in our study using a rat-derived clonal cell line seems unlikely for the following reasons. 1) Tolbutamide gives complete blockade of K+ channel activity. 2) The I/V relationship in Fig. 3F is typical of the KATP channel and is not distorted as would be the case if two channels were operating simultaneously. 3) There is no evidence in the I/V relationship in Fig. 3D for a "classical" steep inwardly rectifying channel, such as Kir3.1, even when studied down to –100 mV (data not shown). 4) The frequency distribution histograms in Fig. 3, C and D, provide evidence for only one channel. 5) The G protein-gated inwardly-rectifying K+ channels are activated by β{gamma}, and we would have detected any effect of NE on G protein-gated inwardly-rectifying K+ channel activity during our experiments with the anti-β antibody. However, in a final test for the presence of Kir3.1, an inhibitor of the channel SCH23390 was used (20). These experiments were carried out in the same series as the peptide inhibitor studies, so that NE increased NPo to 226 ± 6%. In the presence of SCH23390 at the high concentration of 150 µM, NE increased NPo to 204 ± 7%, not significantly different from the control value (n = 8, p > 0.05) and nowhere near the significant reductions to 144 and 143% seen with the individual peptides for G{alpha}i2 and G{alpha}o2, respectively.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The effects of NE on the electrical activity of the INS-1-derived clone 832/13 have been investigated. The INS 832/13 cells exhibit markedly enhanced and stable responses to glucose and several of its known potentiators (21) and have been used extensively as a model system for mammalian β-cells (22–26). We first studied the effects of NE on the membrane potential. The standard whole cell configuration has the disadvantage of disturbing the metabolic equilibrium of the cell (27) and may attenuate or prevent glucose-evoked effects. For this reason, we started our experiments with the perforated whole cell configuration, which keeps the cells relatively intact and efficiently prevents the loss of macromolecules from the cytosol. NE-induced hyperpolarization of the cells was completely reversible, inhibited by tolbutamide, and abolished by pretreatment with PTX, indicating the involvement of Gi/Go proteins. NE hyperpolarized the cells via a Gi/Go protein coupling of the {alpha}2-adrenergic receptors, as described previously in HIT-T15 cells (5). Sieg et al. (6) and Rorsman et al. (5) also reported that catecholamines hyperpolarized mouse pancreatic β-cells. However, they attributed the changes of membrane voltage to the activation of ATP-insensitive K+ channels. These results are not necessarily controversial, because the expression of K+ channels in clonal cells derived from the rat and hamster may well be different. In primary cells also there is evidence for differences between mouse and rat β-cells, and human β-cells may be different again. Therefore, it is important to point out that the KATP channel-dependent mechanism by which norepinephrine hyperpolarizes the INS 832/13 cell may be different from that of other clonal and primary cells, which may also differ among themselves. Nevertheless, the use of a model system, such as the 832/13 cell, to study the G protein interactions with an individual ion channel is valid.

It is generally accepted that glucose-stimulated biphasic insulin secretion involves at least two signaling pathways (26), the KATP channel-dependent and KATP channel-independent pathways, respectively. In the former, enhanced glucose metabolism increases the cellular ATP/ADP ratio, closes KATP channels, and depolarizes the cell. According to our results, we assume that the KATP channels in the study are directly or indirectly regulated by activated Gi/Go proteins upon the binding of NE to the {alpha}2-adrenergic receptors. In an indirect way, Gi/Go could relieve the inhibition of KATP channels from ATP by triggering a series of downstream reactions that either impair glucose metabolism and thus reduce the production of ATP or result in a change of cytosolic second messengers (e.g. decreased activity of adenylyl cyclase). Alternatively, it is also possible that the closure of the KATP channels is impaired due to the direct interaction between Gi/Go proteins and the KATP channels. Because the perforated whole cell configuration applied does not provide further information about the mechanisms underlying the action of NE, the same experiments were repeated, except that the standard whole cell configuration was applied. It is assumed that intracellular glucose metabolism is impaired after the connection between cytosol and pipette solution is established by mechanically breaking the cell membrane. However, in the presence of 2 mM ATP in the pipette solution, the cell membrane was still depolarized under control conditions and hyperpolarized upon challenge with NE, indicating that the effect of NE on the membrane potential is independent of glucose metabolism. As anticipated, when ATP was omitted from the pipette solution, the membrane voltage remained at ~–60 mV in the absence and presence of NE. Next, we examined the effects of NE on single KATP channels by using the outside-out membrane patch. Single channel analysis revealed that, in the presence of ATPi, NE enhanced the open activity of KATP channels by more than 2-fold without changing the single channel conductance. There are many examples of ion channels that are regulated by G protein coupled receptors. The signaling pathways used include second messengers, phosphorylation, and direct regulation by G proteins (28–30). In our studies, the regulation of KATP channels by NE is clearly a membrane-delimited phenomenon, since we show that NE can modulate the channel activity when applied to excised membrane patches in a cell-free environment. The membrane-delimited effect of NE on the KATP channels is also consistent with the observation that NE can induce membrane hyperpolarization even under the conditions of the standard whole cell configuration. In this regard, the action of NE resembles the neurohormonal modulation of other K+ channels that can also proceed without the participation of cytosolic second messengers (31). Considering the rapid onset and recovery of KATP channels from NE-induced activation, two possibilities exist. Either the G proteins act directly on the subunits of the KATP channels, or the functional G proteins trigger some unknown factors, which are located in the cell membrane and can directly activate the KATP channels. Closely comparing the current traces in Fig. 3, A and B, we found that NE, under the experimental conditions employed, antagonized the ATPi-mediated inhibition of KATP channels. However, the action of NE was not sufficient to restore channel activity to the spontaneous level seen in the absence of ATPi. This could be due to an insufficient amount of NE applied, to insufficient G proteins to activate all of the channels, or to the simple inability of norepinephrine to activate the channels as completely as occurs in the absence of ATP. The first possibility is unlikely, because we used super-maximal concentrations of NE (even 10 µM did not increase its effect on NPo. Specific antisera raised against various {alpha} subunits of G proteins (G{alpha}common) and against Gβ have been used to examine which subunits of the pertussis toxin-sensitive Gi/Go proteins are involved in NE-induced electrical modulation. Anti-G{alpha}common was able to completely inhibit the effects of NE on both the KATP channels and the membrane potential (Fig. 4); however, neither effect was influenced by anti-Gβ under the same experimental conditions (Fig. 6). Previous studies have shown that KATP channels in insulin-secreting cell lines are activated by physiological inhibitors other than norepinephrine. Dunne et al. (32) found that galanin enhanced KATP channel activity in the insulinoma cell line, RINm5F, via pertussis toxin-sensitive G proteins. de Weille et al. (33) reported that somatostatin regulated KATP channels in RINm5F cells, and the presence of intracellular GTP was required. Although these observations clearly indicated that G proteins played a role in channel modulation, they did not provide evidence as to which G proteins and corresponding subunits were involved. This problem was approached by Ito et al. (34), who reported that G{alpha} activated KATP channels and Gβ activated KAch channels in cardiac cell membranes, and by Ribalet and Eddlestone (35), who found that {alpha} subunits of Gi or Go proteins stimulated the KATP channels in HIT-T15 and RINm5F cell lines. Our findings agree with these observations but go further. In this study, the activation of KATP channels is mediated by G{alpha} subunits, not by Gβ. Furthermore, only when anti-G{alpha}i1/2 and anti-G{alpha}o were applied together did the antibodies achieve complete blockade of the effects of NE on KATP channels as well as on membrane potential. Additional compelling evidence that both Gi and Go are required for full activation of the channel was gained from the C-terminal peptide studies. Blocking activation of the G proteins by the norepinephrine-occupied {alpha}2-adrenergic receptor with peptides specific for individual G protein C termini provided data nearly identical to data obtained by the antibody studies together with the additional information that G{alpha}i3 and G{alpha}o1 are not involved in activating the channel. An analogous observation that two Gi proteins (Gi2 and Gi3) were required for the full inhibition of adenylyl cyclase has been made in RINm5F cells (36). In that study, it was concluded that individual Gi2 and Gi3 proteins only partially mediated the inhibition of adenylyl cyclase by galanin, whereas the combination of the two G proteins functioned additively. However, since there are at least two isoforms of adenylyl cyclase present in β-cells (37), an alternative explanation may be that the two enzymes are affected separately by Gi2 and Gi3.

Although the current study strongly suggests that different targeting sites of Gi or Go are involved in the NE-mediated activation of the KATP channels, the localization of these targeting sites and the underlying molecular mechanisms of the actions of Gi/Go proteins on these sites are not known. Most likely, the sites are located on the KATP channels. However, we cannot exclude the possibility that one or both of the Gi/Go proteins regulate the KATP channels by affecting other unknown mediators, which are tightly embedded in the cell membrane and modify the channel activity upon the interaction of Gi/Go proteins.

KATP channels are formed from an inward rectifier potassium channel (Kir6.x) and a sulfonylurea receptor (SUR) subunit, arranged with 4:4 stoichiometry (38, 39). The different isoforms of Kir6.x (Kir6.1 or Kir6.2) and SUR (SUR1, SUR2A, or SUR2B) show distinct expression patterns. For insulin-secreting cells, the KATP channels are assembled from Kir6.2 and SUR1. Kir6.2 forms the channel pore and provides the ATP-binding site, whereas the SUR1 acts as a regulatory subunit, endowing the channel with sensitivity to high affinity sulfonylurea inhibition and activation by Mg-nucleotides and channel openers (40–45). If we hypothesize that Gi/Go proteins directly interact with KATP channels, both subunits of Kir6.2 and SUR1 can be the candidates of the targeting sites. Cloning of the KATP channel subunits has already shown that a mutant Kir6.2 lacking the 26 C-terminal residues forms functional channels in the absence of SUR, thus indicating that the ATP-inhibitory site may be located on Kir6.2 (46). Subsequently, by mutagenesis studies, several regions in the cytoplasmic N and C termini of Kir6.2 that profoundly affect the ATP inhibition of the KATP channels have been located (47–50). Based on these studies, our electrophysiological observations can be accounted for in at least three ways: Gi/Go proteins may 1) directly interact with the ATP-binding site and decrease the channel sensitivity to ATP; 2) affect certain regions on Kir6.2 and thus impair channel closure; 3) interfere with the transduction mechanism via which ATP-binding initiates pore closure. The second transmembrane domain of Kir6.2 (TM2) has been proposed to line the intracellular mouth of the pore, since mutations within this region affect the single channel conductance, ion selectivity, and sensitivity to the blocking effects of intracellular cations, such as Mg2+. The fact that the single channel conductance of the KATP channels was not changed by NE may hint that the targeting sites of Gi/Go proteins are not located on TM2. However, this conclusion is not supported by recent mutagenesis studies showing that many of the mutations relevant to the ATP-inhibitory effect are located in the cytoplasmic end of TM2 (43, 44). Additional experiments are needed to resolve this. The alternative candidate for the targeting site of Gi/Go proteins is the SUR1 subunit. The sulfonylurea receptor modulates the activity of Kir6.2, and binding of sulfonylureas or channel openers to SUR results in a decrease or increase of the channel activity, respectively. It is possible that Gi/Go proteins directly affect SUR1 and consequently increase the channel activity. Since the effects of NE on the KATP channels were fully blocked by tolbutamide, the potency of the influences of Gi/Go proteins on SUR1 itself or on the transduction from SUR1 to Kir6.2 must be much weaker, compared with that of tolbutamide, or the effect of the latter prevents NE from acting.

In summary, we have shown that NE stimulates the KATP channel activity in INS 832/13 cells and hyperpolarizes the membrane potential. This modulation is PTX-sensitive and mediated by a combination of the G{alpha}i and G{alpha}o subunits, both of which are required for full activation of the channel.


   FOOTNOTES
 
Note Added in Proof—Since the submission of this manuscript, a related article has been published (Katsuya, D., Kakei, M., and Yada, T. (2007) Diabetes 56, 2319–2327).

* This work was supported by National Institutes of Health Grants DK54243 and DK56737 (to G. W. G. S.) and a Career Development Award from the Juvenile Diabetes Foundation International (to S. G. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. Tel.: 607-253-3875; Fax: 607-253-3659; E-mail: gws2{at}cornell.edu.

2 The abbreviations used are: PTX, pertussis toxin; NE, norepinephrine; ATPi, intracellular free ATP; SUR, sulfonylurea receptor. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Troitza Bratanova-Tochkova for expert tissue culture work and to Drs. Manfred Lindau, Jennifer Mulvaney-Musa, and Linda Nowak for excellent advice and assistance.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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