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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.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-cells (5, 6), and GLP-1 from entero-endocrine cells (7). KATP channels are composed of a homotetramer of four pore-forming Kir6.x subunits surrounded by four sulfonylurea receptor (SUR) subunits. The ß-cell KATP 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 KATP 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 KATP channel more sensitive to activation by acyl CoAs (16).
The molecular mechanism by which acyl CoAs stimulate KATP 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 KATP channel activity has been proposed (19).
The structure of acyl CoAs is related to phosphatidylinositol 4,5-bisphosphate (PIP2), another activator of KATP channels; both possess three negatively charged phosphate groups and one or two long hydrophobic acyl chains. The effects of acyl CoAs and PIP2 on KATP 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 KATP 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.
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RESULTS |
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Previous studies have suggested that KATP channels containing any one of the four mutants (R176A, R192A, R301A, or R314A) possess a reduced intrinsic open probability in the absence of ATP (PO,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 PO,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 PO,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 KATP 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.
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compares the magnitude (A) and rate (B) of activation of current by 2 µM palmitoyl CoA in these two channels in the presence and absence of Mg2+. The presence of Mg2+ had no significant effect on these parameters.
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). The time to 95% activation was also significantly longer than that of wild-type channels, taking 70.1 ± 12.2 sec (vs. 36.4 ± 5 sec for WT; Fig. 4C), again implicating decreased apparent palmitoyl CoA sensitivity of this residue.
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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 KATP 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 KATP channels by palmitoyl CoA.
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, B and C). These findings are in concordance with previous reports (22, 26). |
DISCUSSION |
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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 KATP 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 KATP 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 KATP 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 PO,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 PO,zero by noise analysis or a PIP2 method are correlated, indicating a lower intrinsic PO,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 KATP channel may show how such residues interact with acyl CoAs and lead to activation of the KATP 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.
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In conclusion, the results of this study now assign specific residues within the ß-cell KATP channel complex that underlie the molecular mechanism by which acyl CoAs activate KATP 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 KATP channel polymorphism, E23K, increases the sensitivity of KATP 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 KATP channel sensitivity to acyl CoAs may have important consequences for the coupling of metabolic stimuli to hormone secretion in endocrine tissues expressing KATP channels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (PO,zero) was then calculated using the following equation: PO,zero = I - [2/(iI)].
Structural analysis.
Models of Kir channel structure based on KirBac1.1 (30) were visualized and manipulated using WebLab ViewerLite software.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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; KATP, 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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, 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.
) or Absence (
) of Mg2+
