Evidence for a Unique Long Chain Acyl-CoA Ester Binding Site on the ATP-regulated Potassium Channel in Mouse Pancreatic Beta Cells*

(Received for publication, April 7, 1997)

Robert Bränström , Barbara E. Corkey Dagger , Per-Olof Berggren and Olof Larsson §

From The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 76 Stockholm, Sweden and Dagger  Diabetes and Metabolism Unit, Evans Department of Medicine, Boston University Medical Center, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

The mechanism by which long chain acyl-CoA (LC-CoA) esters affect the ATP-regulated potassium channel (KATP channel) was studied in inside-out patches isolated from mouse pancreatic beta cells. Addition of LC-CoA esters dramatically increased KATP channel activity. The stimulatory effect of the esters could be explained by the induction of a prolonged open state of the channel and did not involve alterations in single channel unitary conductance. Under control conditions, absence of adenine nucleotides, the distribution of KATP channel open time could be described by a single exponential, with a time constant of about 25 ms. Exposing the same patch to LC-CoA esters resulted in the appearance of an additional component with a time constant of >150 ms, indicating a conformational change of the channel protein. LC-CoA esters were also able to potently activate channel activity at different ratios of ATP/ADP. Simultaneous additions of MgADP and LC-CoA esters resulted in a supra-additive effect on channel mean open time, characterized by openings of very long duration. Following modification of the KATP channel by a short exposure of the patch to the protease trypsin, the stimulatory effect of ADP on channel activity was lost while activation by LC-CoA esters still persisted. This indicates that LC-CoA esters and MgADP do not bind to the same site. We conclude that LC-CoA esters may play an important role in the physiological regulation of the KATP channel in the pancreatic beta cell by binding to a unique site and thereby inducing repolarization of the beta cell-membrane potential.


INTRODUCTION

Potassium channels that are ATP-sensitive (KATP)1 are found in many types of cells and serve to couple metabolic state to electrical activity. In the pancreatic beta cell the KATP channel provide a critical link between changes in blood glucose concentration and insulin secretion (1, 2). The initial step in the stimulus-secretion-coupling in the beta cell is closure of the KATP channel subsequent to a rise in the ATP/ADP ratio, resulting in depolarization, activation of voltage-dependent Ca2+ channels and thereby triggering of insulin secretion (3). Stimulation of the beta cell with intermediate glucose concentrations results in a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed (4). Intracellular free Ca2+ concentration ([Ca2+]i) oscillates in synchrony with electrical activity (5). We recently showed that fluctuations in the activity of the KATP channel underlie the oscillations in electrical activity and [Ca2+]i in single pancreatic beta cells (6). A possible mechanism underlying such oscillations in KATP channel activity could be metabolism-driven oscillations in the ATP/ADP ratio (7). Because of the close relation between KATP channel activity and beta cell electrical activity, it is essential to study mechanisms which control or modulate the activity of this channel.

We have recently shown that long-term exposure to free fatty acid increases cellular levels of LC-CoA esters in the beta cell and that these esters are able to directly stimulate KATP channel activity (8). This indicates that increased steady-state content of cytosolic LC-CoA esters could affect glucose-induced closure of the KATP channel. In the present study, we have investigated in detail the mechanisms by which LC-CoA esters exert their stimulatory action and to what extent they interact with ATP and ADP in modulating KATP channel activity in the pancreatic beta cell. These findings show that LC-CoA induces a distinct open state leading to increased channel activity, characterized by openings of long duration which does not require the presence of Mg2+. Thus, binding of LC-CoA induces a conformational change of the KATP channel protein. A potent stimulatory effect of LC-CoA esters also occurred in the presence of different ratios of ATP/ADP, indicating that the esters may play an important role in modulating the channel under physiological conditions.


EXPERIMENTAL PROCEDURES

Animals and Preparation of Cells

Adult obese mice (gene ob/ob) of both sexes were obtained from a local noninbred colony (9). The mice were fasted for 24 h and then killed by decapitation. The islets of these mice contain more than 90% beta cells (10). Dispersed islets were isolated by a collagenase technique (11). Collagenase was obtained from Boehringer Mannheim GmbH, Germany. A cell suspension was prepared and washed essentially as described previously (12). The cells were resuspended in RPMI 1640 culture medium (Flow Laboratories, Scotland, UK), containing 11 mM glucose, supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 60 µg/ml gentamycin. The cell suspension was seeded into Petri dishes (Corning Glass Works, Corning, NY) and incubated at 37 °C in 5% CO2 for 1-3 days.

Solutions

The standard extracellular solution contained (in mM): 138 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, and 5 HEPES-NaOH at pH 7.4. The intracellular solution (i.e. the bath solution) consisted of (in mM): 125 KCl, 1 MgCl2, 10 EGTA, 25 KOH, and 5 HEPES-KOH at pH 7.15. ATP was added as the Mg2+ salt to the intracellular solution as shown in the text and figures. Cis-9-monounsaturated oleoyl-CoA (C18:1), myristoyl-CoA (C14:0), and malonyl-CoA (C3:0) were prepared as stock solutions with a concentration ranging from 1 to 5 mM in deionized water (Millipore) and then added to the intracellular solution with final concentrations as indicated in the figures. Nucleotides and CoA esters were all from Sigma. All reagents were of analytical grade.

Electrophysiology

LC-CoA esters reach their target site by partitioning into the lipid phase of the cell membrane. However, the CoA moiety is hydrophilic and not able to flip across the membrane (13). Like other endogenous modulators of KATP channel activity, such as adenine nucleotides, the CoA esters are not effective when applied to the extracellular face of the cell membrane (14). Therefore, excised inside-out patches from pancreatic beta cells were used to study the effects of the CoA esters on KATP channel activity (15). This type of recording mode allows free access to the cytoplasmic side of the plasma membrane, making it easy to vary the intracellular composition. Pipettes were pulled from alumina- or borosilicate glass (Hilgenberg, Malsfeld, Germany), coated with Sylgard near the tips to reduce electrical noise and then fire-polished. The electrodes had resistances between 3-5 MOmega , when filled with standard extracellular solution. Recordings were made using an Axon patch-clamp amplifier (Axopatch 200, Axon Instruments, Burlingame, CA). During the experiment the current signal was stored on magnetic tape using a VCR (Sony-200, Sony, Tokyo, Japan). Recordings of KATP channel activity were made with the membrane potential (Vm) of the patches clamped at 0 mV. With the solutions used, K+ currents are outward (i.e. into the pipette) and channel records are displayed according to the convention with upward deflections denoting outward ion current. The KATP channel activity was identified based on the unitary amplitude (1.5-2 pA) and the sensitivity to ATP. The experiments were carried out at room temperature (22-24 °C). The zero current potential of the pipette was adjusted with the electrode in the bath, before establishment of the seal. Patches were excised into nucleotide-free solution and 0.1 mM ATP was first applied to test for channel inhibition. After channel inhibition, ATP was removed and the patch was subsequently exposed to the test solutions indicated in text and figures.

Data Analysis

Records were filtered at 200 Hz (-3 db value, 8-pole Bessel filter, Frequency Devices, Haverhill, MA), digitized at 800 Hz using an Axon Instrument analogue digital converter (TL-1) and stored in a computer. For trace figures, digitized recordings were exported into CorelDraw (Corel Inc., Ontario, Canada) for final layout. Digitized segments of current records (30-40 s long) were also used to determine channel activity using in-house software. The mean current (iX) was calculated according to the equation
i<SUB>X</SUB>=<LIM><OP>∑</OP><LL>j<UP>=</UP>i</LL><UL>N</UL></LIM> (I<SUB>j</SUB>−I<SUB>B</SUB>)/N (Eq. 1)
where N is the number of samples, Ij is the current registered in sample j, and IB is the value of a user-defined base line. Unless otherwise indicated, each experimental condition was tested with identical results in at least five (usually more) different patches. The analysis of KATP channel open time was restricted to segments of the experimental records containing a maximum of three active channels. Using the method of maximum likelihood (16), the kinetic constants were derived by approximation of the data to exponential functions. Channel activities were compared using Student's t test.


RESULTS AND DISCUSSION

Recently, we discovered that a new group of substances, LC-CoA esters, can act as potent KATP channel openers and even counteract the blocking effect of ATP (8). Another important finding is that the esters appear to specifically affect the KATP channel, in that channel activity of at least two other K+ channels present in the beta cell are unaltered by the esters, namely the large conductance K+ channel (KBK) and the 8-pS K+ channel (8). However, little is known about the mechanisms underlying the stimulatory action of these esters or how LC-CoA esters interact with ADP, which also stimulates channel activity. The present study therefore focuses on effects of LC-CoA esters on single channel kinetics and on the interaction between the esters and adenine nucleotides.

Effects of LC-CoA Esters on KATP Channel Kinetics

Fig. 1, A and B, show channel activity following administration of a 3-carbon (malonyl), 14-carbon (myristoyl), and 18-carbon (oleoyl) CoA ester to inside-out patches shortly after isolation. It is clear that KATP channel activity, in the presence of myristoyl-CoA and oleoyl-CoA, was increased compared with the activity under control conditions or with malonyl-CoA containing solutions. In Fig. 1, C-F, we have quantified this effect by analyzing the distribution of channel open time during exposure of the patches to oleoyl-, myristoyl-, and malonyl-CoA. In control solution, channel activity consisted of short openings (Fig. 1, C, inset). The distribution of the openings was best described by a single exponential with a time constant (tau ) of 19.8 ms. Mean open time was estimated to be 29.3 ± 4.5 ms (n = 5). In the presence of oleoyl-CoA, there were two types of channel openings, short openings, similar to those observed under control conditions, and long openings, occasionally lasting several hundred milliseconds (Fig. 1D). The distribution of the openings was best described as the sum of two exponentials with tau  values of 49.6 and 260.4 ms, respectively. The slow component comprised 27.6% of the events and the mean open time was increased approximately 3-fold to 84.6 ± 29.0 ms (n = 5). Similar results were obtained when adding myristoyl-CoA to the patch (Fig. 1E), resulting in tau  values of 46.1 and 219.1 ms, respectively, with 19.4% belonging to the slow component. However, when applying malonyl-CoA (Fig. 1F), channel activity did not differ from control conditions with a mean open time of 34.0 ± 9.4 ms (n = 5). The distribution could be described by a single exponential with a time constant of 28.9 ms.


Fig. 1. Effects of oleoyl-, myristoyl-, and malonyl-CoA on KATP channel activity in inside-out patches. A, channel activity during perifusion with 1 µM malonyl-CoA. Malonyl-CoA was withdrawn. and after 90 s the patch was exposed to 1 µM oleoyl-CoA. This resulted in an increase in mean current from 0.2 pA to 1.2 pA. B, in the presence of 1 µM myristoyl-CoA, mean current was increased to 1.4 pA from 0.4 pA, prior to the addition of myristoyl-CoA. C-F, effects of oleoyl-, myristoyl-, and malonyl-CoA on KATP channel open time kinetics. Data are plotted as frequency versus lifetime histograms of KATP channel openings. C, during control conditions, the distribution of channel openings could be described by a single exponential function with a time constant tau  of 19.8 ms. A total number of 2507 events were analyzed from five different patches. D, in the presence of 1 µM oleoyl-CoA the channel open lifetime distribution was best fitted as the sum of a two exponential function with time constants of tau 1 = 49.6 and tau 2 = 260.4 ms, respectively. Six different patches and a total number of 4314 events were analyzed, of which 27.6% of the integrated events belonged to the slow component. The bar at the far right indicates the sum of events exceeding 1000 ms. E, when exposing patches to 1 µM myristoyl-CoA, similar results to those with oleoyl-CoA were obtained. Time constants of tau 1 = 46.1 and tau 2 = 219.1 ms were obtained with 19.4% of the total number of events (3902; n = 4) belonging to the slow component. F, distribution of open times in the presence of 1 µM malonyl-CoA was not significantly different from what was obtained during control conditions. A total of 2379 events were analyzed from three different patches, and the distribution of open times could be described by a single exponential with tau  = 28.9 ms. Insets show typical channel activity during the different experimental conditions. Arrowheads show current level when the channel is closed.
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We have previously reported comparable effects on KATP channel open time induced by ADP and diazoxide (17). Thus, ADP and diazoxide increase channel activity by promoting a similar long lasting open state. We now demonstrate that, when measured under physiological ionic conditions, LC-CoA esters also induced a conformational change of the KATP channel, leading to a prolonged open state. Another possibility could be that LC-CoA esters affect single channel unitary conductance. We therefore estimated KATP channel conductance in the absence and presence of LC-CoA esters. Under control conditions, the channel conductance was 18.0 ± 0.97 pS (n = 6). The conductance was not significantly changed in the presence of oleoyl- and myristoyl-CoA, 17.7 ± 0.73 pS (n = 7) and 18.1 ± 0.22 pS (n = 4), respectively. Exposing patches to malonyl-CoA was also without effect on channel conductance (17.9 ± 1.2 pS; n = 4). Thus, the stimulatory effect of LC-CoA esters on KATP channel mean current can not be explained by effects on single channel unitary conductance.

Do ADP and LC-CoA Bind to the Same Site?

In many respects, the LC-CoA esters seem to affect KATP channel activity in a manner similar to ADP. Thus, the ability to counteract the blocking effect of ATP, prevent channel run down (8), and increase channel open time without affecting single channel conductance are characteristics shared by the two compounds. In this context it should be noted that the CoA moity has a close structural resemblance to ADP, suggesting the possibility of competition for a common binding site. However, on a molar basis, LC-CoA esters are considerably more potent than ADP, and the esters also induce a significantly higher degree of channel stimulation. To what extent this difference between ADP and LC-CoA esters can be accounted for by an additional 3'-phosphate group on the CoA moity is not clear. Nevertheless, this may indicate that LC-CoA esters interact at a site different from that of ADP on the KATP channel complex. In Fig. 2A, perifusions with 1 µM oleoyl-CoA enhanced channel activity 5-fold. An addition of 0.1 mM MgADP, in the continuous presence of oleoyl-CoA, further increased mean current by 720 ± 220% (n = 4; p < 0.001). Noteworthy is that channel openings were characterized by long openings. As MgADP was withdrawn, the long openings of the channel disappeared. In trace B, addition of 0.1 mM MgADP led to an increase in mean current of 320 ± 100% (n = 3). Channel activity declined significantly as the MgADP concentration was increased to 0.5 mM. Inhibition of KATP channel activity at higher concentrations (>0.3 mM) of MgADP is a well documented effect (18-20). Exposing the same patch to oleoyl-CoA, in the continuous presence of MgADP, induced a dramatic augmentation in channel activity with a mean current increase of 690 ± 140% (n = 4). In C, we have quantified the effects by making amplitude-histograms under the different conditions tested in the recording of trace B. Compiled data on mean open time clearly show that a combination of MgADP and LC-CoA ester led to a supra additive effect (Fig. 3).


Fig. 2. Simultaneous presence of MgADP and oleoyl-CoA results in long channel openings. A, inside-out patch exposed to 1 µM oleoyl-CoA led to a mean current of 3.3 pA. Further addition of 0.1 mM MgADP to the patch resulted in a channel activity characterized by long openings and a mean current of 4.4 pA. B, exposure of a patch to 0.1 mM MgADP increased mean current from 0.7 to 1.7 pA. As the MgADP concentration was further increased to 0.5 mM, KATP channel mean current decreased to 0.8 pA. Adding 1 µM oleoyl-CoA to the 0.5 mM MgADP-containing solution induced a mean current of 4.9 pA. C, amplitude histograms of data shown in panel B. Currents were filtered at 0.5 kHz and sampled at 1 kHz. A total number of about 48 × 103 events were obtained for each experimental condition. C denotes closed channel, and the z axis indicates the order of changes of solutions.
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Fig. 3. The effects of ADP and LC-CoA on channel open time. Compiled data on the effects of ADP and oleoyl-CoA, alone and in combination, on KATP channel open time. Each value represents mean ± S.E. for control (n = 15), 100 µM MgADP (n = 4), 1 µM oleoyl-CoA (n = 6) and the combination of MgADP + oleoyl-CoA (n = 6). ***p < 0.001.
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The fact that a combination of ADP and LC-CoA esters activate the channel to a larger extent than administration of either of the two substances alone, suggests that they interact at distinct binding sites. In an attempt to obtain more information on this matter, we have tried modifying the KATP channel by applying a short pulse of trypsin. This approach was used earlier in studies of various types of ion channels including Na+ (21), Ca2+ (22), and K+ channels (23). Although the technique seems crude, it modifies the KATP channel in very specific ways (24). There is a resulting complete loss of the stimulatory effect of ADP as well as of the inhibitory effect of sulfonylurea on channel activity. However, inhibition of channel activity by ATP remains intact, although with slightly decreased sensitivity (24). One interpretation is that the binding sites for ADP and sulfonylurea are lost due to alterations of the channel proteins as a result of proteolytic effects of trypsin, which has a primary affinity for arginine and lysine residues (25). Interestingly, in trypsin modified patches, where ADP was totally ineffective in altering channel activity, addition of LC-CoA esters induced a pronounced increase in channel activity. LC-CoA esters were also able to potently counteract ATP-induced inhibition of channel activity in modified patches (Fig. 4). A possible explanation for these results is that trypsin alters or removes the site to which ADP binds to exert activation, whereas the site involved in LC-CoA-induced stimulation remains. These data further support the notion that LC-CoA esters interact at a unique binding site, separate from that of ADP.


Fig. 4. The effect of trypsin-induced modification of the KATP channel. Following a pulse of trypsin, the stimulatory effect of ADP is lost. In the same patch, oleoyl-CoA still exerts a pronounced stimulatory effect on KATP currents.
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To further study the interaction between LC-CoA esters and ADP, we performed a series of experiments under Mg2+-free conditions, since the ability of ADP to open channels requires Mg2+. As shown in Fig. 5A, LC-CoA activated the KATP channel in the absence of Mg2+, whereas ADP3- not only failed to activate but had an inhibitory effect on KATP channel activity (Fig. 5B) (17). This blocking effect of ADP3- is well documented and it has been proposed that ADP3- binds to the ATP site thereby explaining the inhibitory effect on the channel (20). Addition of oleoyl-CoA to the patch, in the continuous presence of ADP3-, still evoked a dramatic elevation in channel activity (Fig. 5B). Analyzing the effects of simultaneous additions of oleoyl-CoA and ADP3- in the absence of Mg2+ in five patches showed an increase of 590 ± 280% in mean currents (p < 0.01). Inclusion of Mg2+ in the perifusion medium caused the KATP channel activity to display openings of long duration. Together these observations lend strong support to the idea that simultaneous exposure of the KATP channel to a combination of LC-CoA esters and MgADP, results in a unique activity pattern with extremely long open times, not previously observed with either substance alone. All effects of the LC-CoA esters were fully reversible upon withdrawal. Due to the extreme pattern and the resulting high number of open channels in the patches, a precise determination of channel open time was not possible.


Fig. 5. Effects of Mg2+ on ADP and oleoyl-CoA induced KATP channel activity. A, in the absence of Mg2+, mean current increased 5.5-fold to 2.2 pA in the presence of 1 µM oleoyl-CoA. B, addition of ADP, in the absence of Mg2+, induced a decrease in mean current to 0.4 pA, compared with 2.0 pA obtained prior to the inclusion of ADP3-. Adding 1 µM oleoyl-CoA, in the presence of ADP3-, still increased channel activity significantly to 9.6 pA. C, oleoyl-CoA ester increased the mean current from 0.19 to 1.02 pA, in the absence of Mg2+. Inclusion of ADP in the medium did not significantly alter mean current. Inclusion of 1 mM Mg2+ increased the mean current to 1.32 pA and also dramatically increased channel open time. The vertical calibration bar represents 5 pA in A and C and 10 pA in B.
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Earlier studies suggested that the physiological regulation of the KATP channel results from changes in the ATP/ADP ratio (3), changes in ADP exerting the major influence (1). This implies that ATP-induced blockade of the channel is potently counteracted by intracellular ADP. We therefore assessed the extent to which LC-CoA esters were able to further activate channel activity in the presence of fixed ATP/ADP ratios. Fig. 6A shows the effect of 100 µM ATP and ADP on channel activity, a ratio which has been reported to give maximal stimulation of KATP currents. Subsequent addition of 1 µM oleoyl-CoA, in the continuous presence of nucleotides, resulted in an augmentation of the KATP currents. In six out of six patches we found that addition of oleoyl-CoA, in the presence of 100 µM ATP and ADP, increased mean currents significantly (380 ± 140%; p < 0.01; n = 5). Adding LC-CoA esters to 500 µM ATP and ADP also induced an increase in KATP channel activity (Fig. 6B). It should, however, be pointed out that the most dramatic effects were seen when the nucleotides and CoA ester were washed out. Thus, just after withdrawal of the substances, we repeatedly observed the same channel activity pattern as following the combination of MgADP and LC-CoA ester (see Fig. 3), characterized by channel activity with very long openings. A possible explanation for this phenomena is that ADP is washed-out more slowly than ATP (26), leaving MgADP and LC-CoA at their binding sites. Even at an ATP/ADP ratio of 10, administration of 1 µM oleoyl-CoA potently increased channel activity (Fig. 6C).


Fig. 6. Effects of oleoyl-CoA on KATP currents at different concentrations and ratios of ATP/ADP. A, the effects of 100 µM ADP and ATP, ratio 1, on channel currents. In the presence of both 1 µM oleoyl-CoA and adenine nucleotides, an increase in mean current from 1.3 to 4.6 pA was observed. B, higher concentrations of ATP/ADP (0.5 mM), still at ratio 1, resulted in a mean current of 4.9 pA, compared with 1.2 pA obtained before exposure to the nucleotides. Addition of 1 µM oleoyl-CoA resulted in a strong augmentation in mean current to 11.7 pA. C, 1 mM ATP completely blocked KATP channel currents. ADP (0.1 mM) giving an ATP/ADP ratio of 10, partially prevented the inhibitory effect of ATP and occasional openings were observed. Addition of 1 µM oleoyl-CoA, in the continuous presence of the nucleotides, potently increased KATP currents. The vertical calibration bar represents 5 pA in A and C and 10 pA in B.
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Concluding Remarks

The recent cloning of the rat sulfonylurea receptor (SUR1) (27) combined with the reconstitution of the KATP channel (28) has elegantly shown that the beta cell KATP channel is encoded by the SUR1 and an inwardly rectified K+ channel (Kir6.2) (Fig. 7) with small intrinsic activity. The SUR1 belongs to a superfamily of ATP-binding cassette proteins and comprises a 13-membrane spanning segment and two cytosolic nucleotide binding folds (NBFs) (27). It is not clear which physiological agonists interact with the two NBFs. Recent data indicate that NBF-2 is involved in ADP binding, since mutation of this site leads to a lack of stimulatory effect of ADP which is no longer able to counteract the blocking effect of ATP (29). To what extent LC-CoA esters are interacting with the NBFs is at present not known. Although it is generally believed that the main physiological activator of the KATP channel in the pancreatic beta cell is ADP, we now confirm that LC-CoA esters are potent activators of the channel (8) and that the kinetic effects on channel activity is similar to those of ADP. Furthermore, the LC-CoA esters are able to activate the channel in the absence of nucleotides, at various concentrations of ATP and ADP as well as to counteract the blocking effect of ATP (8).


Fig. 7. Schematic model of the pancreatic beta cell KATP channel, Kir6.2, and the sulfonylurea receptor proteins and their interaction with ATP, ADP, and LC-CoA esters.
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The fact that a combination of ADP and LC-CoA ester resulted in supra-additive effects on mean channel open time and that trypsin fully eliminates the stimulatory effect of ADP, under conditions where CoA esters still stimulated the channel, strongly suggest that ADP and LC-COA esters do not bind to the same site. Thus, LC-CoA esters form a class of substances which with high potency activates the beta cell KATP channel. The fact that most of our results were obtained by CoA esters derived from oleate, which is one of the predominant free fatty acid components in rodent and man plasma, supports the notion that these esters may serve the function of important modulators of beta cell electrical activity and thereby insulin release under physiological conditions.


FOOTNOTES

*   This work was supported in part by grants from the Swedish Medical Research Council (04X-09891, 03X-09890, 19X-00034), the Swedish Diabetes Association, the Nordic Insulin Foundation, funds of the Karolinska Institute, Novo Nordisk Pharma AB, Berth von Kantzows Foundation, and the United States Public Health Service grants DK35914 and DK50662 (to B. E. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: The Rolf Luft Center for Diabetes Research, Department of Molecular Medicine, L1:02, Karolinska Institute, Karolinska Hospital, S-171 76 Stockholm, Sweden. Tel.: 46-8-5177 27 44; Fax: 46-8-30 34 58; E-mail: olof.larsson{at}molmed.ki.se.
1   The abbreviations used are: KATP channel, ATP-regulated K+ channel; LC-CoA, long chain acyl coenzyme A; NBF, nucleotide binding fold.

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