Biomedical subjects

F M Ashcroft

Publications and source records attributed to F M Ashcroft.

At least 37 records · Page 2Linked to original sources

PubMed2000

A novel method for measurement of submembrane ATP concentration.

There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca(2+), capable of resolving the submembrane ATP concentration ([ATP](sm)) in real time within a single cell. We show here that mutant ATP-sensitive K(+) channels can be used to measure [ATP](sm) by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP](sm) was 4.6 +/- 0.3 mm (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mm). In mammalian (COSm6) cells, [ATP](sm) was slightly lower and averaged 1.4 +/- 0.1 mm (n = 66). Metabolic poisoning (10 min of 3 mm azide) produced a significant fall in [ATP](sm) in both types of cells: to 1.2 +/- 0.1 mm (n = 24) in oocytes and 0.8 +/- 0.11 mm for COSm6 cells. We conclude that [ATP](sm) lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].

Adenosine Triphosphate↗

PubMed2000

Direct photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-[gamma]4-azidoanilide.

ATP-sensitive potassium (K(ATP)) channels are under complex regulation by intracellular ATP and ADP. The potentiatory effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We have previously reported that Kir6.2 can be directly labeled by 8-azido-[gamma-(32)P]ATP. However, the binding affinity of 8-azido-ATP to Kir6.2 was low probably due to modification at 8' position of adenine. Here we demonstrate that Kir6.2 can be directly photoaffinity labeled with higher affinity by [gamma-(32)P]ATP-[gamma]4-azidoanilide ([gamma-(32)P]ATP-AA), containing an unmodified adenine ring. Photoaffinity labeling of Kir6.2 by [gamma-(32)P]ATP-AA is not affected by the presence of Mg(2+), consistent with Mg(2+)-independent ATP inhibition of K(ATP) channels. Interestingly, SUR1, which can be strongly and specifically photoaffinity labeled by 8-azido-ATP, was not photoaffinity labeled by ATP-AA. These results identify key differences in the structure of the nucleotide binding sites on SUR1 and Kir6.2.

ATP-Binding Cassette Transporters↗

PubMed2000

New windows on the mechanism of action of K(ATP) channel openers.

K(ATP) channel openers are a diverse group of drugs with a wide range of potential therapeutic uses. Their molecular targets, the K(ATP) channels, exhibit tissue-specific responses because they possess different types of regulatory sulfonylurea receptor subunits. It is well recognized that complex interactions occur between K(ATP) channel openers and nucleotides, but the cloning of the K(ATP) channel has introduced a new dimension to the study of these events and has furthered our understanding of the molecular basis of the action of K(ATP) channel openers.

Adenosine Triphosphate↗

PubMed2000

The antimalarial agent mefloquine inhibits ATP-sensitive K-channels.

The aim of this study was to determine whether antimalarial agents inhibit ATP-sensitive potassium (K(ATP)) channels and thereby contribute to the observed side-effects of these drugs. Mefloquine (10 - 100 microM), but not artenusate (100 microM), stimulated insulin release from pancreatic islets in vitro. Macroscopic K(ATP) currents were studied in inside-out patches excised from Xenopus oocytes expressing cloned K(ATP) channels. Mefloquine (IC(50) approximately 3 microM), quinine (IC(50) approximately 3 microM), and chloroquine inhibited the pancreatic beta-cell type of K(ATP) channel Kir6.2/SUR1. Artenusate (100 microM) was without effect. Mefloquine and quinine also blocked a truncated form of Kir6.2 (Kir6. 2DeltaC36) when expressed in the absence of SUR1. The extent of block was similar to that observed for Kir6.2/SUR1 currents. Our results suggest that inhibition of the beta-cell K(ATP) channel accounts for the ability of quinoline-based antimalarial drugs to stimulate insulin secretion, and thereby produce hypoglycaemia. The results also indicate that quinoline-based antimalarial agents inhibit K(ATP) channels by interaction with the Kir6.2 subunit. This subunit is common to beta-cell, neuronal, cardiac, skeletal muscle, and some smooth muscle K(ATP) channels suggesting that K(ATP) channel inhibition may contribute to the other side effects of these drugs, which include cardiac conduction abnormalities and neuropsychiatric disturbances.

Adenosine Triphosphate↗

PubMed2000

Direct interaction of Na-azide with the KATP channel.

1. The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. 2. In inside-out patches from oocytes expressing Kir6.2 delta C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. 3. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2 delta C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. 4. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. 5. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration.

Adenosine Triphosphate↗

PubMed2000

Differential response of K(ATP) channels containing SUR2A or SUR2B subunits to nucleotides and pinacidil.

ATP-sensitive K-channels (K(ATP) channels) are the target for K(ATP)-channel openers (KCOs), such as pinacidil and P1075. These channels are formed from pore-forming Kir6.2 and regulatory sulfonylurea receptors (SUR2A in heart and skeletal muscle; SUR2B in smooth muscle). The two isoforms of SUR2 differ only in their final 42 amino acids, a region that includes neither the Walker A and B nucleotide binding motifs nor the proposed KCO binding site, yet channels containing SUR2A or SUR2B respond differently to both nucleotides and KCOs. We explored the basis for this difference by expressing Kir6.2/SUR2A and Kir6.2/SUR2B currents in Xenopus laevis oocytes. Kir6.2/SUR2B but not Kir6.2/SUR2A currents were activated by the Mg-nucleoside triphosphates MgATP and MgGTP, whereas both channel types responded to the diphosphates MgADP and MgGDP. This activation of Kir6.2/SUR2B currents by MgATP explains how the ATP concentration-response curve is shifted to the right in the presence of Mg(2+). In the absence of nucleotide, pinacidil and P1075 activated Kir6.2/SUR2B and Kir6.2/SUR2A currents, but the presence of nucleotide slowed the drug off-rates. In the presence of MgATP, the response to pinacidil reversed approximately 14 times more slowly with SUR2B than SUR2A. The EC(50) for ATP, measured by its ability to slow the pinacidil off-rate, was also approximately 20 times higher for channels containing SUR2A than SUR2B. Our findings suggest that nucleotide binding and/or hydrolysis is enhanced in SUR2B compared with SUR2A, and that the greater KCO-affinities of SUR2B compared with SUR2A may be a consequence of this altered nucleotide handling.

Adenosine Triphosphate↗

PubMed2000

Nucleotide modulation of pinacidil stimulation of the cloned K(ATP) channel Kir6.2/SUR2A.

ATP-sensitive K(+) channels are the target for K(+) channel openers such as pinacidil. These channels are formed from pore-forming Kir6. 2 and regulatory sulfonylurea receptor (SUR) subunits. Pinacidil activates channels containing SUR2A (heart, skeletal muscle), but not those containing SUR1 (beta cells). Surprisingly, binding of the pinacidil analog [(3)H]P1075 is dependent on added nucleotides, yet in electrophysiological studies, pinacidil is effective in the absence of intracellular nucleotides. To determine the reason for this anomaly, we examined the functional interactions between pinacidil (or P1075) and nucleotides by expressing cloned Kir6. 2/SUR2A channels in Xenopus laevis oocytes. Both pinacidil and P1075 activated macroscopic Kir6.2/SUR2A currents in the absence of added nucleotide, but the presence of intracellular ATP or ADP slowed the off-rate of the response. Mutation of the Walker A lysine in a single nucleotide binding domain (NBD) of SUR2A (K707A in NBD1, K1348A in NBD2), abolished this action of nucleotide. The K1348A mutation prevented stimulation by MgADP but had little effect on the amplitude of the pinacidil response. In contrast, Kir6.2/SUR2A-K707A currents were activated by MgADP, but only responded to pinacidil in the presence of Mg-nucleotide. Off-rates in the absence (or presence) of nucleotide were slower for the pinacidil analog P1075 than for pinacidil, consistent with the higher affinity of P1075. We suggest that slowing of P1075 dissociation by nucleotide enables binding to be detected.

Adenosine Diphosphate↗

PubMed2000

Sulfonylurea sensitivity of adenosine triphosphate-sensitive potassium channels from beta cells and extrapancreatic tissues.

Sulfonylureas are widely used to stimulate insulin secretion in type 2 diabetic patients because they close adenosine triphosphate-sensitive potassium (K(ATP)) channels in the pancreatic beta-cell membrane. This action is mediated by binding of the drug to the sulfonylurea receptor (SUR1) subunit of the channel. K(ATP) channels are also present in a range of extrapancreatic tissues, but many of these contain an alternative type of SUR subunit (SUR2A in heart and SUR2B in smooth muscle). The sulfonylurea-sensitivity of K(ATP) channels containing the different types of SUR is variable: gliclazide and tolbutamide block the beta cell, but not the cardiac or smooth muscle types of K(ATP) channels with high affinity. Glibenclamide and glimepiride, on the other hand, block channels containing SUR1 and SUR2 with similar affinity. The reversibility of the different sulfonylureas also varies. Tolbutamide and gliclazide produce a reversible inhibition of Kir6.2/SUR1 and Kir6.2/SUR2 channels, whereas glibenclamide has a reversible effect on cardiac, but not beta-cell, K(ATP) channels. In this article, we summarize current knowledge of how sulfonylureas act on K(ATP) channels containing the different types of sulfonylurea receptor, and discuss the implications of these findings for the use of sulfonylureas in the treatment of diabetes mellitus.

Adenosine Triphosphate↗

PubMed1999

Quantal analysis of 5-hydroxytryptamine release from mouse pancreatic beta-cells.

1. A combination of patch-clamp, amperometric and fluorimetric methods were used to investigate the Ca2+ dependence and kinetics of secretion from pancreatic beta-cells elicited by voltage-gated Ca2+ entry. 2. Whether measured by the change in cell capacitance or by amperometric detection of 5-hydroxytryptamine (5-HT) release, the voltage dependence of the amount of secretion mirrored that of both the peak Ca2+ current and Ca2+ entry. 3. The magnitude of secretion elicited by a single pulse could be entirely accounted for by a readily releasable pool of approximately 200 vesicles. Neither depression nor potentiation of release was observed with 0.1 Hz pulse trains. 4. Transient amperometric currents were detected, which occurred independently of each other and were attributed to the fusion of single vesicles. 5. The time course of the macroscopic amperometric current could be accurately reconstructed by convolution of the all-events latency distribution and the unitary amperometric current. 6. In response to membrane depolarisation, secretion was initiated with a variable latency: approximately 95 % of the first secretory events occurred at least 50 ms after the start of the voltage pulse (and Ca2+ influx). Secretion fell rapidly on membrane repolarisation, even though the average intracellular calcium concentration ([Ca2+]i) was still elevated. 7. The [Ca2+] in the locality of the release site was estimated from the all-events latency distribution. [Ca2+] rose during a voltage pulse and secretion was elicited at > 0.4 microM and peaked at approximately 2-10 microM.

Algorithms↗

PubMed1999

Altered functional properties of KATP channel conferred by a novel splice variant of SUR1.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming (Kir6.x) and regulatory sulphonylurea receptor (SURx) subunits. We have isolated a novel SUR variant (SUR1bDelta33) from a hypothalamic cDNA library. This variant lacked exon 33 and introduced a frameshift that produced a truncated protein lacking the second nucleotide binding domain (NBD2). It was expressed at low levels in hypothalamus, midbrain, heart and the insulin-secreting beta-cell line MIN6. 2. We examined the properties of KATP channels composed of Kir6.2 and SUR1bDelta33 by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing these subunits. We also investigated the effect of truncating SUR1 at either the start (SUR1bT1) or end (SUR1bT2) of exon 33 on KATP channel properties. 3. Kir6.2/SUR1bDelta33 showed an enhanced open probability (Po = 0.6 at -60 mV) and a reduced ATP sensitivity (Ki, 86 microM), when compared with wild-type channels (Po = 0.3; Ki, 22 microM). However, Kir6.2/SUR1bT1 and Kir6.2/SUR1bT2 resembled the wild-type channel in their Po and ATP sensitivity. 4. Neither MgADP, nor the K+ channel opener diazoxide, enhanced Kir6.2/SUR1bDelta33, Kir6.2/SUR1bT1 or Kir6.2/SUR1bT2 currents, consistent with the idea that these agents require an intact NBD2 for their action. Sulphonylureas blocked KATP channels containing any of the three SUR variants, but in excised patches the extent of block was less than that for the wild-type channel. In intact cells, the extent of sulphonylurea block of Kir6.2/SUR1bDelta33 was greater than that in excised patches and was comparable to that found for wild-type channels. 5. Our results demonstrate that NBD2 is not essential for functional expression or sulphonylurea block, but is required for KATP channel activation by K+ channel openers and nucleotides. Some of the unusual properties of Kir6.2/SUR1bDelta33 resemble those reported for the KATP channel of ventromedial hypothalamic (VMH) neurones, but the fact that this mRNA is expressed at low levels in many other tissues makes it less likely that SUR1bDelta33 serves as the SUR subunit for the VMH KATP channel.

ATP-Binding Cassette Transporters↗

PubMed1999

Mapping of the physical interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2.

The amino-terminal and carboxyl-terminal domains of inwardly rectifying potassium (Kir) channel subunits are both intracellular. There is increasing evidence that both of these domains are required for the regulation of Kir channels by agents such as G-proteins and nucleotides. Kir6.2 is the pore-forming subunit of the ATP-sensitive K(+) (K(ATP)) channel. Using an in vitro protein-protein interaction assay, we demonstrate that the two intracellular domains of Kir6.2 physically interact with each other, and we map a region within the N terminus that is responsible for this interaction. "Cross-talk" through this interaction may explain how mutations in either the N or C terminus can influence the intrinsic ATP-sensitivity of Kir6.2. Interestingly, the "interaction domain" is highly conserved throughout the superfamily of Kir channels. The N-terminal interaction domain of Kir6.2 can also interact with the C terminus of both Kir6.1 and Kir2.1. Furthermore, a mutation within the conserved region of the N-terminal interaction domain, which disrupts its interaction with the C terminus, severely compromised the ability of both Kir6.2 and Kir2.1 to form functional channels, suggesting that this interaction may be a feature common to all members of the Kir family of potassium channels.

Amino Acid Sequence↗

PubMed1999

The role of lysine 185 in the kir6.2 subunit of the ATP-sensitive channel in channel inhibition by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, forms ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on Kir6.2. 2. Previous studies have shown that mutation of the lysine residue at position 185 (K185) in the C-terminus of Kir6.2 to glutamine, decreased the channel sensitivity to ATP without affecting the single-channel conductance or the intrinsic channel kinetics. This mutation also impaired 8-azido[32P]-ATP binding to Kir6.2. 3. To determine if K185 interacts directly with ATP, we made a range of mutations at this position, and examined the effect on the channel ATP sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 4. Substitution of K185 by a positively charged amino acid (arginine) had no substantial effect on the sensitivity of the channel to ATP. Mutation to a negatively charged residue markedly decreased the channel ATP sensitivity: the Ki for ATP inhibition increased from 85 microM to >30 mM when arginine was replaced with aspartic acid. Substitution of neutral residues had intermediate effects. 5. The inhibitory effects of ADP, ITP and GTP were also reduced when K185 was mutated to glutamine or glutamate. 6. The results indicate that a positively charged amino acid at position 185 is required for high-affinity ATP binding to Kir6.2. Our results demonstrate that ATP does not interact with the side-chain of K185. It remains unclear whether ATP interacts with the backbone of this residue, or whether its mutation influences ATP binding allosterically.

Adenosine Triphosphate↗

PubMed1999

NEM modification prevents high-affinity ATP binding to the first nucleotide binding fold of the sulphonylurea receptor, SUR1.

Pancreatic beta-cell ATP-sensitive potassium channels, composed of SUR1 and Kir6.2 subunits, serve as a sensor for intracellular nucleotides and regulate glucose-induced insulin secretion. To learn more about the interaction of SUR1 with nucleotides, we examined the effect of N-ethylmaleimide (NEM) modification. Photoaffinity labeling of SUR1 with 5 microM 8-azido-[alpha-32P]ATP or 8-azido-[gamma-32P]ATP was inhibited by NEM with Ki of 1.8 microM and 2.4 microM, and Hill coefficients of 0.94 and 1.1, respectively. However, when the cysteine residue in the Walker A motif of the first nucleotide binding fold (NBF1) of SUR1 was replaced with serine (C717S), photoaffinity labeling was not inhibited by 100 microM NEM. These results suggest that NBF1 of SUR1 has a NEM-sensitive structure similar to that of NBF1 of MDR1, a multidrug transporter, and confirm NBF1 as the high-affinity ATP binding site on SUR1.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

Interaction of vanadate with the cloned beta cell K(ATP) channel.

Vanadate is used as a tool to trap magnesium nucleotides in the catalytic site of ATPases. However, it has also been reported to activate ATP-sensitive potassium (K(ATP)) channels in the absence of nucleotides. K(ATP) channels comprise Kir6.2 and sulfonylurea receptor subunits (SUR1 in pancreatic beta cells, SUR2A in cardiac and skeletal muscle, and SUR2B in smooth muscle). We explored the effect of vanadate (2 mM), in the absence and presence of magnesium nucleotides, on different types of cloned K(ATP) channels expressed in Xenopus oocytes. Currents were recorded from inside-out patches. Vanadate inhibited Kir6.2/SUR1 currents by approximately 50% but rapidly activated Kir6.2/SUR2A ( approximately 4-fold) and Kir6. 2/SUR2B ( approximately 2-fold) currents. Mutations in SUR that abolish channel activation by magnesium nucleotides did not prevent the effects of vanadate. Studies with chimeric SUR indicate that the first six transmembrane domains account for the difference in both the kinetics and the vanadate response of Kir6.2/SUR1 and Kir6. 2/SUR2A. Boiling the vanadate solution, which removes the decavanadate polymers, largely abolished both stimulatory and inhibitory actions of vanadate. Our results demonstrate that decavanadate modulates K(ATP) channel activity via the SUR subunit, that this modulation varies with the type of SUR, that it differs from that produced by magnesium nucleotides, and that it involves transmembrane domains 1-6 of SUR.

Adenosine Triphosphate↗

PubMed1999

Involvement of the n-terminus of Kir6.2 in coupling to the sulphonylurea receptor.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. ATP inhibits the channel by interacting with Kir6.2, while sulphonylureas block channel activity by interaction with a high-affinity site on SUR1 and a low-affinity site on Kir6.2. MgADP and diazoxide interact with SUR1 to promote channel activity. 2. We examined the effect of N-terminal deletions of Kir6.2 on the channel open probability, ATP sensitivity and sulphonylurea sensitivity by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2/SUR1. 3. A 14 amino acid N-terminal deletion (DeltaN14) did not affect the gating, ATP sensitivity or tolbutamide block of a truncated isoform of Kir6.2, Kir6.2DeltaC26, expressed in the absence of SUR1. Thus, the N-terminal deletion does not alter the intrinsic properties of Kir6.2. 4. When Kir6.2DeltaN14 was coexpressed with SUR1, the resulting KATP channels had a higher open probability (Po = 0.7) and a lower ATP sensitivity (Ki = 196 microM) than wild-type (Kir6.2/SUR1) channels (Po = 0.32, Ki = 28 microM). High-affinity tolbutamide block was also abolished. 5. Truncation of five or nine amino acids from the N-terminus of Kir6.2 also enhanced the open probability, and reduced both the ATP sensitivity and the fraction of high-affinity tolbutamide block, although to a lesser extent than for the DeltaN14 deletion. Site-directed mutagenesis suggests that hydrophobic residues in Kir6. 2 may be involved in this effect. 6. The reduced ATP sensitivity of Kir6.2DeltaN14 may be explained by the increased Po. However, when the Po was decreased (by ATP), tolbutamide was unable to block Kir6. 2DeltaN14/SUR1-K719A,K1385M currents, despite the fact that the drug inhibited Kir6.2-C166S/SUR1-K719A,K1385M currents (which in the absence of ATP have a Po of > 0.8 and are not blocked by tolbutamide). Thus the N-terminus of Kir6.2 may be involved in coupling sulphonylurea binding to SUR1 to closure of the Kir6.2 pore.

ATP-Binding Cassette Transporters↗

PubMed1999

Miniglucagon (glucagon 19-29), a potent and efficient inhibitor of secretagogue-induced insulin release through a Ca2+ pathway.

Using the MIN6 B-cell line, we investigated the hypothesis that miniglucagon, the C-terminal () fragment processed from glucagon and present in pancreatic A cells, modulates insulin release, and we analyzed its cellular mode of action. We show that, at concentrations ranging from 0.01 to 1000 pM, miniglucagon dose-dependently (ID50 = 1 pM) inhibited by 80-100% the insulin release triggered by glucose, glucagon, glucagon-like peptide-1-(7-36) amide (tGLP-1), or glibenclamide, but not that induced by carbachol. Miniglucagon had no significant effects on cellular cAMP levels. The increase in 45Ca2+ uptake induced by depolarizing agents (glucose or extracellular K+), by glucagon, or by the Ca2+channel agonist Bay K-8644 was blocked by miniglucagon at the doses active on insulin release. Electrophysiological experiments indicated that miniglucagon induces membrane hyperpolarization, probably by opening potassium channels, which terminated glucose-induced electrical activity. Pretreatment with pertussis toxin abolished the effects of miniglucagon on insulin release. It is concluded that miniglucagon is a highly potent and efficient inhibitor of insulin release by closing, via hyperpolarization, voltage-dependent Ca2+ channels linked to a pathway involving a pertussis toxin-sensitive G protein.

Calcium↗

PubMed1999

Direct photoaffinity labeling of the Kir6.2 subunit of the ATP-sensitive K+ channel by 8-azido-ATP.

ATP-sensitive potassium channels are under complex regulation by intracellular ATP and ADP. The potentiating effect of MgADP is conferred by the sulfonylurea receptor subunit of the channel, SUR, whereas the inhibitory effect of ATP appears to be mediated via the pore-forming subunit, Kir6.2. We determined whether ATP directly interacts with a binding site on the Kir6.2 subunit to mediate channel inhibition by analyzing binding of a photoaffinity analog of ATP (8-azido-[gamma-32P]ATP) to membranes from COS-7 cells transiently expressing Kir6.2. We demonstrate that Kir6.2 can be directly labeled by 8-azido-[gamma-32P]ATP but that the related subunit Kir4.1, which is not inhibited by ATP, is not labeled. Photoaffinity labeling of Kir6.2 is reduced by approximately 50% with 100 microM ATP. In addition, mutations in the NH2 terminus (R50G) and the COOH terminus (K185Q) of Kir6.2, which have both been shown to reduce the inhibitory effect of ATP upon Kir6.2 channel activity, reduced photoaffinity labeling by >50%. These results demonstrate that ATP binds directly to Kir6.2 and that both the NH2- and COOH-terminal intracellular domains may influence ATP binding.

Adenosine Triphosphate↗

PubMed1999

Involvement of the N-terminus of Kir6.2 in the inhibition of the KATP channel by ATP.

1. ATP-sensitive potassium (KATP) channels are composed of pore-forming Kir6.2 and regulatory SUR subunits. A truncated isoform of Kir6.2, Kir6.2DeltaC26, expresses ATP-sensitive channels in the absence of SUR1, suggesting the ATP-inhibitory site lies on the Kir6. 2 subunit. 2. We examined the effect on the channel ATP sensitivity of mutating the arginine residue at position 50 (R50) in the N-terminus of Kir6.2, by recording macroscopic currents in membrane patches excised from Xenopus oocytes expressing wild-type or mutant Kir6.2DeltaC26. 3. Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 microM to 800 microM, 1.1 mM and 3.8 mM, respectively. The single-channel conductance and kinetics were unaffected by any of these mutations. Mutation to glutamate, lysine, asparagine, glutamine or leucine had a smaller effect (Ki, approximately 300-400 microM). The results indicate that the side chain of the arginine residue at position 50 is unlikely to contribute directly to the binding site for ATP, and suggest it may affect ATP inhibition by allosteric interactions. 4. Mutation of the isoleucine residue at position 49 to glycine (I49G) reduced the channel ATP sensitivity, while the mutation of the glutamate residue at position 51 to glycine (E51G) did not. 5. When a mutation in the N-terminus of Kir6.2DeltaC26 that alters ATP sensitivity (R50S; Ki, 800 microM) was combined with one in the C-terminus (E179Q; Ki, 300 microM), the Ki for the apparent ATP sensitivity was increased to 2.8 mM. The Hill coefficient was also increased. This suggests that the N- and C-termini of Kir6.2 may co-operate to influence channel closure by ATP.

Adenosine Triphosphate↗