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Activation of Adenosine Triphosphate-Sensitive Potassium Channels by Acyl Coenzyme A Esters Involves Multiple Phosphatidylinositol 4,5-Bisphosphate-Interacting Residues

Department of Pharmacology, University of Alberta (J.E.M.F., P.E.L.), Edmonton, Alberta, Canada T6G 2H7; and Department of Cell Biology and Physiology, Washington University School of Medicine (C.G.N.), St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. Jocelyn Manning Fox, 9-58 Medical Sciences Building, Edmonton, Alberta, Canada T6G 2H7. E-mail: jmanningfox{at}pmcol.ualberta.ca.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
ATP-sensitive potassium (K_ATP) channels are crucial to pancreatic endocrine function and their activation by acyl coenzyme A esters (acyl CoAs) may disrupt hormone secretion, contributing to the pathophysiology of type 2 diabetes. The molecular mechanism of this activation is potentially important in our further understanding of this disease. We use excised patch-clamp techniques to assess the effects of N- and C-terminal Kir6.2 mutations on the activation of recombinant K_ATP channels by palmitoyl CoA. We demonstrate that several residues previously shown to be involved in channel activation by the structurally related lipid phosphatidylinositol 4,5-bisphosphate (PIP₂) also play a role in activation by acyl CoAs, including R54, R176, R192, and R301. Mutation of these residues caused decreased open probability in the absence of ATP and slower and greater relative activation by both PIP₂ and acyl CoAs. By contrast, K185Q, which probably alters ATP binding, had no effect on either PIP₂ or palmitoyl CoA activation. These findings suggest that activation by the two classes of lipids involves multiple common residues. We use the crystal structure of a related channel, KirBac1.1, as a template to locate the residues of interest in this study within a putative three-dimensional model of Kir6.2. We propose a model in which these residues mediate both direct electrostatic interactions and allosteric modulations of open state stability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
POTASSIUM CHANNELS THAT are sensitive to intracellular ATP, known as K_ATP channels, are found in many tissues and function to couple the metabolic state of a cell to its electrical excitability and subsequent processes such as hormone secretion (1, 2, 3). They play an important role in regulating the release of insulin from ß-cells (4), glucagon from -cells (5, 6), and GLP-1 from entero-endocrine cells (7).

K_ATP channels are composed of a homotetramer of four pore-forming Kir6.x subunits surrounded by four sulfonylurea receptor (SUR) subunits. The ß-cell K_ATP channels consist of Kir6.2 subunits that are thought to interact directly with ATP to mediate inhibition by this molecule (8, 9, 10). The SUR subunits confer the sensitivity of the channel to the stimulatory effects of ADP (magnesium salt) and various pharmacological agents (10, 11, 12), and in the ß-cell are believed to be the SUR1 isoform.

Acyl coenzyme A esters (acyl CoAs) have been demonstrated to decrease the ATP sensitivity of K_ATP channels, producing a higher level of channel activity at any given ATP concentration. Such activation by acyl CoAs may contribute to the decline of insulin secretion and other endocrine dysfunction observed in type 2 diabetes, as cellular acyl CoA levels are known to be raised in obese and diabetic patients (13, 14). Indeed, common Kir6.2 polymorphisms that are thought to predispose subjects to the development of type 2 diabetes (15) render the ß-cell K_ATP channel more sensitive to activation by acyl CoAs (16).

The molecular mechanism by which acyl CoAs stimulate K_ATP channels has yet to be fully elucidated and is therefore of considerable importance in our understanding of how the metabolites of dietary fats may affect endocrine function. Current evidence suggests that the Kir6.2 subunit is sufficient for activation by long-chain acyl CoAs (17), although the presence of SUR2A enhances this effect and allows activation by shorter acyl CoAs (18, 19). A complex mechanism of action requiring both the hydrophilic CoA and hydrophobic acyl side-chain to elicit the full stimulatory effects on cardiac K_ATP channel activity has been proposed (19).

The structure of acyl CoAs is related to phosphatidylinositol 4,5-bisphosphate (PIP₂), another activator of K_ATP channels; both possess three negatively charged phosphate groups and one or two long hydrophobic acyl chains. The effects of acyl CoAs and PIP₂ on K_ATP channels also share some properties, including shifting ATP sensitivity and preventing inhibition by sulfonylureas (18). However, significant differences in the effects of the two compounds have led to the suggestion that they act via independent mechanisms (17, 18). It is only relatively recently that Rohacs et al. (20) investigated the activation of several members of the inwardly rectifying potassium channel (Kir) family by oleoyl CoA and phosphoinositides and concluded that PIP₂ and oleoyl CoA exert their effects on Kir6.2 channels through a common mechanism. PIP₂ has been shown to interact directly with the C terminus of Kir6.2 (21), and several positively charged residues have been demonstrated to play a role in activation of the channel by PIP₂ (22). One of these residues has also recently been shown to play a role in activation of K_ATP channels by oleoyl CoA (23). Similarly, a Kir6.2 N-terminal residue, R54, has been suggested to be involved in both PIP₂ and oleoyl CoA activation (23, 24).

It was the aim of this study to further identify the molecular mechanism by which acyl CoAs activate Kir6.2 channels. Using site-directed mutagenesis of multiple candidate residues, depicted in Fig. 1, we show that several intracellular residues are important in mediating the effects of acyl CoAs, and that the mechanism of action is probably similar to that of PIP₂.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Schematic Representation of Kir6.2 Residues Mutated in this Study , Residues known to be involved in channel activation by PIP2; , residue known to be involved in ATP sensitivity but not important for activation by PIP2.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

Previous studies have suggested that K_ATP channels containing any one of the four mutants (R176A, R192A, R301A, or R314A) possess a reduced intrinsic open probability in the absence of ATP (P_O,zero) due to their decreased sensitivity to ambient PIP₂ (and possibly also acyl CoA) levels in the membrane. We used stationary fluctuation analysis of macroscopic currents to assess P_O,zero for channels generated by each mutant. It is apparent that the open probability of each mutant channel is indeed significantly lower than that of the WT channel. Apparent P_O,zero values calculated were: WT, 0.93 ± 0.03; R176A, 0.54 ± 0.05; R192A, 0.81 ± 0.04; R301A, 0.77 ± 0.03; and R314A, 0.78 ± 0.04.

In each case, 2 µM palmitoyl CoA was coapplied to the patch with 0.1 mM ATP. The presence of ATP has been demonstrated to maintain the phosphorylation of membrane lipid phosphatidylinositol by phosphoinositol kinases, thus preventing the depletion of PIPs and the rundown of current that are commonly observed in the absence of ATP (25). Indeed, no rundown of K_ATP current was observed over the time course of our experiments (typically 3–5 min). The addition of palmitoyl CoA led to a significant increase in the level of activation of all channels, as shown by the representative traces in Fig. 2A. In each case the increase in current was significantly greater than that of the WT channel (R176A, 911 ± 262%; R192A, 316 ± 41%; R301A, 434 ± 62%; R314A, 727 ± 128%; WT, 150 ± 11%; Fig. 2B). For three of the four residues (R176A, R192A, and R301A), the time taken to produce 95% activation was also significantly increased (R176A, 71 ± 10 sec; R192A, 48 ± 3 sec; R301A, 63 ± 6 sec; WT, 36 ± 5 sec; Fig. 2C), again taken to indicate a lower apparent affinity (22). These findings suggest that R176, R192, and R301 are all involved in the mechanism of activation of Kir6.2 channels by palmitoyl CoA.

View larger version (29K): [in this window] [in a new window]   Fig. 2. The Effects of 2 µM Palmitoyl CoA on Current from WT (SUR1/Kir6.2) and Four Kir6.2 C-Terminal Mutant Channels Representative traces of the effect of 2 µM palmitoyl CoA on SUR1/Kir6.2 (Ai) and SUR1/Kir6.2,R176A (Aii) channels. Grouped data show the percent increase in current (B) and the time to 95% activation of that current (C) evoked by 2 µM palmitoyl CoA in WT and mutant channels. *, P < 0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 3. The Effects of 2 µM Palmitoyl CoA on Current from WT and SUR1/Kir6.2R176A Channels in the Presence () or Absence () of Mg2+ Neither the level of current activation (A) nor the time to 95% current activation (B) was significantly altered by the change in magnesium concentration.

View larger version (26K): [in this window] [in a new window]   Fig. 4. The Effects of 2 µM Palmitoyl CoA on Current from SUR1/Kir6.2,R54E Channels A, Representative trace of the effect of 2 µM palmitoyl CoA on SUR1/Kir6.2,R54E channels. Grouped data show the percent increase in current (B) and the time to 95% activation of that current (C) evoked by 2 µM palmitoyl CoA in mutant channels. *, P < 0.05.

In the absence of ATP, 2 µM palmitoyl CoA increased current levels in R176A and R54E channels to a greater extent than in wild-type channels (Fig. 5, A and C). The time courses of activation could not be compared due to the lack of apparent effect of palmitoyl CoA on WT current in the absence of ATP. However, repeated applications of 1 mM ATP during these experiments demonstrated that palmitoyl CoA was decreasing the ATP sensitivity of WT channels, and that the activating effect was not apparent over a background of rundown in the absence of ATP (Fig. 5Ai). The data are consistent with the results acquired in the presence 0.1 mM ATP, suggesting that R176 and R54 are indeed involved in the activation of K_ATP channels by palmitoyl CoA. The level of activation of K185Q channels produced by 2 µM palmitoyl CoA showed a slight increase in some cases, but this did not reach statistical significance compared with wild-type channels (Fig. 5, Aiv and C). This suggests that K185 is not involved in the activation of K_ATP channels by palmitoyl CoA.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Effects of Palmitoyl CoA and PIP2 on KATP Channels in the Absence of Magnesium and ATP A, Representative traces of the effect of 2 µM palmitoyl CoA on WT and mutant SUR1/Kir6.2 channels. B, Representative traces of the effect of 5 µg/ml PIP2 on WT and mutant SUR1/Kir6.2 channels. C, Grouped data show the percent increase in current evoked by 2 µM palmitoyl CoA or 5 µg/ml PIP2 in WT and mutant channels. *, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Our data demonstrate that four Kir6.2 mutations known to be involved in PIP₂ activation (R54E, R176A, R192A, and R301A) cause an increase in the time course and extent of activation by palmitoyl CoA. This represents a decreased sensitivity of these channels to acyl CoAs. It instinctively seems counterintuitive to view a relative increase in current as indicative of reduced sensitivity to acyl CoAs. However, it is important to recognize that these values represent a percent change in current. A higher percent increase in current is taken to indicate a lower intrinsic open probability (i.e. in the presence of endogenous phospholipids) and thus a decreased apparent palmitoyl CoA sensitivity. Such decreased intrinsic open probability has previously been largely assumed. We now provide definitive measurements of P_O,zero for these mutants and show that each one is reduced, confirming the validity of the above interpretation of the data.

The finding that acyl CoA activation appears to involve at least four of the same residues that are required for activation by PIP₂ sheds new light on a question that has been under debate recently: whether PIP₂ and acyl CoAs share a common mechanism of activation of K_ATP channels. Similarities and differences in the activating properties of these lipids have been previously described (9, 18), and these researchers, as well as others (17), have concluded that long-chain acyl CoA esters and PIP₂ modulate K_ATP channels via different mechanisms. However, more recently, Rohacs et al. (20) reported that the specificity of activation of Kir channels by phosphoinositides also determines their activation by acyl CoAs, implicating at least a common final action.

In our study we investigated a number of candidate residues and demonstrate that several of those implicated in the binding or transduction of PIP₂ to activation are also involved in activation by acyl CoAs. These findings extend the results reported by Schulze et al. (23), who very recently used a different methodology to indicate involvement of two residues (R54 and R176) in channel activation by 20 µM oleoyl CoA. Here we report novel findings of two additional residues, R192 and R301, that are important for activation of channels by acyl CoAs. Previous studies of the activation of channels by PIP₂ and acyl CoAs have employed different experimental systems and methodologies. We clarify the interpretation of these findings by demonstrating that R54E and R176A have similar effects on activation by either palmitoyl CoA or PIP₂ when directly compared in the same experimental system. K185Q, on the other hand, has been reported to have no effect on PIP₂ interaction despite its involvement in ATP sensitivity and location within the same region as the PIP₂-modulating residues (22). We now show that this residue does not affect palmitoyl CoA activation, lending further credence to the suggestion that PIP₂ and palmitoyl CoA activation of K_ATP channels involves interaction with a binding region involving specific positively charged residues rather than a generalized electrostatic attraction to any positively charged residue,

The apparent P_O,zero value for WT SUR1/Kir6.2 channels obtained from noise analysis is larger than previously reported (0.93 vs. 0.2–0.6) (17, 27, 28), and this is consistent with the lack of overt stimulation by PIP₂ and acyl CoA in the absence of ATP. A possible explanation is that the endogenous levels of activating lipids in tsA201 cells may be higher than those in other commonly used oocyte or mammalian expression systems. As we previously demonstrated (29), estimates of P_O,zero by noise analysis or a PIP₂ method are correlated, indicating a lower intrinsic P_O,zero for each of the PIP₂-dependent mutants examined above.

Knowledge of where the residues identified in this study lie within the three-dimensional structure of the K_ATP channel may show how such residues interact with acyl CoAs and lead to activation of the K_ATP channel complex. Although a high resolution structure of the Kir6.2 channel has yet to be obtained, the recently published high resolution structures of the cytoplasmic termini of the G protein-coupled, inwardly rectifying potassium channel (GIRK1, Kir3.1) and the bacterial inward rectifier channel, KirBac1.1, are of relevant use (30, 31). The KirBac1.1 channel has approximately 50% sequence similarity to Kir6.2, and the inclusion of a more complete N terminus than that of GIRK1 prompts the use of this structure as a template for the N- and C-terminal structural interactions in the Kir6.2 channel.

Figure 6A shows the KirBac1.1 structure (30) as a ribbon diagram with opposite subunits depicted. Labeled on the model are the likely corresponding residues to those considered in the present study, many of which appear to lie on the outer surface of the cytoplasmic domain. Also shown is residue F168, suggested by Doyle and colleagues (30) to be important in channel gating. Of particular interest are the equivalents of C-terminal residues R176 and R301, which lie in close proximity to the N-terminal R54 residue from the adjacent subunit (Fig. 6B). These three residues, when mutated to alanine, were the most effective in modulating activation by acyl CoA. It is plausible that these form a positively charged pocket that interacts directly with the negatively charged acyl CoA. It is interesting to note that this putative binding pocket is in close proximity to the plasma membrane. Although the charged residues may interact with the CoA headgroup, it is possible to imagine how the acyl side-chain may be incorporated into the cell membrane. Figure 7 depicts how this tethering to the membrane may produce a conformational change in the channel, opening the channel at the bundle crossing of the M2 helixes (32, 33). Such a mechanism may also explain the differences in potency of different side-chain length acyl CoAs in activating the channel (19), as shorter acyl chains may be less effective in reaching and interacting with the plasma membrane.

View larger version (41K): [in this window] [in a new window]   Fig. 6. Locations of Important Residues A, Side view of the structure of opposite subunits of KirBac1.1 indicating the locations of residues within the protein corresponding to those examined in this study. B, Close up view of adjacent subunits of KirBac1.1 indicating the proximity of the N and C termini of adjacent residues.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Schematic Diagram Schematic diagram of the proposed mechanism by which binding of acyl CoAs may tether the cytoplasmic region of the channel to the plasma membrane, causing a conformational change in the channel and opening the gating region.

In conclusion, the results of this study now assign specific residues within the ß-cell K_ATP channel complex that underlie the molecular mechanism by which acyl CoAs activate K_ATP channels, including two novel residues, R192 and R301. Our construction of a three-dimensional model of the Kir6.2 channel provides visualization of likely residue locations, which may promote our understanding of the mechanism by which acyl CoAs and PIP₂ modulate channel activity. From a pathophysiological perspective, identification of a positively charged N-terminal residue, R54, is in accordance with our previous work indicating that a commonly found N-terminal K_ATP channel polymorphism, E23K, increases the sensitivity of K_ATP channels to acyl CoAs (16). The presence of the E23K polymorphism has been shown to predispose Caucasians to type 2 diabetes (15). Therefore, it is likely that alterations in K_ATP channel sensitivity to acyl CoAs may have important consequences for the coupling of metabolic stimuli to hormone secretion in endocrine tissues expressing K_ATP channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Mutagenesis
The Kir6.2,K185Q and Kir6.2,R54E mutants were created using the QuikChange site-directed mutagenesis protocol (Stratagene, La Jolla, CA) with pCDNA3Kir6.2 as the template. Other constructs containing point mutations were prepared by overlap extension at the junctions of relevant residues by sequential PCR and subsequent cloning into pCMV6b. Before transfection, all constructs were sequenced to verify the correct mutation.

Cell Culture
TsA201 and COS-1 cells were maintained in DMEM supplemented with 2 mM L-glutamine, 10% fetal calf serum, and 0.1% penicillin/streptomycin at 37 C (10% CO₂). Cells were plated at 50–70% confluence on 35-mm culture dishes 6 h before transfection. The hamster SUR1 subunit was donated by Drs. L. Aguilar-Bryan and J. Bryan (35) (Baylor College of Medicine, Houston, TX). The SUR clone was inserted into the mammalian expression vector pCDNA3.1 and was cotransfected with a vector encoding the green fluorescent protein (pGL, Life Technologies, Inc., Gaithersburg, MD) and the relevant Kir clone into tsA201 cells using the calcium phosphate precipitation method. Recordings were made from cells 48–72 h after transfection.

Electrophysiology
Standard patch-clamp techniques were used to record single-channel currents in the inside-out configuration, so that the internal face of membrane patches could be exposed directly to test solutions using a multi-input perfusion pipette. The time required for solution change at the tip of the recording pipette was less than 1 sec. Single-channel currents were recorded at a holding potential of -60 mV, amplified (Axopatch 200B, Axon Instruments, Foster City, CA), and then digitized and analyzed using Axoscope version 8.0 and pClamp 8.0 software. Data were sampled at 2.5 kHz and filtered at 1 kHz.

The pipette/bath solution used for excised patch recordings contained the following: 110 mM KCl, 30 mM KOH, 10 mM EGTA, 5 mM HEPES, and 1 mM MgCl₂. The pH of the solution was adjusted to 7.4 using HCl. Experiments performed under Mg²⁺-free conditions used the following pipette/bath solution: 107 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 10 mM EDTA, and 10 mM HEPES, pH 7.4, with KOH (600 nM free-Mg²⁺).

Experimental Compounds
ATP (as MgATP or K₂ATP where indicated; Sigma-Aldrich Corp., St. Louis, MO) was added as required from a 10-mM stock, which was prepared immediately before use. Palmitoyl CoA was obtained from Sigma-Aldrich Corp. and stored as a 5-mM stock in water at -20 C before use. PIP₂ was purchased from Calbiochem (Mississauga, Canada), used at a final concentration of 5 µg/ml (5 µM) in Mg²⁺-free solution and bath-sonicated on ice for 15 min before use.

Data Analysis
Statistics.
Statistical significance was evaluated using unpaired t test. Differences with P < 0.05 were considered significant. All values in the text are the mean ± SEM (n = 6–14 in each case).

Signal-noise analysis.
Single-channel properties can be estimated by analyzing the signal to noise ratio of current recordings (36). Mean channel current (I) and variance (²) in the absence of ATP were obtained by subtraction of mean patch current and variance in 1 mM ATP (all channels assumed closed) from patch current and variance calculated in zero ATP. Single-channel current (i) was assumed to be constant at 3.27 pA, a value measured for SUR1/Kir6.2 channels in single-channel recordings at -60 mV. The mean peak open probability in the absence of ATP (P_O,zero) was then calculated using the following equation: P_O,zero = I - [²/(iI)].

Structural analysis.
Models of Kir channel structure based on KirBac1.1 (30) were visualized and manipulated using WebLab ViewerLite software.

	ACKNOWLEDGMENTS

 
We thank Dr. Declan Doyle for providing the Kirbac1.1 coordinates ahead of publication, Diana Steckley and Lynn Jones for their expert technical assistance, and Daniel Brewster for his assistance with the computer modeling.

	FOOTNOTES

 
This work was supported by an operating grant from the Canadian Diabetes Association in honor of Gordon M. Stevenson.

J.E.M.F. is an Alberta Heritage Foundation for Medical Research Postdoctoral Fellow.

P.E.L. is a Canadian Institutes of Health Research New Investigator and Alberta Heritage Foundation for Medical Research Scholar.

Abbreviations: CoA, Coenyzme A; K_ATP, ATP-sensitive potassium; Kir, inwardly rectifying potassium channel; PIP₂, phosphatidylinositol 4,5-bisphosphate; SUR, sulfonylurea receptor; WT, wild-type.

Received for publication November 5, 2003. Accepted for publication December 16, 2003.

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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