F M Gribble

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle.

A cDNA clone encoding an inwardly‐rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole‐cell Kir6.2/SUR currents were K+‐selective, time‐independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (K d = 4.5μM) or quinine (20μM) and by 5 mM intracellular ATP. The single‐channel conductance was 73 pS. Single‐channel activity was voltage‐independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP‐sensitive K‐channel.

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP‐sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2‐SUR1 currents resembled those of native beta‐cell ATP‐sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8‐fold by diazoxide (340 microM), 1.4‐fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2‐SUR1 currents in inside‐out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1‐SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide‐induced metabolic inhibition.

The interaction of nucleotides with the tolbutamide block of cloned ATP-sensitive K+ channel currents expressed in Xenopus oocytes: a reinterpretation.

1 We have examined the mechanism by which nucleotides modulate the tolbutamide block of the β‐cell ATP‐sensitive K+ channel (KATP channel), using wild‐type and mutant KATP channels heterologously expressed in Xenopus oocytes. This channel is composed of sulphonylurea receptor (SUR1) and pore‐forming (Kir6.2) subunits. 2 The dose–response relation for tolbutamide block of wild‐type KATP currents in the absence of nucleotide showed both a high‐affinity (Ki=2.0 μm) and a low‐affinity (Ki=1.8 mm) site. 3 The dose–response relation for tolbutamide block of Kir6.2ΔC36 (a truncated form of Kir6.2 which is expressed independently of SUR1) was best fitted with a single, low‐affinity site (Ki=1.7 mm). This indicates that the high‐affinity site resides on SUR1, whereas the low‐affinity site is located on Kir6.2. 4 ADP (100 μM) had a dual effect on wild‐type KATP currents: the nucleotide enhanced the current in the presence of Mg2+, but was inhibitory in the absence of Mg2+. Kir6.2ΔC36 currents were blocked by 100 μM ADP in the presence of Mg2+. 5 For wild‐type KATP currents, the blocking effect of 0.5 mm tolbutamide appeared greater in the presence of 100 μm MgADP (84 ± 2%) than in its absence (59 ± 4%). When SUR1 was mutated to abolish MgADP activation of KATP currents (K719A or K1384M), there was no difference in the extent of tolbutamide inhibition in the presence or absence of MgADP. 6 The ki for tolbutamide interaction with either the high‐ or low‐affinity site was unaffected by 100 μM MgADP, for both wild‐type and K719A‐K1384M currents. 7 MgGDP (100μM) enhanced wild‐type KATP currents and was without effect on K719A– K1384M currents. It did not affect the ki for tolbutamide block at either the high‐ or low‐affinity site. 8 Our results indicate that interaction of tolbutamide with the high‐affinity site (on SUR1) abolishes the stimulatory action of MgADP. This unmasks the inhibitory effect of ADP and leads to an apparent increase in channel inhibition. Under physiological conditions, abolition of MgADP activation is likely to constitute the principal mechanism by which tolbutamide inhibits the KATP channel.

Mechanism of Cloned ATP-sensitive Potassium Channel Activation by Oleoyl-CoA

Insulin secretion from pancreatic beta cells is coupled to cell metabolism through closure of ATP-sensitive potassium (KATP) channels, which comprise Kir6.2 and sulfonylurea receptor (SUR1) subunits. Although metabolic regulation of KATP channel activity is believed to be mediated principally by the adenine nucleotides, other metabolic intermediates, including long chain acyl-CoA esters, may also be involved. We recorded macroscopic and single-channel currents from Xenopusoocytes expressing either Kir6.2/SUR1 or Kir6.2ΔC36 (which forms channels in the absence of SUR1). Oleoyl-CoA (1 μm) activated both wild-type Kir6.2/SUR1 and Kir6.2ΔC36 macroscopic currents, ∼2-fold, by increasing the number and open probability of Kir6.2/SUR1 and Kir6.2ΔC36 channels. It was ineffective on the related Kir subunit Kir1.1a. Oleoyl-CoA also impaired channel inhibition by ATP, increasing the Ki values for both Kir6.2/SUR1 and Kir6.2ΔC36 currents by ∼3-fold. Our results indicate that activation of KATP channels by oleoyl-CoA results from an interaction with the Kir6.2 subunit, unlike the stimulatory effects of MgADP and diazoxide which are mediated through SUR1. The increased activity and reduced ATP sensitivity of KATP channels by oleoyl-CoA might contribute to the impaired insulin secretion observed in non-insulin-dependent diabetes mellitus.

Tissue-specific effects of sulfonylureas: lessons from studies of cloned K(ATP) channels.

Sulfonylureas stimulate insulin secretion in type-2 diabetic patients by blocking ATP-sensitive (K(ATP)) potassium channels in the pancreatic beta-cell membrane. This effect is mediated by the binding of the drug to the sulfonylurea receptor (SUR) subunit of the channel. K(ATP) channels are also present in other tissues, but often contain different types of SUR subunits (e.g., SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). The sensitivity of these different types of K(ATP) channels to sulfonylureas is variable: gliclazide and tolbutamide block the beta-cell, but not the cardiac or smooth muscle, types of K(ATP) channel. In contrast, glibenclamide blocks all three types of channel with similar affinity. The reversibility of the drugs also varies, with tolbutamide and gliclazide being reversible on all three types of K(ATP) channel, while glibenclamide is reversible on cardiac, but not beta-cell, K(ATP) channels. This review summarizes current knowledge of how sulfonylureas act on the different types of K(ATP) channel found in beta-cells and in extrapancreatic tissues, and discusses the implications of these findings for their use as therapeutic agents.

Differential sensitivity of beta-cell and extrapancreatic K(ATP) channels to gliclazide.

Aims/hypothesis. To investigate the tissue specificity of gliclazide for cloned beta-cell, cardiac and smooth muscle ATP-sensitive K-channels (KATP channels). These channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart, SUR2B in smooth muscle). Methods. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes, and channel activity was measured by recording macroscopic currents in giant inside-out membrane patches. Gliclazide was added to the intracellular membrane surface. Results. We reported previously that Kir6.2-SUR1 currents are blocked at two sites by tolbutamide: a high-affinity site on SUR1 and a low-affinity site on Kir6.2. We now show that gliclazide also inhibits beta-cell KATP channels at two sites: a high-affinity site, which is half-maximally blocked (Ki) at 50 ± 7 nmol/l (n = 8) and a low-affinity site with a Ki of 3.0 ± 0.6 mmol/l (n = 4). The high-affinity site on SUR1 was thus about 40-fold more sensitive to gliclazide than to tolbutamide (Ki∼ 2 μmol/l). Cloned cardiac and smooth muscle KATP channels did not show high-affinity block by gliclazide. Kir6.2-SUR2A currents exhibited a single low-affinity site with a Ki of 0.8 ± 0.1 mmol/l (n = 5), which is likely to reside on the Kir6.2 subunit. Conclusion/interpretation. Our results show that gliclazide is a sulphonylurea with high affinity and strong selectivity for the beta-cell type of KATP channel. [Diabetologia (1999) 42: 845–848]

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, Kir6.2ΔC26, 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 Kir6.2ΔC26. 3 Substitution of R50 by serine, alanine or glycine reduced the Ki for ATP inhibition from 117 μm to 800 μm, 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, ∼300–400 μm). 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 Kir6.2ΔC26 that alters ATP sensitivity (R50S; Ki, 800 μm) was combined with one in the C‐terminus (E179Q; Ki, 300 μm), 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.

The antimalarial agent mefloquine inhibits ATP-sensitive K-channels

The aim of this study was to determine whether antimalarial agents inhibit ATP‐sensitive potassium (KATP) channels and thereby contribute to the observed side‐effects of these drugs. Mefloquine (10–100 μM), but not artenusate (100 μM), stimulated insulin release from pancreatic islets in vitro. Macroscopic KATP currents were studied in inside‐out patches excised from Xenopus oocytes expressing cloned KATP channels. Mefloquine (IC50 ∼3 μM), quinine (IC50 ∼3 μM), and chloroquine inhibited the pancreatic β‐cell type of KATP channel Kir6.2/SUR1. Artenusate (100 μM) was without effect. Mefloquine and quinine also blocked a truncated form of Kir6.2 (Kir6.2ΔC36) 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 β‐cell KATP 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 KATP channels by interaction with the Kir6.2 subunit. This subunit is common to β‐cell, neuronal, cardiac, skeletal muscle, and some smooth muscle KATP channels suggesting that KATP channel inhibition may contribute to the other side effects of these drugs, which include cardiac conduction abnormalities and neuropsychiatric disturbances.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic β-cells and in the brain

A cDNA clone encoding an inwardly‐rectifying K‐channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP‐sensitive K‐channel rcKATP (KATP‐1; [6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K+‐selective, time‐independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside‐out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP‐1 suggests that BIR1 is a subunit of a brain and β‐cell KATP channel. However, pharmacological differences and the lack of ATP‐sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

In vitro mechanism of action on insulin release of S-22068, a new putative antidiabetic compound.

The MIN6 cell line derived from in vivo immortalized insulin‐secreting pancreatic β cells was used to study the insulin‐releasing capacity and the cellular mode of action of S‐22068, a newly synthesized imidazoline compound known for its antidiabetic effect in vivo. S‐22068, was able to release insulin from MIN6 cells in a dose‐dependent manner with a half‐maximal stimulation at 100 μM. Its efficacy (8 fold over the basal value), which did not differ whatever the glucose concentration (stimulatory or not), was intermediate between that of sulphonylurea and that of efaroxan. Similarly to sulphonylureas and classical imidazolines, S‐22068 blocked KATP channels and, in turn, opened nifedipine‐sensitive voltage‐dependent Ca2+ channels, triggering Ca2+ entry. Similarly to other imidazolines, S‐22068 induced a closure of cloned KATP channels injected to Xenopus oocytes by interacting with the pore‐forming Kir6.2 moiety. S‐22068 did not interact with the sulphonylurea binding site nor with the non‐I1 and non‐I2 imidazoline site evidenced in the β cells that is recognized by the imidazoline compounds efaroxan, phentolamine and RX821002. We conclude that S‐22068 is a novel imidazoline compound which stimulates insulin release via interaction with an original site present on the Kir6.2 moiety of the β cell KATP channels.