Glucagon-Like Peptide-1 Inhibits Pancreatic ATP-Sensitive Potassium Channels via a Protein Kinase A- and ADP-Dependent Mechanism

Peter E. Light, Jocelyn E. Manning Fox, Michael J. Riedel and Michael B. Wheeler

Department of Pharmacology (P.E.L., J.E.M.F., M.J.R.), Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7; and Department of Physiology (M.B.W.), University of Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: Peter E. Light, Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, 9-58 Medical Science Building, Alberta, Canada T6G 2H7. E-mail: peter.light{at}ualberta.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Glucagon-like peptide-1 (GLP-1) elicits a glucose-dependent insulin secretory effect via elevation of cAMP and activation of protein kinase A (PKA). GLP-1-mediated closure of ATP-sensitive potassium (KATP) channels is involved in this process, although the mechanism of action of PKA on the KATP channels is not fully understood. KATP channel currents and membrane potentials were measured from insulin-secreting INS-1 cells and recombinant ß-cell KATP channels. 20 nM GLP-1 depolarized INS-1 cells significantly by 6.68 ± 1.29 mV. GLP-1 reduced recombinant KATP channel currents by 54.1 ± 6.9% in mammalian cells coexpressing SUR1, Kir6.2, and GLP-1 receptor clones. In the presence of 0.2 mM ATP, the catalytic subunit of PKA (cPKA, 20 nM) had no effect on SUR1/Kir6.2 activity in inside-out patches. However, the stimulatory effects of 0.2 mM ADP on SUR1/Kir6.2 currents were reduced by 26.7 ± 2.9% (P < 0.05) in the presence of cPKA. cPKA increased SUR1/Kir6.2 currents by 201.2 ± 20.8% (P < 0.05) with 0.5 mM ADP present. The point mutation S1448A in the ADP-sensing region of SUR1 removed the modulatory effects of cPKA. Our results indicate that PKA-mediated phosphorylation of S1448 in the SUR1 subunit leads to KATP channel closure via an ADP-dependent mechanism. The marked alteration of the PKA-mediated effects at different ADP levels may provide a cellular mechanism for the glucose-sensitivity of GLP-1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
ATP-SENSITIVE POTASSIUM (KATP) channels serve to couple cellular metabolism to electrical excitability in many different tissues including pancreatic ß-cells, heart, smooth muscle, skeletal muscle, and brain (1, 2, 3, 4). ß-Cell KATP channels act as metabolic sensors coupling glucose metabolism to insulin secretion. At low plasma glucose, KATP channels permit a small efflux of potassium ions, keeping the ß-cell in a hyperpolarized inexcitable state. At elevated plasma glucose levels, KATP channels are inhibited via increases in the intracellular ATP/ADP ratio upon changes in glucose metabolism (4, 5) and the resultant membrane depolarization leads to an increase in ß-cell excitability, calcium influx, and subsequent insulin release.

The ß-cell KATP channel complex consists of two subunits, the sulphonylurea receptor (SUR1) and the inwardly rectifying pore forming K+ channel subunit Kir6.2. SUR1 and Kir6.2 are coassembled in a stoichiometry of (SUR1)4,(Kir6.2)4 to form the hetero-octameric channel complex (1, 4, 6). The Kir6.2 subunit forms the potassium-conducting pore of the KATP channel and is largely responsible for the ATP-sensing properties of the channel complex (4, 6, 7, 8). SUR1 is responsible for conferring the unique pharmacological characteristics to the channel complex, including inhibition by antidiabetic sulphonylurea compounds such as glibenclamide and activation by diazoxide. SUR1 is predicted to contain 17 trans-membrane domains and possesses two nucleotide-binding folds (NBFs), the second of which appears to act as the ADP sensor (2, 7, 9). ADP antagonizes the inhibitory action of ATP and fluctuations in intracellular ADP serve to couple KATP channel activity to membrane excitability and subsequent insulin secretion (4, 9).

KATP channels have recently been found to be cellular targets for receptor-mediated signal transduction pathways that modulate insulin release including the cAMP/protein kinase A (PKA) pathway (10, 11), protein kinase C (12), and tyrosine kinase (13, 14).

Glucagon-like peptide-1 (GLP-1) is a gut hormone released from intestinal L-cells in response to food ingestion and stimulates insulin secretion from pancreatic ß-cells in a glucose-dependent manner (15, 16). GLP-1-stimulated insulin release is mediated via activation of the GLP-1 receptor (GLP-1R), G protein-coupled production of cAMP, and subsequent activation of PKA (17). The cellular targets for PKA-mediated phosphorylation include the KATP channel, the L-type calcium channel, intracellular calcium stores, and the exocytotic machinery (10). It has been demonstrated that GLP-1 facilitates closure of ß-cell KATP channels, leading to membrane depolarization and triggering of the insulin secretory pathway (10, 11, 18). It is proposed that the PKA-induced closure of KATP channels is the initial step in GLP-1-mediated insulin secretion (10). The insulin stimulatory effects of GLP-1 are glucose dependent, such that GLP-1 only stimulates insulin release when glucose levels are elevated. However, the underlying mechanism for this phenomenon is still unclear.

Recent studies have shown that PKA may phosphorylate residues at positions 372 (19) or 224 (20) in the pore-forming Kir6.2 subunit. However, both of these studies observed activation of KATP channels with PKA in the presence of ATP only. This situation is somewhat unphysiological as in intact ß-cells and cell lines (11, 18, 21) intracellular nucleotides such as ATP and ADP are preserved. Moreover, recent evidence suggests that intracellular ADP levels fluctuate much more than ATP levels at different glucose concentrations (22).

It was therefore the aim of this study to further investigate the mechanisms by which GLP-1, acting through PKA, modulates the ß-cell KATP channel via changes in nucleotide sensitivity.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
The Effects GLP-1 on the Insulin-Secreting Cell Line INS-1 Cell
The resting membrane potential of INS-1 cells after more than 15 min exposure to 5 mM glucose was -53.01 ± 2.20 mV (n = 11 cells). In the presence of 5 mM glucose, application of 20 nM GLP-1 caused a 6.68 ± 1.29 mV (n = 11 cells) depolarization of the membrane potential (Fig. 1Go, A and B). This depolarization resulted in the initiation of action potential firing in 80% of the cells tested (Fig. 1AGo). To test whether the effects of GLP-1 on membrane excitability are dependent on PKA activity, a 5-min pretreatment with the selective PKA inhibitor H-89 (1 µM) was used. H-89 prevented the GLP-1-induced depolarization (-0.55 ± 0.46 mV change, n = 5 cells, Fig. 1Go, B and C). To confirm that these cells were receptive to KATP channel inhibition, the sulphonylurea tolbutamide was used. Application of 20 µM tolbutamide led to membrane depolarization and the firing of action potentials (Fig. 1BGo). The addition of GLP-1 in the presence of the GLP-1R antagonist exendin (9–39) (50 nM) caused a slight membrane hyperpolarization (-4.9 ± 0.8 mV, n = 5 cells, data not shown).



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Figure 1. The Effects of GLP-1 on Insulin-Secreting INS-1 Cells

The glucose concentration in all experiments was 5 mM. A and B, Representative membrane potential measurements, made using the perforated whole-cell patch-clamp technique in current-clamp mode (see Materials and Methods for details). GLP-1 was superfused over cells via a fast-switching perfusion device. The application of H-89 to cells was started 5 min before addition of GLP-1. Dashed lines denote initial membrane potential. C, Grouped data from membrane experiments represented in A and B. Asterisk denotes a statistically significant difference (P < 0.05) compared with control (no GLP-1).

 
In a parallel set of experiments, INS-1 cell cAMP content was measured in response to the application of GLP-1. In resting INS-1 cells at 5 mM glucose, the cAMP concentration was 10.5 pmol/ml, upon 2-min and 10-min exposure to 20 nM GLP-1, cAMP increased to 36 and 48 pmol/ml, respectively.

Reconstitution of the GLP-1R/cAMP Pathway in a Recombinant System
The GLP-1R, SUR1, and Kir6.2 clones were coexpressed in tsA201 cells to directly confirm the coupling of GLP-1R activation, cAMP, PKA, and the ß-cell KATP channel isoform. Whole-cell perforated patch-clamp experiments were performed on cells that exhibited a barium-sensitive inward current (basal KATP current) and fluoresced green ({lambda} = 520 nm) upon excitation with 488 nm epifluorescent light, indicating expression of the green fluorescent protein (GFP)-tagged GLP-1R. In positively identified cells, application of 100 nM GLP-1 caused a time-dependent decrease in whole-cell KATP channel current (54.1 ± 6.9% reduction (P < 0.05), n = 6 cells, Fig. 2Go, A and C). In cells expressing SUR1 and Kir6.2 only, 100 nM GLP-1 had no effect (data not shown). To test the PKA dependence of the observed reduction in current, cells expressing SUR1, Kir6.2, and GFP-tagged GLP-1R were incubated for 5 min before recording with the selective PKA inhibitor H-89 (1 µM). In these cells, application of 100 nM GLP-1 did not cause any reduction in the observed whole-cell KATP channel current [5.2 ± 4.9%, n = 5 cells, (P > 0.95), Fig. 2Go, B and C]. In a parallel series of experiments, cAMP content was measured in this recombinant cellular system. Nontransfected tsA201 cells had a resting cAMP content of 7.9 ± 4.0 pmol/ml and addition of 20 nM GLP-1 did not increase this value (8.2 ± 4.6 pmol/ml). In tsA201 cells expressing SUR1, Kir6.2, and GLP-1R, the resting cAMP content was 10.2 ±3.3 pmol/ml, a value not significantly different to that observed in nontransfected cells. However, in cells expressing SUR1, Kir6.2 and GLP-1R, 10-min exposure to 20 nM GLP-1, increased the cAMP content significantly [60.3 ± 7.1 pmol/ml (P < 0.01), Fig. 2EGo]. Taken together, these data indicate that, in an intact cellular system, activation the GLP-1 receptor leads to a significant cAMP/PKA dependent decrease in recombinant KATP channel activity.



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Figure 2. The Effects of GLP-1 on KATP Channels in a Recombinant Intact Cell System

A–C, Whole-cell KATP channel currents measured using the perforated patch-clamp technique in voltage-clamp mode. tsA201 cells were transiently transfected with GFP-tagged GLP-1R, SUR1, and Kir6.2 clones 48–72 h before recording. Currents were recorded with 140 mM K+ in the bath solution and were held at 0 mV and stepped to -100 mV every 10 sec. H-89 was applied to cells 5 min before application of GLP-1. Barium chloride (Ba2+, 2 mM) was used as a fully washable potassium current blocker to confirm the amount of recombinant inward KATP channel current present. Arrows in A and B denote zero current level. In Aii, whole-cell current traces labeled a–d are derived from the respective time points labeled in Ai. C, Grouped data from the experiments illustrated in A and B. D, Representative whole-cell recording of recombinant KATP channel current expressed with the GLP-1R (i) and the lack of current in a nontransfected cell (ii). Cells were dialyzed with an ATP-free pipette solution to elicit any ATP-sensitive current. E, Grouped data from cAMP assay experiments on tsA201 cells transfected with SUR1, Kir6.2, and GLP-1R. *, Statistically significant difference (P < 0.05).

 
PKA Modulates Recombinant KATP Channels in an ADP-Dependent Manner
To investigate the molecular mechanism by which PKA inhibits ß-cell KATP channels, the effects of the catalytic subunit of PKA (cPKA) were tested on inside-out membrane patches containing recombinant KATP channels. Figure 3AGo shows a representative recording of macroscopic Kir6.2/SUR1 currents. In all experiments, the measured inward current was maximally active in the absence of internal ATP, inhibited by 1 mM ATP and was partially restored upon exposure to intermediate levels of ATP such as 0.1 or 0.2 mM. Application of 20 nM cPKA had no significant effect on macroscopic KATP channel current in the presence of 0.2 mM ATP alone (Irel = 109.8 ± 11.0%, n = 8 patches, P > 0.05). In the presence of 0.2 mM ATP, the addition of 0.2 mM ADP partially relieved the ATP inhibition (0.2 mM ATP; Irel = 10.15 ± 1.23%, n = 9 patches: 0.2 mM ATP and 0.2 mM ADP; Irel = 65.64 ± 9.6%, n = 9 patches, Fig. 3BGo). In the presence of both ATP and ADP, application of 20 nM cPKA caused a significant decrease in current (Irel = 73.34 ± 2.9%, n = 9 patches, P < 0.05, Fig. 3BGo). However, in the presence of 0.2 mM ATP and elevated ADP (0.5 mM), application of cPKA caused a significant increase in KATP channel activity [Irel = 201.16 ± 20.18%, n = 19 patches, (P < 0.05), Fig. 3CGo]. The grouped results from this series of experiments are presented in Fig. 3DGo.



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Figure 3. The Effects of Purified PKA on Recombinant KATP Channels

A–C, Representative recordings of SUR1/Kir6.2 currents from excised inside-out membrane patches from tsA201 cells. The purified cPKA was applied to the internal face of the membrane patch at a concentration of 20 nM. The functional expression of recombinant KATP channels was confirmed by the presence of large macroscopic currents that were inhibited by 1 mM ATP. Steady-state currents were recorded at a holding potential of -60 mV under symmetrical K+ conditions (140 mM). Dotted lines denote the zero current level. B and C, Arrows show the inhibitory (B) or stimulatory (C) effects of cPKA are dependent on internal ADP levels. D, Grouped data from the representative experiments illustrated in A–C. Asterisks denote a statistically significant difference (P < 0.05) compared with control.

 
A Single Residue Substitution in the SUR1 Subunit Abolishes the Effects of cPKA
The results from the section above indicate that PKA reduces the ADP-induced release of ATP inhibition leading to a more pronounced channel closure in the presence of ATP and 0.2 mM ADP. These findings suggest that the molecular site of action of PKA may reside in the ADP-sensing region of SUR1. The second nucleotide binding fold (NBF2) of SUR1 has been identified as the intracellular binding site for ADP (2, 7, 9). Consensus PKA phosphorylation motifs typically consist of two basic residues such as arginine or lysine, a residue that is not critical followed by a phospho-acceptor serine or threonine (23). Sequence analysis of NBF2 in the hamster SUR1 subunit revealed four potential consensus PKA motif sequences that are conserved in human and rat. One of these, a KKCS motif, is located in the linker between the Walker A and B motifs of NBF2 (Fig. 4Go). The putative serine phospho-acceptor residue at position 1448 (S1448) is in close proximity to G1479, a residue found to be important in the ADP-sensing properties of the KATP channel (9). In the following set of experiments, we set out to determine if substitution of the serine at 1448 prevented the effects of cPKA observed in wild-type (WT) KATP channels comprised of SUR1 and Kir6.2. We therefore generated a S1448A substitution mutant in the hamster SUR1 sequence and coexpressed this mutant with Kir6.2. Inside-out patch experiments revealed that the S1448A mutant was functionally expressed and possessed similar ATP and ADP-sensing properties when compared with WT SUR1/Kir6.2 channels (Fig. 5DGo). Under the identical conditions that produced a cPKA-induced significant reduction in WT KATP channel current (0.2 mM ATP and 0.2 mM ADP), SUR1(S1448A)/Kir6.2 current was not significantly changed in the presence of 20 nM cPKA [95.40 ± 2.8%, n = 8 patches (P > 0.05), Fig. 5Go, A and C]. At higher levels of ADP (0.5 mM), where WT KATP current is enhanced by cPKA, the SUR1(S1448A)/Kir6.2 current was also unaffected by application of cPKA (95.31 ± 5.94, n = 13 patches, Fig. 5BGo).



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Figure 4. SUR1 Nucleotide Binding Fold 2 and PKA Phosphorylation Sites

A, Amino acid sequence alignment of the NBF2 of SUR1 from the hamster, human and rat clones; residues 1358-end (K1582) are shown. The nucleotide binding Walker A and B motifs are underlined. The PKA consensus phosphorylation site at position 1448, G1479 and the human clone-specific PKA consensus site (S1571) are highlighted in reverse type. B, Schematic representation of the 17 trans-membrane domains of the SUR1 clone and the Kir6.2 subunits of the KATP channel. The nucleotide binding folds 1 and 2 (NBF1, NBF2) and the Walker A and B motifs (A and B) are illustrated. The relative positions of residues S1448, G1479, and S1571 in SUR1 and T224 and S372 in Kir6.2 are marked.

 


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Figure 5. The Effects of Purified PKA on Recombinant WT or KATP Channels Expressing the SUR1 Substitution Mutant, SUR1(S1448A)

A and B, Representative recordings of Kir6.2/SUR1(S1448A) currents from excised inside-out membrane patches from tsA201 cells in response to application of 20 nM cPKA in the presence of either 0.2 mM ADP (A) or 0.5 mM ADP (B). Dotted line denotes zero current level. C, Grouped data from WT (Kir6.2/SUR1) or mutated (S1448A) KATP channels in response to application of cPKA, either in the presence or absence of ADP (0.2 mM) and ATP (0.2 mM) ATP continuously present. Asterisk denotes a statistically significant difference (P < 0.05) compared with control (WT). D, Grouped data, from inside-out patch experiments with WT and S1448A mutant KATP channels, showing a similar response to ATP alone (0.2 mM) and a release of this ATP-induced inhibition by ADP (0.2 mM). There were no statistical differences in nucleotide sensitivities between WT and S1448A KATP channels.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Glucose-mediated depolarization of the ß-cell membrane potential is a key event that occurs before the initiation of insulin secretion (10, 24, 25). Nucleotide-mediated closure of KATP channels is thought to account for the observed depolarization via alterations in the intracellular ATP and ADP levels (7, 8, 9) as glucose levels increase. Therefore, receptor-linked pathways such as GLP-1, that facilitate KATP channel closure in ß-cells will promote insulin secretion. In accordance with previous work, our current study confirms that GLP-1 induces closure of ß-cell KATP channels via a cAMP/PKA-dependent process. More importantly, our data suggest that the mechanism of KATP channel inhibition by PKA is dependent on the presence of intracellular ADP. Such a nucleotide-dependent mechanism may contribute to the observed glucose-dependent GLP-1-induced insulin secretory response.


    GLP-1 and ß-Cell KATP Channels
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Previous patch-clamp studies have suggested that GLP-1 inhibits KATP channels from rodent ß-cells in a reversible manner (10, 11, 18). The effects of GLP-1 observed in this present study were measured at an intermediate glucose concentration of 5 mM. This concentration of glucose was chosen as it has previously been shown to be just subthreshold for the maintenance of sustained depolarization and action potential firing in INS-1 cells (26), although occasional firing was observed. In all experiments, a GLP-1-induced membrane depolarization preceded increases in action potential firing. Our data demonstrate that 1) GLP-1 significantly depolarizes INS-1 cells; 2) GLP-1 increases cAMP content in INS-1 cells; and 3) the observed effects are blocked by the PKA inhibitor H-89 (Fig. 1Go). These findings are in accordance to the majority of previous studies (Refs. 10, 11 and 18 , but see Refs. 27 and 28) and further confirm that GLP-1 acts by a cAMP/PKA-dependent membrane depolarization via inhibition of KATP channels. Physiologically, the GLP-1-mediated depolarization would likely have several effects. Firstly, it may trigger glucose-unresponsive cells to become excitable, therefore increasing the percentage of ß-cells secreting insulin. Secondly, GLP-1 could further depolarize ß-cells already in an excitable state, leading to increased action potential firing and augmented insulin secretion.

In addition to its effects on KATP channels, GLP-1 likely modulates membrane excitability via changes in the activity of other ion channels. For example, GLP-1 has been shown to regulate nonspecific cation channels (29) and voltage gated L-type calcium channels (28, 30). GLP-1 also directly modulates release of calcium from intracellular stores (31) and the exocytotic release of insulin-containing granules directly (11). Because of the complexity of GLP-1 signaling in intact ß-cells, we decided to reconstitute this pathway in an intact cellular system. This approach has the advantage of isolating and amplifying the signal transduction pathway, while maintaining the integrity of the intracellular environment. This was achieved by coexpression of the recombinant GLP-1 receptor and the KATP channel subunits SUR1 and Kir6.2 in tsA201 cells. GLP-1 was used at 100 nM, a concentration that has previously been shown to maximally activate recombinant GLP-1R in mammalian cell lines (30, 32), while having no effect on other related receptors such as the glucose-dependent insulinotropic polypeptide receptor (33). Results from this set of experiments demonstrate that stimulation of the GLP-1R increases intracellular cAMP content and leads to a marked PKA-dependent reduction in whole-cell KATP channel current. The reconstitution of the signaling pathway using recombinant overexpression of the respective components of the signaling cascade has limitations, for example, the limited availability of endogenous signaling components such as adenylyl cyclase to couple with overexpressed receptors. Nevertheless, these results demonstrate directly that simulation of GLP-1R can functionally modulate KATP channel activity via a cAMP/PKA-dependent pathway.


    The Effects of PKA on the ß-Cell KATP Channel
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Evidence from this study clearly demonstrates a modulatory effect of PKA on recombinant KATP channels in the presence of ATP and ADP. At lower levels of ADP, PKA inhibits KATP channels, while at elevated ADP levels PKA augments KATP channel activity. Previous data suggest that intracellular ATP levels in insulin secreting cells remain relatively unchanged upon increasing glucose, whereas the ADP levels fluctuate to a greater extent (22). Regarding the nucleotide-dependent control of ß-cell KATP channels, it is generally accepted that intracellular ADP is a key component in regulating channel activity and therefore ß-cell excitability and insulin release (7, 8, 9). These findings, taken together with the data presented in this study, suggest the effects of PKA, mediated through GLP-1-R activation, may be dependent on the metabolic status of the cell.

It should be mentioned that, while our findings are in accordance with the majority of previous studies, it has been suggested by Suga et al. (27) that the effects of GLP-1 on the ß-cell KATP channel are cAMP/PKA independent. In our study, we used the cell-permeant specific PKA inhibitor H-89 at a concentration of 1 µM (IC50 48 nM). In the study by Suga et al. (27), the cAMP analog Rp-cAMPs (100 µM) was used to inhibit the cAMP pathway; however, Rp-cAMPs possesses limited membrane-permeability and was used at 100 µM (only 9-fold the IC50 of 11 µM). Suga et al. (27) also showed that forskolin significantly stimulated insulin secretion despite the presence of Rp-cAMPs. Therefore, incomplete inhibition of the cAMP/PKA pathway may account for the apparent discrepancy. Mechanistically, Suga et al. also observed that GLP-1 directly increases ATP-sensitivity of the KATP channel reducing the IC50 for ATP-inhibition from to 12 to 6 µM, with no change in the Hill coefficient. Physiologically, this is likely to have negligible effects on KATP channel activity, as intracellular ATP is in the millimolar range and KATP channel open probability, in the presence or absence of GLP-1, will be virtually identical at these physiological ATP levels. In contrast, data from our own study link PKA-induced modulation of KATP channel activity to the more metabolically variable ADP concentration under more physiological conditions.

The molecular site of PKA action on the KATP channel is of considerable importance. Although the possibility exists that PKA acts indirectly upon the KATP channel via phosphorylation of an accessory protein, the observed effects of the purified catalytic subunit of PKA on the recombinant KATP channel in excised patches argues against this notion (Fig. 3Go and Refs. 19 and 20). The location of the phosphorylation site on the KATP channel complex has been the subject of recent research in several laboratories. In the study by Béguin et al. (19), it is suggested that S372 in the Kir6.2 subunit and the S1571 residue (found only in the human SUR1 subunit) are the targets for PKA. In another study by Lin et al. (20), T224 in Kir6.2 was identified as another putative molecular target for PKA. In both of these studies, inside-out excised patch experiments were performed in the presence of ATP only, and a PKA-induced increase in KATP channel activity was observed. In direct contrast to their findings, our own observations in intact cells and in excised inside-out patches, in the presence of ATP and ADP, demonstrate that PKA modulates KATP channel activity under more physiological conditions in an ADP-dependent fashion. Moreover, the inhibitory effects of GLP-1 and PKA have been previously observed in several species (10, 11, 18) suggesting the human specific S1571 residue found by Béguin et al. (19) is unlikely to play a significant physiological role in any common GLP-1-mediated cross-species mechanism.

Data from our study now suggest that a serine located at position 1448 in SUR1 also contributes to the PKA-mediated regulation of KATP channel activity, as substitution of the serine with an alanine, prevented ADP-dependent PKA-mediated KATP channel modulation (Fig. 5Go). S1448 is found in the linker between the Walker A and B motifs in NBF2 and is highly conserved among different species (Fig. 4Go). The importance of NBF2 in the ADP-sensing properties of the KATP channel is well established (2, 7, 9). For example, mutations in the human SUR1 gene in NBF2 lead to persistent hyperinsulinemic hypoglycemia of infancy characterized by very low plasma glucose resulting from uncontrolled insulin release (9, 34, 35, 36). Taken together, the ADP dependence and the involvement of S1448 in the action of PKA on the KATP channel suggest a novel molecular mechanism for the regulation of ß-cell KATP channel activity.

It is worth noting that mutagenic substitution of residues T224 and S372 to alanines in Kir6.2 resulted in PKA-induced inhibition of SUR1/Kir6.2 currents in the presence of ATP alone (20). This finding and the data from our study suggest that PKA-mediated control of KATP channel activity is probably dependent on the phosphorylation of one or more phospho-acceptor residues on either SUR1 or Kir6.2. It is likely that, depending on the metabolic status of the cell (ATP:ADP ratio), phosphorylation of respective residues may lead to markedly different modulation of KATP channel activity.


    Physiological Consequences
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Understanding the effects of GLP-1 on ß-cell ionic currents such as KATP channels has direct relevance to insulin secretion in vivo. GLP-1 is known to promote insulin secretion in a glucose-dependent manner (18) (for reviews see Refs. 10 and 15). The mechanisms of GLP-1 action seem to be predominately mediated via elevation of cAMP and activation of PKA (10, 15). It has also been suggested that calmodulin may play a role in mediating the effects of GLP-1 in ß-cells (37).

The cellular sites of action of PKA in the pancreatic ß-cell include classes of ion channels such as KATP channels, nonspecific cation channels, and voltage-gated calcium and potassium channels (for review see Ref. 10). PKA is also known to facilitate the exocytotic release of insulin-containing vesicles (11). To date, the cellular mechanism for the observed glucose-sensitivity of GLP-1 is poorly understood, although it has been suggested that glucose-induced increases in intracellular ATP prime insulin-containing vesicles for release (38). However, recent data suggest that, in the ßHC9 insulin-secreting cell line, intracellular ATP does not fluctuate greatly (~2 mM) in response to elevated glucose, whereas intracellular free ADP decreases exponentially as glucose levels increase (22). Glucose-dependent increases in the ATP/ADP ratio have also been observed in INS-1 cells (39). Data from our study demonstrate that PKA inhibits KATP channels in an ADP-dependent manner, and we suggest that at low glucose concentrations, when intracellular ADP is elevated, the effect of GLP-1 (via PKA) on KATP channel is negligible or even stimulatory. However, as intracellular ADP drops in response to increased glucose, GLP-1 elicits a more pronounced closure of KATP channels. The physiological consequence of such a pathway would be a GLP-1-facilitated membrane depolarization and subsequent initiation of ß-cell excitability only when glucose levels are elevated. We therefore propose this sequence of events as a possible mechanism for the glucose-dependent action of GLP-1. Our data also indicate that GLP-1-induced membrane depolarization, via closure of KATP channels, may be the initial trigger for a chain of inter-related events that promote insulin release (for review see Ref. 10).

KATP channels are also found in other tissues, and PKA has been shown to activate these channels in smooth muscle (40) and neurons (41). These documented tissue-specific effects may be accounted for by a combination of the following three observations: 1) the existence of multiple phosphorylation sites in both the SUR and Kir6.x subunits; 2) KATP channel isoform-specific PKA phosphorylation sites, for example the equivalent consensus PKA site at S1448 is not found in the SUR2A or SUR2B isoforms (42); and 3) the different metabolism and glucose/substrate sensitivity of the cell-type in question. Further studies are required to elucidate the molecular mechanisms underlying PKA-mediated regulation of KATP channel activity in a variety of other cell types.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture and Transfection
The insulin-secreting ß-cell line INS-1 was maintained in culture with Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 11 mM glucose, 2 mM L-glutamine, 10% fetal calf serum, and 0.1% penicillin/streptomycin. tsA201 cells (a SV40-transformed variant of the HEK293 human embryonic kidney cell line) were maintained in DMEM supplemented with 25 mM glucose, 2 mM L-glutamine, 10% fetal calf serum, and 0.1% penicillin/streptomycin. INS-1 and tsA201 cells were kept at 37 C with 5% CO2. The KATP channel Kir6.2 subunit clone from mouse was generously provided by Dr. S. Seino (43). The SUR1 subunit clone from hamster was generously provided by Drs. L. Aguilar Bryan and J. Bryan (44). Clones were inserted into the mammalian expression vector pCDNA3. The green fluorescent protein tagged GLP-1 receptor clone was created by us (45). tsA201 cells were plated at 50–70% confluency on 35-mm culture dishes 4 h before transfection. Clones were then transfected into tsA201 cells using the calcium phosphate precipitation technique. Transfected cells were identified using fluorescence optics in combination with either coexpression of the green fluorescent protein plasmid (pGreenLantern, Life Technologies, Inc., Gaithersburg, MD) or the GFP-tagged GLP-1 receptor. Recordings were made from cells 48–72 h after transfection. In contrast to cells expressing SUR1/Kir6.2 and GLP-1R, nontransfected cells did not exhibit any inward barium-sensitive potassium current when dialyzed with pipette solution containing no ATP (see Fig. 2DGo).

Molecular Biology
The S1448A point mutation was introduced into the hamster SUR1 clone, using the Unique Site Elimination (U.S.E.) Mutagenesis Kit as per the manufacturer’s instructions (Amersham Pharmacia Biotech, Piscataway, NJ) and confirmed by sequence analysis.

Patch-Clamp Experiments
The perforated patch technique (46) was used to measure whole-cell currents and membrane potentials from INS-1 cells whilst maintaining the intracellular signaling environment. Amphotericin (Sigma) was dissolved in dimethylsulfoxide (DMSO) (40 mg/ml) and diluted into the pipette solution immediately before use to give a final concentration of 80 µg/ml. Pipettes were then back-filled with this solution containing Amphotericin. The pipette solution used for all whole-cell recordings contained the following (in mM): KCl 10, K Aspartate 130, HEPES 10, MgCl2 1.4, EGTA 1, glucose 10. The pH of the solution was adjusted to 7.4 with KOH. Patch pipettes were pulled using borosilicate glass (G85150T, Warner Instrument Corp., Hamden, CT) to yield pipettes with a resistance of 2–6 M{Omega} when filled with pipette solution. Once a G{Omega} seal was formed, series resistance was monitored and a perforation access of less than 20 M{Omega} was deemed acceptable. All membrane potential recordings were made in current-clamp mode. Cells were not used in which the resting membrane potential was more positive than -40 mV. In order not to overestimate the GLP-1-induced depolarization, the interburst membrane potential was measured during the period of action potential firing. Cells were superfused with control and test solutions containing (in mM) NaCl 140, KCl 5, HEPES 10, CaCl2 1.0, and MgCl2 1.4.

Whole-cell currents were recorded in voltage-clamp mode under symmetrical [K+] conditions (140 mM). The holding potential was 0 mV and inward currents were elicited using 200 msec long voltage steps to -100 mV every 10 sec. The amplitude of potassium current was estimated as the current blocked by 2 mM barium chloride. In several instances, 20 µm tolbutamide (Sigma) was also used to estimate the KATP channel current present. An Axopatch 200B patch-clamp amplifier and Clampex 8.0 software (Axon Instruments, Foster City, CA) were used for data acquisition and analysis. Cells were superfused with control and test solutions containing (in mM) NaCl 140, KCl 5, HEPES 10, CaCl2 1.0, and MgCl2 1.4.

Standard patch-clamp techniques were used to record macroscopic single-channel currents in the inside-out patch configuration. Single channel currents were recorded at fixed holding potentials, amplified, digitized, and acquired using pClamp 8.0 software. Data were sampled at 1000 Hz and filtered at 400 Hz except where otherwise stated. The pipette solution used for all inside-out patch recordings contained the following (in mM): KCl 140, HEPES 10, MgCl2 1.4, EGTA 1, and glucose 10. The pH of the solution was adjusted to 7.4 with KOH. This solution was also used in the recording chamber to superfuse the cells/patches for experiments using symmetrical [K+].

Cells or membrane patches were directly exposed to test solutions via a multi-input perfusion pipette (time to change solution at the tip of the recording pipette was less than 2 sec). All patch clamp experiments were performed at room temperature (20–22 C).

Experimental Compounds
MgATP or K2ADP (Sigma, St. Louis, MO) was added as required from a 10 mM stock, which was prepared immediately before use. Tolbutamide (Sigma) was stored as a 100 mM stock in DMSO at 4 C. H-89 (Calbiochem, La Jolla, CA) was stored as a 1 mM stock in DMSO at 4 C. The catalytic subunit of PKA (cPKA) was generously provided by Dr. Michael P. Walsh (University of Calgary, Calgary, Alberta, Canada) (47) and was used at a final concentration of 20 nM. GLP-1 (7–36) and Exendin (9–39) (Sigma) were stored frozen as 100 µM stock solutions in Tris-HCl buffered solution (pH 7.4) and added to the experimental solution immediately before use.

cAMP Assay
Intracellular cAMP levels were determined in tsA201 cells that had been cultured in 35-mm plates. Cells were transfected with SUR1, Kir6.2, and the GFP-tagged GLP-1R. Transfection efficiency was 70–80% in all experiments as denoted by the number of cells fluorescing green. GLP-1 (20 nM) was added to tsA201 cells for 10 min before the assay. Cells were then washed three times in ice-cold PBS, cAMP was then extracted with 5% trichloroacetic acid and measured using an enhanced immunoassay kit as per the manufacturer’s instructions (Biomedical Technologies Inc., Stoughton, MA).

Analysis and Statistics
Recombinant single-channel current data were normalized to yield Irel where Irel is the current under test conditions relative to the maximal control current observed and was expressed as a percentage i.e. I(test)/I(control) x 100. Statistical significance was evaluated by Student’s paired t test. Differences with values of probability P < 0.05 were considered to be significant. All values in the text are given as mean ± SEM.


    ACKNOWLEDGMENTS
 
We would like to thank Lynn Eisner and Diana Steckley for their expert technical assistance.


    FOOTNOTES
 
This study was supported by grants from the Canadian Diabetes Association in honor of Violet D. Mulcahy (to P.E.L.) and the Canadian Institutes of Health Research (to M.B.W. M.O.P. 12898). P.E.L. received salary support as an Alberta Heritage Foundation for Medical Research (AHFMR) Scholar and Canadian Institutes of Health Research (CIHR) New Investigator. M.B.W. is a CIHR Investigator. J.E.M.F. is an AHFMR Postdoctoral Fellow.

Abbreviations: cPKA, Catalytic subunit of PKA; DMSO, dimethylsulfoxide; GFP, green fluorescence protein; GLP-1, glucagon-like peptide 1; GLP-1R, GLP-1 receptor; KATP, ATP-sensitive potassium; NBF, nucleotide-binding fold; NBF2, second NBF; PKA, protein kinase A; SUR1, sulphonylurea receptor; WT, wild-type.

Received for publication February 26, 2002. Accepted for publication June 3, 2002.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 GLP-1 and ß-Cell KATP...
 The Effects of PKA...
 Physiological Consequences
 MATERIALS AND METHODS
 REFERENCES
 

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