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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

Analysis of the differential modulation of sulphonylurea block of β-cell and cardiac ATP-sensitive K+ (KATP) channels by Mg-nucleotides

Sulphonylureas stimulate insulin secretion by binding with high-affinity to the sulphonylurea receptor (SUR) subunit of the ATP-sensitive potassium (K_ATP) channel and thereby closing the channel pore (formed by four Kir6.2 subunits). In the absence of added nucleotides, the maximal block is around 60–80 %, indicating that sulphonylureas act as partial antagonists. Intracellular MgADP modulated sulphonylurea block, enhancing inhibition of Kir6.2/SUR1 (β-cell type) and decreasing that of Kir6.2/SUR2A (cardiac-type) channels. We examined the molecular basis of the different response of channels containing SUR1 and SUR2A, by recording currents from inside-out patches excised from Xenopus oocytes heterologously expressing wild-type or chimeric channels. We used the benzamido derivative meglitinide as this drug blocks Kir6.2/SUR1 and Kir6.2/SUR2A currents, reversibly and with similar potency. Our results indicate that transfer of the region containing transmembrane helices (TMs) 8–11 and the following 65 residues of SUR1 into SUR2A largely confers a SUR1-like response to MgADP and meglitinide, whereas the reverse chimera (SUR128) largely endows SUR1 with a SUR2A-type response. This effect was not specific for meglitinide, as tolbutamide was also unable to prevent MgADP activation of Kir6.2/SUR128 currents. The data favour the idea that meglitinide binding to SUR1 impairs either MgADP binding or the transduction pathway between the NBDs and Kir6.2, and that TMs 8–11 are involved in this modulatory response. The results provide a basis for understanding how β-cell K_ATP channels show enhanced sulphonylurea inhibition under physiological conditions, whereas cardiac K_ATP channels exhibit reduced block in intact cells, especially during metabolic inhibition.     

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.     

Diverse Kir modulators act in close proximity to residues implicated in phosphoinositide binding

Inwardly rectifying potassium (Kir) channels were the first shown to be directly activated by phosphoinositides in general and phosphatidylinositol bisphosphate (PIP₂) in particular. Atomic resolution structures have been determined for several mammalian and bacterial Kir channels. Basic residues, identified through mutagenesis studies to contribute to the sensitivity of the channel to PIP₂, have been mapped onto the three dimensional channel structure and their localization has given rise to a plausible model that can explain channel activation by PIP₂. Moreover, mapping onto the three-dimensional channel structure sites involved in the modulation of Kir channel activity by a diverse group of regulatory molecules, revealed a striking proximity to residues implicated in phosphoinositide binding. These observations support the hypothesis that the observed dependence of diverse modulators on channel–PIP₂ interactions stems from their localization within distances that can affect PIP₂-interacting residues.     

Signal transduction pathway for the substance P-induced inhibition of rat Kir3 (GIRK) channel

Certain transmitters inhibit Kir3 (GIRK) channels, resulting in neuronal excitation. We analysed signalling mechanisms for substance P (SP)-induced Kir3 inhibition in relation to the role of phosphatidylinositol 4,5-bisphosphate (PIP₂). SP rapidly – with a half-time of ∼10 s with intracellular GTPγS and ∼14 s with intracellular GTP – inhibits a robustly activated Kir3.1/Kir3.2 current. A mutant Kir3 channel, Kir3.1(M223L)/Kir3.2(I234L), which has a stronger binding to PIP₂ than does the wild type Kir3.1/Kir3.2, is inhibited by SP as rapidly as the wild type Kir3.1/Kir3.2. This result contradicts the idea that Kir3 inhibition originates from the depletion of PIP₂. A Kir2.1 (IRK1) mutant, Kir2.1(R218Q), despite having a weaker binding to PIP₂ than wild type Kir3.1/Kir3.2, shows a SP-induced inhibition slower than the wild type Kir3.1/Kir3.2 channel, again conflicting with the PIP₂ theory of channel inhibition. Co-immunoprecipitation reveals that Gα_q binds with Kir3.2, but not with Kir2.2 or Kir2.1. These functional results and co-immunoprecipitation data suggest that G_q activation rapidly inhibits Kir3 (but not Kir2), possibly by direct binding of Gα_q to the channel.     

PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of two-pore domain K+ channels

Two-pore (2-P) domain potassium channels are implicated in the control of the resting membrane potential, hormonal secretion, and the amplitude, frequency and duration of the action potential. These channels are strongly regulated by hormones and neurotransmitters. Little is known, however, about the mechanism underlying their regulation. Here we show that phosphatidylinositol 4,5-bisphosphate (PIP₂) gating underlies several aspects of 2-P channel regulation. Our results demonstrate that all four 2-P channels tested, TASK1, TASK3, TREK1 and TRAAK are activated by PIP₂. We show that mechanical stimulation may promote PIP₂ activation of TRAAK channels. For TREK1, TASK1 and TASK3 channels, PIP₂ hydrolysis underlies inhibition by several agonists. The kinetics of inhibition by the PIP₂ scavenger polylysine, and the inhibition by the phosphatidylinositol 4-kinase inhibitor wortmannin correlated with the level of agonist-induced inhibition. This finding suggests that the strength of channel PIP₂ interactions determines the extent of PLC-induced inhibition. Finally, we show that PIP₂ hydrolysis modulates voltage dependence of TREK1 channels and the unrelated voltage-dependent KCNQ1 channels. Our results suggest that PIP₂ is a common gating molecule for K⁺ channel families despite their distinct structures and physiological properties.     

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.     

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.     

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.     

Functional modulation of the ATP-sensitive potassium channel by extracellular signal-regulated kinase-mediated phosphorylation

ATP-sensitive potassium (K(ATP)) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K(ATP) channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K(atp) channel remained unknown. To investigate how ERK modulates the function of K(ATP) channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K(ATP) channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K(ATP) channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K(ATP) channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K(ATP) channels, ERK2-mediated phosphorylation may represent a common mechanism for K(ATP) channel regulation in different tissues.     

Target-specific PIP2 signalling: how might it work?

Phosphatidylinositol 4,5-bisphosphate (PIP₂)-mediated signalling is a new and rapidly developing area in the field of cellular signal transduction. With the extensive and growing list of PIP₂-sensitive membrane proteins (many of which are ion channels and transporters) and multiple signals affecting plasma membrane PIP₂ levels, the question arises as to the cellular mechanisms that confer specificity to PIP₂-mediated signalling. In this review we critically consider two major hypotheses for such possible mechanisms: (i) clustering of PIP₂ in membrane microdomains with restricted lateral diffusion, a hypothesis providing a mechanism for spatial segregation of PIP₂ signals and (ii) receptor-specific buffering of the global plasma membrane PIP₂ pool via Ca²⁺-mediated stimulation of PIP₂ synthesis or release, a concept allowing for receptor-specific signalling with free lateral diffusion of PIP₂. We also discuss several other technical and conceptual intricacies of PIP₂-mediated signalling.