Pyridine nucleotide regulation of the KATP channel Kir6.2/SUR1 expressed in Xenopus oocytes

The pancreatic β-cell type of ATP-sensitive potassium (K_ATP) channel (Kir6.2/SUR1) is inhibited by intracellular ATP and ADP, which bind to the Kir6.2 subunit, and is activated by Mg-nucleotide interaction with the regulatory sulphonylurea receptor subunits (SUR1). The nicotinamide adenine dinucleotides NAD and NADP consist of an ADP molecule with a ribose group and a nicotinamide moiety attached to the terminal phosphate. Both these molecules block native K_ATP channels in pancreatic β-cells at concentrations above 500 μM, and activate them at lower concentrations. We therefore investigated whether NAD and NADP interact with both Kir6.2 and SUR1 subunits of the K_ATP channel by comparing the potency of these agents on recombinant Kir6.2ΔC and Kir6.2/SUR1 channels expressed in Xenopus oocytes. Our results show that, at physiological concentrations, NAD and NADP interact with the nucleotide inhibitory site of Kir6.2 to inhibit Kir6.2/SUR1 currents. They may therefore contribute to the resting level of channel inhibition in the intact cell. Importantly, our data also reveal that this interaction is dependent on the presence of SUR1, which may act by increasing the width of the nucleotide-binding pocket of Kir6.2.     

A cytosolic factor that inhibits KATP channels expressed in Xenopus oocytes by impairing Mg-nucleotide activation by SUR1

ATP-sensitive K⁺ (K_ATP) channels couple cell metabolism to cell electrical activity. Wild-type (Kir6.2/SUR1) K_ATP channels heterologously expressed in Xenopus oocytes give rise to very small inward currents in cell-attached patches. A large increase in the current is observed on patch excision into zero ATP solution. This is presumably due to loss of intracellular ATP leading to unblock of K_ATP channels. In contrast, channels containing Kir6.2 mutations associated with reduced ATP-sensitivity display non-zero cell-attached currents. Unexpectedly, these cell-attached currents are significantly smaller (by ∼40%) than those observed when excised patches are exposed to physiological ATP concentrations (1–10 m m ). Cramming the patch back into the oocyte cytoplasm restores mutant K_ATP current amplitude to that measured in the cell-attached mode. This implies that the magnitude of the cell-attached current is regulated not only by intracellular ATP but also by another cytoplasmic factor/s. This factor seems to require the nucleotide-binding domains of SUR1 to be effective. Thus a mutant Kir6.2 (Kir6.2ΔC-I296L) expressed in the absence of SUR1 exhibited currents of similar magnitude in cell-attached patches as in inside-out patches exposed to 10 m m MgATP. Similar results were found when Kir6.2-I296L was coexpressed with an SUR1 mutant that is insensitive to MgADP or MgATP activation. This suggests the oocyte contains a cytoplasmic factor that reduces nucleotide binding/hydrolysis at the NBDs of SUR1. In conclusion, our results reveal a novel regulatory mechanism for the K_ATP channel. This was not evident for wild-type channels because of their high sensitivity to block by ATP.     

Functional effects of naturally occurring KCNJ11 mutations causing neonatal diabetes on cloned cardiac KATP channels

ATP-sensitive K⁺ (K_ATP) channels are hetero-octamers of inwardly rectifying K⁺ channel (Kir6.2) and sulphonylurea receptor subunits (SUR1 in pancreatic β-cells, SUR2A in heart). Heterozygous gain-of-function mutations in Kir6.2 cause neonatal diabetes, which may be accompanied by epilepsy and developmental delay. However, despite the importance of K_ATP channels in the heart, patients have no obvious cardiac problems. We examined the effects of adenine nucleotides on K_ATP channels containing wild-type or mutant (Q52R, R201H) Kir6.2 plus either SUR1 or SUR2A. In the absence of Mg²⁺, both mutations reduced ATP inhibition of SUR1- and SUR2A-containing channels to similar extents, but when Mg²⁺ was present ATP blocked mutant channels containing SUR1 much less than SUR2A channels. Mg-nucleotide activation of SUR1, but not SUR2A, channels was markedly increased by the R201H mutation. Both mutations also increased resting whole-cell K_ATP currents through heterozygous SUR1-containing channels to a greater extent than for heterozygous SUR2A-containing channels. The greater ATP inhibition of mutant Kir6.2/SUR2A than of Kir6.2/SUR1 can explain why gain-of-function Kir6.2 mutations manifest effects in brain and β-cells but not in the heart.     

The Kir6.2-F333I mutation differentially modulates KATP channels composed of SUR1 or SUR2 subunits

Mutations in Kir6.2, the pore-forming subunit of the K_ATP channel, that reduce the ability of ATP to block the channel cause neonatal diabetes. The stimulatory effect of MgATP mediated by the regulatory sulphonylurea receptor (SUR) subunit of the channel may also be modified. We compared the effect of the Kir6.2-F333I mutation on K_ATP channels containing SUR1, SUR2A or SUR2B. The open probability of Kir6.2/SUR1 channels, or a C-terminally truncated form of Kir6.2 expressed in the absence of SUR, was unaffected by the mutation. However, that of Kir6.2/SUR2A and Kir6.2/SUR2B channels was increased. In the absence of Mg²⁺, ATP inhibition of all Kir6.2-F333I/SUR channel types was reduced, although SUR1-containing channels were reduced more than SUR2-containing channels. These results suggest F333 is involved in differential coupling of Kir6.2 to SUR1 and SUR2. When Mg²⁺ was present, ATP blocked SUR2A channels but activated SUR2B and SUR1 channels. Activation by MgGDP (or MgADP) was similar for wild-type and mutant channels and was independent of SUR. This indicates Mg-nucleotide binding to SUR and the transduction of binding into opening of the Kir6.2 pore are unaffected by the mutation. The data further suggest that MgATP hydrolysis by the nucleotide-binding domains of SUR1 and SUR2B, but not SUR2A, is enhanced by the F333I mutation in Kir6.2. Taken together, our data suggest the region of the C terminus within which F333 lies is involved in more than one type of functional interaction with SUR, and that F333 interacts differentially with SUR1 and SUR2.     

A mutation in the ATP-binding site of the Kir6.2 subunit of the KATP channel alters coupling with the SUR2A subunit

Mutations in the pore-forming subunit of the ATP-sensitive K⁺ (K_ATP) channel Kir6.2 cause neonatal diabetes. Understanding the molecular mechanism of action of these mutations has provided valuable insight into the relationship between the structure and function of the K_ATP channel. When Kir6.2 containing a mutation (F333I) in the putative ATP-binding site is coexpressed with the cardiac type of regulatory K_ATP channel subunit, SUR2A, the channel sensitivity to ATP inhibition is reduced and the intrinsic open probability ( P _o ) is increased. However, the extent of macroscopic current activation by MgADP was unaffected. Here we examine rundown and MgADP activation of wild-type and Kir6.2-F333I/SUR2A channels using single-channel recording, noise analysis and spectral analysis. We also compare the effect of mutating the adjacent residue, G334, on rundown and MgADP activation. All three approaches indicated that rundown of Kir6.2-F333I/SUR2A channels is due to a reduction in the number of active channels in the patch and that MgADP reactivation involves recruitment of inactive channels. In contrast, rundown and MgADP reactivation of wild-type and Kir6.2-G334D/SUR2A channels, and of Kir6.2-F333I/SUR1 channels, involve a gradual change in P _o . Our results suggest that F333 in Kir6.2 interacts functionally with SUR2A to modulate channel rundown and MgADP activation. This interaction is fairly specific as it is not disturbed when the adjacent residue (G334) is mutated. It is also not a consequence of the enhanced P _o of Kir6.2-F333I/SUR2A channels, as it is not found for other mutant channels with high P _o (Kir6.2-I296L/SUR2A).     

Mapping the architecture of the ATP-binding site of the KATP channel subunit Kir6.2

ATP-sensitive potassium (K_ATP) channels comprise Kir6.2 and SUR subunits. The site at which ATP binds to mediate K_ATP channel inhibition lies on Kir6.2, but the potency of block is enhanced by coexpression with SUR1. To assess the structure of the ATP-binding site on Kir6.2, we used a range of adenine nucleotides as molecular measuring sticks to map the internal dimensions of the binding site. We compared their efficacy on Kir6.2–SUR1, and on a truncated Kir6.2 (Kir6.2ΔC) that expresses in the absence of SUR. We show here that SUR1 modifies the ATP-binding pocket of Kir6.2, by increasing the width of the groove that binds the phosphate tail of ATP, without changing the length of the groove, and by enhancing interaction with the adenine ring.     

Three C-terminal residues from the sulphonylurea receptor contribute to the functional coupling between the K(ATP) channel subunits SUR2A and Kir6.2

Cardiac ATP-sensitive potassium (K_ATP) channels are metabolic sensors formed by the association of the inward rectifier potassium channel Kir6.2 and the sulphonylurea receptor SUR2A. SUR2A adjusts channel gating as a function of intracellular ATP and ADP and is the target of pharmaceutical openers and blockers which, respectively, up- and down-regulate Kir6.2. In an effort to understand how effector binding to SUR2A translates into Kir6.2 gating modulation, we examined the role of a 65-residue SUR2A fragment linking transmembrane domain TMD2 and nucleotide-binding domain NBD2 that has been shown to interact with Kir6.2. This fragment of SUR2A was replaced by the equivalent residues of its close homologue, the multidrug resistance protein MRP1. The chimeric construct was expressed in Xenopus oocytes and characterized using the patch-clamp technique. We found that activation by MgADP and synthetic openers was greatly attenuated although apparent affinities were unchanged. Further chimeragenetic and mutagenetic studies showed that mutation of three residues, E1305, I1310 and L1313 (rat numbering), was sufficient to confer this defective phenotype. The same mutations had no effects on channel block by the sulphonylurea glibenclamide or by ATP, suggesting a role for these residues in activatory – but not inhibitory – transduction processes. These results indicate that, within the K_ATP channel complex, the proximal C-terminal of SUR2A is a critical link between ligand binding to SUR2A and Kir6.2 up-regulation.     

Zinc is both an intracellular and extracellular regulator of KATP channel function

Extracellular Zn²⁺ has been identified as an activator of pancreatic K_ATP channels. We further examined the action of Zn²⁺ on recombinant K_ATP channels formed with the inward rectifier K⁺ channel subunit Kir6.2 associated with either the pancreatic/neuronal sulphonylurea receptor 1 (SUR1) subunit or the cardiac SUR2A subunit. Zn²⁺, applied at either the extracellular or intracellular side of the membrane appeared as a potent, reversible activator of K_ATP channels. External Zn²⁺, at micromolar concentrations, activated SUR1/Kir6.2 but induced a small inhibition of SUR2A/Kir6.2 channels. Cytosolic Zn²⁺ dose-dependently stimulated both SUR1/Kir6.2 and SUR2A/Kir6.2 channels, with half-maximal effects at 1.8 and 60 μ m , respectively, but it did not affect the Kir6.2 subunit expressed alone. These observations point to an action of both external and internal Zn²⁺ on the SUR subunit. Effects of internal Zn²⁺ were not due to Zn²⁺ leaking out, since they were unaffected by the presence of a Zn²⁺ chelator on the external side. Similarly, internal chelators did not affect activation by external Zn²⁺. Therefore, Zn²⁺ is an endogenous K_ATP channel opener being active on both sides of the membrane, with potentially distinct sites of action located on the SUR subunit. These findings uncover a novel regulatory pathway targeting K_ATP channels, and suggest a new role for Zn²⁺ as an intracellular signalling molecule.     

Remodelling of the SUR–Kir6.2 interface of the KATP channel upon ATP binding revealed by the conformational blocker rhodamine 123

ATP-sensitive K⁺ channels (K_ATP channels) are metabolic sensors formed by association of a K⁺ channel, Kir6, and an ATP-binding cassette (ABC) protein, SUR, which allosterically regulates channel gating in response to nucleotides and pharmaceutical openers and blockers. How nucleotide binding to SUR translates into modulation of Kir6 gating remains largely unknown. To address this issue, we have used a novel conformational K_ATP channel inhibitor, rhodamine 123 (Rho123) which targets the Kir6 subunit in a SUR-dependent manner. Rho123 blocked SUR-less Kir6.2 channels with an affinity of ∼1 μ m , regardless of the presence of nucleotides, but it had no effect on channels formed by the association of Kir6.2 and the N-terminal transmembrane domain TMD0 of SUR. Rho123 blocked SUR + Kir6.2 channels with the same affinity as Kir6.2 but this effect was antagonized by ATP. Protection from Rho123 block by ATP was due to direct binding of ATP to SUR and did not entail hydrolysis because it was not mimicked by AMP, did not require Mg²⁺ and was reduced by mutations in the nucleotide-binding domains of SUR. These results suggest that Rho123 binds at the TMD0–Kir6.2 interface and that binding of ATP to SUR triggers a change in the structure of the contact zone between Kir6.2 and domain TMD0 of SUR that causes masking of the Rho123 site on Kir6.2.     

Protein kinase C modulation of recombinant ATP-sensitive K+ channels composed of Kir6.1 and/or Kir6.2 expressed with SUR2B

The molecular identity of smooth muscle ATP-sensitive K⁺ channels (K_ATP) is not established with certainty. Patch clamp methods were employed to determine if recombinant K_ATP channels composed of Kir6.1 and SUR2B subunits expressed by human embryonic kidney (HEK293) cells share an identical modulation by protein kinase C (PKC) with the vascular K_NDP subtype of K_ATP channel. The open probability of Kir6.1/SUR2B channels was determined before and after sequential exposure to pinacidil (50 μM) and the combination of pinacidil and phorbol 12,13-dibutyrate (PdBu; 50 n m ). Treatment with PdBu caused a decline in channel activity, but this was not seen with an inactive phorbol ester, 4α-phorbol 12,13-didecanoate (PdDe; 50 n m ). Angiotensin II (0.1 μM) induced a similar inhibition of Kir6.1/SUR2B channels in cells expressing angiotensin AT₁ receptors. The effects of PdBu and angiotensin II were blocked by the PKC inhibitor, chelerythrine (3 μM). Purified PKC inhibited Kir6.1/SUR2B activity (in 0.5 m m ATP/ 0.5 m m ADP), and the inhibition was blocked by a specific peptide inhibitor of PKC, PKC(19-31). In contrast, PdBu increased the activity of recombinant K_ATP channels composed of Kir6.2 and SUR2B, or the combination of Kir6.1, Kir6.2 and SUR2B subunits. The results indicate that the modulation by PKC of Kir6.1/SUR2B, but not Kir6.2/SUR2B or Kir6.1-Kir6.2/SUR2B channel gating mimics that of native vascular K_NDP channels. Physiological inhibition of vascular K_ATP current by vasoconstrictors which utilize intracellular signalling cascades involving PKC is concluded to involve the modulation of K_NDP channel complexes composed of four Kir6.1 and their associated SUR2B subunits.     

Allosteric modulation of the mouse kir6.2 channel by intracellular H+ and ATP

The ATP-sensitive K⁺ (K_ATP) channels are regulated by intracellular H⁺ in addition to ATP, ADP, and phospholipids. Here we show evidence for the interaction of H⁺ with ATP in regulating a cloned K_ATP channel, i.e. Kir6.2 expressed with and without the SUR1 subunit. Channel sensitivity to ATP decreases at acidic pH, while the pH sensitivity also drops in the presence of ATP. These effects are more evident in the presence of the SUR1 subunit. In the Kir6.2 + SUR1, the pH sensitivity is reduced by about 0.4 pH units with 100 μM ATP and 0.6 pH units with 1 m m ATP, while a decrease in pH from 7.4 to 6.8 lowers the ATP sensitivity by about fourfold. The Kir6.2 + SUR1 currents are strongly activated at pH 5.9-6.5 even in the presence of 1 m m ATP. The modulations appear to take place at His175 and Lys185 that are involved in proton and ATP sensing, respectively. Mutation of His175 completely eliminates the pH effect on the ATP sensitivity. Similarly, the K185E mutant-channel loses the ATP-dependent modulation of the pH sensitivity. Thus, allosteric modulations of the cloned K_ATP channel by ATP and H⁺ are demonstrated. Such a regulation allows protons to activate directly the K_ATP channels and release channel inhibition by intracellular ATP; the pH effect is further enhanced with a decrease in ATP concentration as seen in several pathophysiological conditions.     

The N-terminal transmembrane domain (TMD0) and a cytosolic linker (L0) of sulphonylurea receptor define the unique intrinsic gating of KATP channels

ATP-sensitive potassium (K_ATP) channels comprise four pore-forming Kir6 and four regulatory sulphonylurea receptor (SUR) subunits. SUR, an ATP-binding cassette protein, associates with Kir6 through its N-terminal transmembrane domain (TMD0). TMD0 connects to the core domain of SUR through a cytosolic linker (L0). The intrinsic gating of Kir6.2 is greatly altered by SUR. It has been hypothesized that these changes are conferred by TMD0. Exploiting the fact that the pancreatic (SUR1/Kir6.2) and the cardiac (SUR2A/Kir6.2) K_ATP channels show different gating behaviours, we have tested this hypothesis by comparing the intrinsic gating of Kir6.2 with the last 26 residues deleted (Kir6.2Δ26) co-expressed with SUR1, S1-TMD0, SUR2A and S2-TMD0 at −40 and −100 mV (S is an abbreviation for SUR; TMD0/Kir6.2Δ26, but not TMD0/Kir6.2, can exit the endoplastic reticulum and reach the cell membrane). Single-channel kinetic analyses revealed that the mean burst and interburst durations are shorter for TMD0/Kir6.2Δ26 than for the corresponding SUR channels. No differences were found between the two TMD0 channels. We further demonstrated that in isolation even TMD0-L0 (SUR truncated after L0) cannot confer the wild-type intrinsic gating to Kir6.2Δ26 and that swapping L0 (SUR truncated after L0)between SUR1 and SUR2A only partially exchanges their different intrinsic gating. Therefore, in addition to TMD0, L0 and the core domain also participate in determining the intrinsic gating of Kir6.2. However, TMD0 and L0 are responsible for the different gating patterns of full-length SUR1 and SUR2A channels. A kinetic model with one open and four closed states is presented to explain our results in a mechanistic context.     

Gene targeting approach to clarification of ion channel function: studies of Kir6.x null mice

ATP-sensitive potassium (K_ATP) channels are present in many tissues, including pancreatic β-cells, heart, skeletal muscle, vascular smooth muscle and brain, in which they couple the cell metabolic state to membrane potential. K_ATP channels are hetero-octameric proteins composed of the pore-forming subunits Kir6.x (Kir6.1 or Kir6.2) of the inwardly rectifying K⁺ channel family and the regulatory subunits SURx (SUR1, SUR2A or SUR2B), the receptor of the sulphonylureas widely used in treatment of type 2 diabetes mellitus. Different combinations of Kir6.x and SURx comprise K_ATP channels with distinct electrophysiological and pharmacological properties, but their physiological functions in the various tissues are unclear. Our studies of Kir6.2 null (knockout) and Kir6.1 null mice have shown that K_ATP channels are critical metabolic sensors in protection against acute metabolic stress such as hyperglycaemia, hypoglycaemia, ischaemia and hypoxia.     

SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K⁺ channels (K_ATP). To investigate the protective role of K_ATP channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294–1358, the A-fragment, reduced sarcolemmal K_ATP currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 ± 1.22; and A-fragment 0.94 ± 0.07 pA pF⁻¹, n = 6,6, P < 0.001). An inactive fragment (1358–1545, current density 6.30 ± 0.85 pA pF⁻¹, n = 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca²⁺ loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca²⁺ and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% ( P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca²⁺ loading, was abolished by the A-fragment. Our data are consistent with a role for K_ATP channels in causing action potential failure and reduced Ca²⁺ loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K_ATP channel, but we also discuss the possibility of mitochondrial effects.     

Physiological roles of ATP-sensitive K+ channels in smooth muscle

Potassium channels that are inhibited by intracellular ATP (ATP_i) were first identified in ventricular myocytes, and are referred to as ATP-sensitive K⁺ channels (i.e. K_ATP channels). Subsequently, K⁺ channels with similar characteristics have been demonstrated in many other tissues (pancreatic β-cells, skeletal muscle, central neurones, smooth muscle). Approximately one decade ago, K_ATP channels were cloned and were found to be composed of at least two subunits: an inwardly rectifying K⁺ channel six family (Kir6.x) that forms the ion conducting pore and a modulatory sulphonylurea receptor (SUR) that accounts for several pharmacological properties. Various types of native K_ATP channels have been identified in a number of visceral and vascular smooth muscles in single-channel recordings. However, little attention has been paid to the molecular properties of the subunits in K_ATP channels and it is important to determine the relative expression of K_ATP channel components which give rise to native K_ATP channels in smooth muscle. The aim of this review is to briefly discuss the current knowledge available for K_ATP channels with the main interest in the molecular basis of native K_ATP channels, and to discuss their possible linkage with physiological functions in smooth muscle.     

Cytoplasmic accumulation of long-chain coenzyme A esters activates KATP and inhibits Kir2.1 channels

Long-chain fatty acids acyl coenzyme A esters (LC-CoA) are obligate intermediates of fatty acid metabolism and have been shown to activate K_ATP channels but to inhibit most other Kir channels (e.g. Kir2.1) by direct channel binding. The activation of K_ATP channels by elevated levels of LC-CoA may be involved in the pathophysiology of type 2 diabetes, the hypothalamic sensing of circulating fatty acids and the regulation of cardiac K_ATP channels. However, LC-CoA are effectively buffered in the cytoplasm and it is currently not clear whether their free concentration can reach levels sufficient to affect Kir channels in vivo . Here, we report that extracellular oleic acid complexed with albumin at an unbound concentration of 81 ± 1 n m strongly activated K_ATP channels and inhibited Kir2.1 channels in Chinese hamster ovary (CHO) cells as well as endogenous Kir currents in human embryonic kidney (HEK293) cells. These effects were only seen in the presence of a high concentration of glucose (25 m m ), a condition known to promote the accumulation of LC-CoA by inhibiting their mitochondrial uptake via carnitine-palmitoyl-transferase-1 (CPT1). Accordingly, pharmacological inhibition of CPT1 by etomoxir restored the effects of oleic acid under low glucose conditions. Finally, triacsin C, an inhibitor of the acyl-CoA synthetase, which is necessary for LC-CoA formation, abolished the effects of extracellular oleic acid on the various Kir channels. These results establish the direct regulation of Kir channels by the cytoplasmic accumulation of LC-CoA, which might be of physiological and pathophysiological relevance in a variety of tissues.     

Role of sarcolemmal ATP-sensitive K+ channels in the regulation of sinoatrial node automaticity: an evaluation using Kir6.2-deficient mice

The role of cardiac sarcolemmal ATP-sensitive K⁺ (K_ATP) channels in the regulation of sinoatrial node (SAN) automaticity is not well defined. Using mice with homozygous knockout (KO) of the Kir6.2 (a pore-forming subunit of cardiac K_ATP channel) gene, we investigated the pathophysiological role of K_ATP channels in SAN cells during hypoxia. Langendorff-perfused mouse hearts were exposed to hypoxic and glucose-free conditions (hypoxia). After 5 min of hypoxia, sinus cycle length (CL) was prolonged from 207 ± 10 to 613 ± 84 ms ( P < 0.001) in wild-type (WT) hearts. In Kir6.2 KO hearts, CL was slightly prolonged from 198 ± 17 to 265 ± 32 ms. The CL of spontaneous action potentials of WT SAN cells, recorded in the current-clamp mode, was markedly prolonged from 410 ± 56 to 605 ± 108 ms ( n = 6, P < 0.05) with a decrease of the slope of the diastolic depolarization (SDD) after the application of the K⁺ channel opener pinacidil (100 μ m ). Pinacidil induced a glibenclamide (1 μ m )-sensitive outward current, which was recorded in the voltage-clamp mode, only in WT SAN cells. During metabolic inhibition by 2,4-dinitrophenol, CL was prolonged from 292 ± 38 to 585 ± 91 ms ( P < 0.05) with a decrease of SDD in WT SAN cells but not in Kir6.2 KO SAN cells. Diastolic Ca²⁺ concentration, measured by fluo-3 fluorescence, was decreased in WT SAN cells but increased in Kir6.2 KO SAN cells after short-term metabolic inhibition. In conclusion, the present study using Kir6.2 KO mice indicates that, during hypoxia, activation of sarcolemmal K_ATP channels in SAN cells inhibits SAN automaticity, which is important for the protection of SAN cells.     

Long-chain acyl-CoA esters and phosphatidylinositol phosphates modulate ATP inhibition of Katp channels by the same mechanism

Phosphatidylinositol phosphates (PIPs, e.g. PIP₂) and long-chain acyl-CoA esters (e.g. oleoyl-CoA) are potent activators of K _atp channels that are thought to link K _atp channel activity to the cellular metabolism of PIPs and fatty acids. Here we show that the two types of lipid act by the same mechanism: oleoyl-CoA potently reduced the ATP sensitivity of cardiac (Kir6.2/SUR2A) and pancreatic (Kir6.2/SUR1) K _atp channels in a way very similar to PIP₂. Mutations (R54Q, R176A) in the C- and N-terminus of Kir6.2 that greatly reduced the PIP₂ modulation of ATP sensitivity likewise reduced the modulation by oleoyl-CoA, indicating that the two lipids interact with the same site. Polyvalent cations reduced the effect of oleoyl-CoA and PIP₂ on the ATP sensitivity with similar potency suggesting that electrostatic interactions are of similar importance. However, experiments with differently charged inhibitory adenosine phosphates (ATP^4-, ADP^3- and 2'(3')- O -(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP^2-)) and diadenosine tetraphosphate (Ap₄A^5-) ruled out a mechanism where oleoyl-CoA or PIP₂ attenuate ATP inhibition by reducing ATP binding through electrostatic repulsion. Surprisingly, CoA (the head group of oleoyl-CoA) did not activate but inhibited K _atp channels (IC₅₀= 265 ± 33 μM). We provide evidence that CoA and diadenosine polyphosphates (e.g. Ap₄A) are ligands of the inhibitory ATP-binding site on Kir6.2.     

Functional analysis of six Kir6.2 (KCNJ11) mutations causing neonatal diabetes

ATP-sensitive potassium (K_ATP) channels, composed of pore-forming Kir6.2 and regulatory sulphonylurea receptor (SUR) subunits, play an essential role in insulin secretion from pancreatic beta cells. Binding of ATP to Kir6.2 inhibits, whereas interaction of Mg-nucleotides with SUR, activates the channel. Heterozygous activating mutations in Kir6.2 (KCNJ11) are a common cause of neonatal diabetes (ND). We assessed the functional effects of six novel Kir6.2 mutations associated with ND: H46Y, N48D, E227K, E229K, E292G, and V252A. K_ATP channels were expressed in Xenopus oocytes and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the beta cell type of SUR). All mutations reduced the sensitivity of the K_ATP channel to inhibition by MgATP, and enhanced whole-cell K_ATP currents. Two mutations (E227K, E229K) also enhanced the intrinsic open probability of the channel, thereby indirectly reducing the channel ATP sensitivity. The other four mutations lie close to the predicted ATP-binding site and thus may affect ATP binding. In pancreatic beta cells, an increase in the K_ATP current is expected to reduce insulin secretion and thereby cause diabetes. None of the mutations substantially affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting patients carrying these mutations may respond to these drugs.     

Glucose-dependent regulation of rhythmic action potential firing in pancreatic β-cells by kATP-channel modulation

The regulation of a K⁺ current activating during oscillatory electrical activity ( I _K,slow) in an insulin-releasing β-cell was studied by applying the perforated patch whole-cell technique to intact mouse pancreatic islets. The resting whole-cell conductance in the presence of 10 m m glucose amounted to 1.3 nS, which rose by 50 % during a series of 26 simulated action potentials. Application of the K_ATP-channel blocker tolbutamide produced uninterrupted action potential firing and reduced I _K,slow by ≈50 %. Increasing glucose from 15 to 30 m m , which likewise converted oscillatory electrical activity into continuous action potential firing, reduced I _K,slow by ≈30 % whilst not affecting the resting conductance. Action potential firing may culminate in opening of K_ATP channels by activation of ATP-dependent Ca²⁺ pumping as suggested by the observation that the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibitor thapsigargin (4 μ m ) inhibited I _K,slow by 25 % and abolished bursting electrical activity. We conclude that oscillatory glucose-induced electrical activity in the β-cell involves the opening of K_ATP-channel activity and that these channels, in addition to constituting the glucose-regulated K⁺ conductance, also play a role in the graded response to supra-threshold glucose concentrations.