Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.     

On potential interactions between non-selective cation channel TRPM4 and sulfonylurea receptor SUR1

The sulfonylurea receptor SUR1 associates with Kir6.2 or Kir6.1 to form K(ATP) channels, which link metabolism to excitability in multiple cell types. The strong physical coupling of SUR1 with Kir6 subunits appears exclusive, but recent studies argue that SUR1 also modulates TRPM4, a member of the transient receptor potential family of non-selective cation channels. It has been reported that, following stroke, brain, or spinal cord injury, SUR1 is increased in neurovascular cells at the site of injury. This is accompanied by up-regulation of a non-selective cation conductance with TRPM4-like properties and apparently sensitive to sulfonylureas, leading to the postulation that post-traumatic non-selective cation currents are determined by TRPM4/SUR1 channels. To investigate the mechanistic hypothesis for the coupling between TRPM4 and SUR1, we performed electrophysiological and FRET studies in COSm6 cells expressing TRPM4 channels with or without SUR1. TRPM4-mediated currents were Ca(2+)-activated, voltage-dependent, underwent desensitization, and were inhibited by ATP but were insensitive to glibenclamide and tolbutamide. These properties were not affected by cotransfection with SUR1. When the same SUR1 was cotransfected with Kir6.2, functional K(ATP) channels were formed. In cells cotransfected with Kir6.2, SUR1, and TRPM4, we measured K(ATP)-mediated K(+) currents and Ca(2+)-activated, sulfonylurea-insensitive Na(+) currents in the same patch, further showing that SUR1 controls K(ATP) channel activity but not TRPM4 channels. FRET signal between fluorophore-tagged TRPM4 subunits was similar to that between Kir6.2 and SUR1, whereas there was no detectable FRET efficiency between TRPM4 and SUR1. Our data suggest that functional or structural association of TRPM4 and SUR1 is unlikely.     

Molecular characterization of a local sulfonylurea system in human adipose tissue

ATP-sensitive potassium (KATP) channels are present in many cell types and link cellular metabolism to the membrane potential. These channels are heterooctamers composed of two subunits. The sulfonylurea receptor (SUR) subunits are targets for drugs that are inhibitors or openers of the KATP channels, while the inwardly rectifying K+ (Kir) subunits form the ion channel. Two different SUR genes (SUR1 and SUR2) and two different Kir6.x genes (Kir6.1 and Kir6.2) have been identified. In addition, isoforms of SUR2, SUR2A and SUR2B, have been described. We have previously performed expression profiling on pooled human adipose tissue and found high expression of SUR2. Others have reported expression of SUR1 in human adipocytes. The aim of this study was to characterize the expression of the sulfonylurea receptor complex components in human adipose tissue. RT-PCR analysis, verified by restriction enzyme digestions and DNA sequencing, showed that SUR2B, Kir6.1 and alpha-endosulfine, but not SUR1, SUR2A or Kir6.2, are expressed in human adipose tissue. Real-time RT-PCR showed that SUR2B was expressed at higher levels in subcutaneous compared with omental adipose tissue in paired biopsies obtained from seven obese men (p < 0.05). Analysis of tissue distribution showed that SUR2B expression in adipose tissue was lower than that in muscle, similar to that in heart and liver, while the expression in pancreas was lower. The effect of caloric restriction was tested in obese men (n = 10) treated with very low calorie diet for 16 weeks, followed by a gradual reintroduction of ordinary food for 2 weeks. Biopsies were taken at week 0, 8 and 18. There was no consistent effect of weight reduction on SUR2B or Kir6.1 expression. We conclude that the necessary components for a local sulfonylurea system are expressed in human adipose tissue and that the sulfonylurea receptor complex in this tissue is composed of SUR2B and Kir6.1. The expression of SUR2B was higher in subcutaneous compared with omental adipose tissue and was not affected by weight loss.     

Assembly limits the pharmacological complexity of ATP-sensitive potassium channels

ATP-sensitive potassium channels (K(ATP) channels) are formed from an octameric complex of an inwardly rectifying K(+) channel (Kir6.1, Kir6.2) and a sulfonylurea receptor (SUR1, SUR2A, and SUR2B). In this study we have attempted to address the question of whether SUR heteromultimers can form using a combination of biochemical and electrophysiological approaches. We have constructed monoclonal stable lines in HEK293 cells co-expressing Kir6.2 with SUR1 and SUR2A. Using coimmunoprecipitation analysis with SUR isotype-specific antibodies two biochemical populations are distinguished, one containing SUR1 and the other SUR2A. It is not possible to detect immune complexes containing both SUR1 and SUR2A. Functional studies were undertaken and whole cell membrane currents were studied using the patch clamp. Concentrations of sulfonylureas and potassium channel openers were determined that selectively inhibited or activated SUR1/Kir6.2 and SUR2A/Kir6.2. In the cell line expressing SUR1/SUR2AKir6.2 we were unable to demonstrate a population of channels with unique pharmacological properties. Thus we conclude from these studies that heteromultimeric channel complexes containing both SUR1 and SUR2A are not formed, suggesting an incompatibility between different SUR subtypes. This incompatibility limits the pharmacological complexity of K(ATP) channels that may be observed in native tissues.     

Subunit composition of ATP-sensitive potassium channels in mitochondria of rat hearts

Mitochondrial ATP-sensitive potassium (mitoKATP) channels play a pivotal role in early and late ischemic preconditioning, but the subunit composition of mitoKATP channels remains unclear. In this study, we investigated the subunit composition of mitoKATP channels in rat hearts using confocal microscopy, immunofluorescence, and Western blot analysis. The green fluorescent probe glibenclamide-BODIPY was colocalized with the red fluorescent mitochondrial marker MitroTracker Red in isolated ventricular myocytes and in ventricular myocyte mitochondria, indicating the presence of sulfonylurea receptors (SURs) in the mitochondria. Anti-Kir6.1, anti-Kir6.2, and anti-SUR2 immunofluorescence was colocalized with that of MitoTracker Red in isolated mitochondria, suggesting that Kir6.1, Kir6.2, and SUR2 subunits are present in the mitochondria. Similarly, Kir6.1 (approximately 46 kDa), Kir6.2 (approximately 46 and approximately 40 kDa), and SUR2 (approximately 140 kDa) proteins were found to be expressed in mitochondria using Western blot analysis. By contrast, SUR1 was not present in mitochondria. These results suggest that mitoKATP channels in rat hearts might comprise a combination of Kir6.1, Kir6.2, and SUR2 subunits.     

PKA phosphorylation of SUR2B subunit underscores vascular KATP channel activation by beta-adrenergic receptors

ATP-sensitive K(+) (K(ATP)) channels are activated by several vasodilating hormones and neurotransmitters through the PKA pathway. Here, we show that phosphorylation at Ser1387 of the SUR2B subunit is critical for the channel activation. Experiments were performed in human embryonic kidney (HEK) 293 cells expressing the cloned Kir6.1/SUR2B channel. In whole cell patch, the Kir6.1/SUR2B channel activity was stimulated by isoproterenol via activation of beta(2) receptors. This effect was blocked in the presence of inhibitors for adenylyl cyclase or PKA. Similar channel activation was seen by exposing inside-out patches to the catalytic subunit of PKA. Because none of the previously suggested PKA phosphorylation sites accounted for the channel activation, we performed systematic mutational analysis on Kir6.1 and SUR2B. Two serine residues (Ser1351, Ser1387) located in the NBD2 of SUR2B were critical for the channel activation. In vitro phosphorylation experiments showed that Ser1387 but not Ser1351 was phosphorylated by PKA. The PKA-dependent activation of cell-endogenous K(ATP) channels was observed in acutely dissociated mesenteric smooth myocytes and isolated mesenteric artery rings, where activation of these channels contributed significantly to the isoproterenol-induced vasodilation. Taken together, these results indicate that the Kir6.1/SUR2B channel is a target of beta(2) receptors and that the channel activation relies on PKA phosphorylation of SUR2B at Ser1387.     

Expression of ATP sensitive K+ channel subunit Kir6.1 in rat kidney

ATP-sensitive K+ (K(ATP)) channels in kidney are considered to play roles in regulating membrane potential during the change in intracellular ATP concentration. They are composed of channel subunits (Kir6.1, Kir6.2), which are members of the inwardly rectifying K+ channel family, and sulphonylurea receptors (SUR1, SUR2A and SUR2B), which belong to the ATP-binding cassette superfamily. In the present study, we have investigated the expression and localization of Kir6.1 in rat kidney with Western blot analysis, immunohistochemistry, in situ hybridization histochemistry, and immunoelectron microscopy. Western blot analysis showed that Kir6.1 was expressed in the mitochondria and microsome fractions of rat kidney and very weakly in the membrane fractions. Immunohistochemistry revealed that Kir6.1 was widely distributed in renal tubular epithelial cells, glomerular mesangial cells, and smooth muscles of blood vessels. In immunoelectron microscopy, Kir6.1 is mainly localized in the mitochondria, endoplasmic reticulum (ER), and very weakly in cell membranes. Thus, Kir6.1 is contained in the kidney and may be a candidate of mitochondrial K(ATP) channels.     

ATP-sensitive potassium channel opener iptakalim protected against the cytotoxicity of MPP+ on SH-SY5Y cells by decreasing extracellular glutamate level

Mounting evidence reveals that ATP-sensitive potassium (K(ATP)) channel openers (KCOs) exert significant neuroprotection in vivo and in vitro in several models of Parkinson's disease (PD). However, the mechanisms are not well understood. In this study, we demonstrated that SH-SY5Y cells expressed mRNA and proteins for Kir6.1, Kir6.2, SUR1 and SUR2 subunits of K(ATP) channels. Moreover, our results showed that 1-methyl-4-phenyl-pyridinium ion (MPP+) induced up-regulation of mRNA for the Kir6.2 subunit and down-regulation of SUR1. It was further found that pretreatment with iptakalim, a novel K(ATP) channel opener, could attenuate increased extracellular glutamate level and decreased cell survival in SH-SY5Y cell culture after exposure to MPP+. Trans-pyrrolidine-2, 4-dicarboxylic acid (t-PDC), a glutamate transporter inhibitor, partially blocked the effect of iptakalim decreasing extracellular glutamate level. Additionally, iptakalim prevented MPP+-induced inhibition of glutamate uptake in primary cultured astrocytes. The beneficial effects of iptakalim on glutamate uptake of astrocytes were abolished by selective mitochondrial K(ATP) (mitoK(ATP)) channel blocker 5-HD. These results suggest (i) K(ATP) channel dysfunction may be involved in the mechanisms of MPP+-induced cytotoxicity and (ii) iptakalim may modulate glutamate transporters and subsequently alleviate the increase of extracellular glutamate levels induced by MPP+ through opening mitoK(ATP) channels, thereby protecting SH-SY5Y cells against MPP+-induced cytotoxicity.     

Physiological and pathophysiological roles of ATP-sensitive K+ channels

ATP-sensitive potassium (K(ATP)) channels are present in many tissues, including pancreatic islet cells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolic state to its membrane potential, playing a crucial role in various cellular functions. The K(ATP) channel is a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifier K(+) channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologous expression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B) reconstitute different types of K(ATP) channels with distinct electrophysiological properties and nucleotide and pharmacological sensitivities corresponding to the various K(ATP) channels in native tissues. The physiological and pathophysiological roles of K(ATP) channels have been studied primarily using K(ATP) channel blockers and K(+) channel openers, but there is no direct evidence on the role of the K(ATP) channels in many important cellular responses. In addition to the analyses of naturally occurring mutations of the genes in humans, determination of the phenotypes of mice generated by genetic manipulation has been successful in clarifying the function of various gene products. Recently, various genetically engineered mice, including mice lacking K(ATP) channels (knockout mice) and mice expressing various mutant K(ATP) channels (transgenic mice), have been generated. In this review, we focus on the physiological and pathophysiological roles of K(ATP) channels learned from genetic manipulation of mice and naturally occurring mutations in humans.     

A short motif in Kir6.1 consisting of four phosphorylation repeats underlies the vascular KATP channel inhibition by protein kinase C

Vascular ATP-sensitive K(+) channels are inhibited by multiple vasoconstricting hormones via the protein kinase C (PKC) pathway. However, the molecular substrates for PKC phosphorylation remain unknown. To identify the PKC sites, Kir6.1/SUR2B and Kir6.2/SUR2B were expressed in HEK293 cells. Following channel activation by pinacidil, the catalytic fragment of PKC inhibited the Kir6.1/SUR2B currents but not the Kir6.2/SUR2B currents. Phorbol 12-myristate 13-acetate (a PKC activator) had similar effects. Using Kir6.1-Kir6.2 chimeras, two critical protein domains for the PKC-dependent channel inhibition were identified. The proximal N terminus of Kir6.1 was necessary for channel inhibition. Because there was no PKC phosphorylation site in the N-terminal region, our results suggest its potential involvement in channel gating. The distal C terminus of Kir6.1 was crucial where there are several consensus PKC sites. Mutation of Ser-354, Ser-379, Ser-385, Ser-391, or Ser-397 to nonphosphorylatable alanine reduced PKC inhibition moderately but significantly. Combined mutations of these residues had greater effects. The channel inhibition was almost completely abolished when 5 of them were jointly mutated. In vitro phosphorylation assay showed that 4 of the serine residues were necessary for the PKC-dependent (32)P incorporation into the distal C-terminal peptides. Thus, a motif containing four phosphorylation repeats is identified in the Kir6.1 subunit underlying the PKC-dependent inhibition of the Kir6.1/SUR2B channel. The presence of the phosphorylation motif in Kir6.1, but not in its close relative Kir6.2, suggests that the vascular K(ATP) channel may have undergone evolutionary optimization, allowing it to be regulated by a variety of vasoconstricting hormones and neurotransmitters.     

Sulfonylurea receptors type 1 and 2A randomly assemble to form heteromeric KATP channels of mixed subunit composition

ATP-sensitive potassium (K(ATP)) channels play important roles in regulating insulin secretion, controlling vascular tone, and protecting cells against metabolic stresses. K(ATP) channels are heterooctamers of four pore-forming inwardly rectifying (Kir6.2) subunits and four sulfonylurea receptor (SUR) subunits. K(ATP) channels containing SUR1 (e.g. pancreatic) and SUR2A (e.g. cardiac) display distinct metabolic sensitivities and pharmacological profiles. The reported expression of both SUR1 and SUR2 together with Kir6.2 in some cells raises the possibility that heteromeric channels containing both SUR subtypes might exist. To test whether SUR1 can coassemble with SUR2A to form functional K(ATP) channels, we made tandem constructs by fusing SUR to either a wild-type (WT) or a mutant N160D Kir6.2 subunit. The latter mutation greatly increases the sensitivity of K(ATP) channels to block by intracellular spermine. We expressed, individually and in combinations, tandem constructs SUR1-Kir6.2 (S1-WT), SUR1-Kir6.2[N160D] (S1-ND), and SUR2A-Kir6.2[N160D] (S2-ND) in Xenopus oocytes, and studied the voltage dependence of spermine block in inside-out macropatches over a range of spermine concentrations and RNA mixing ratios. Each tandem construct expressed alone supported macroscopic K(+) currents with pharmacological properties indistinguishable from those of the respective native channel types. Spermine sensitivity was low for S1-WT but high for S1-ND and S2-ND. Coexpression of S1-WT and S1-ND generated current components with intermediate spermine sensitivities indicating the presence of channel populations containing both types of Kir subunits at all possible stoichiometries. The relative abundances of these populations, determined by global fitting over a range of conditions, followed binomial statistics, suggesting that WT and N160D Kir6.2 subunits coassemble indiscriminately. Coexpression of S1-WT with S2-ND also yielded current components with intermediate spermine sensitivities, suggesting that SUR1 and SUR2A randomly coassemble into functional K(ATP) channels. Further pharmacological characterization confirmed coassembly of not only S1-WT and S2-ND, but also of coexpressed free SUR1, SUR2A, and Kir6.2 into functional heteromeric channels.     

KATP channels in mesenchymal stromal stem cells: strong up-regulation of Kir6.2 subunits upon osteogenic differentiation

The promising use of mesenchymal stromal cells (MSC) in regenerative technologies accounts for necessity of detailed study of their physiology. Proliferation and differentiation of multipotent cells often involve changes in their metabolic state. In the present study, we analyzed the expression of ATP-sensitive potassium (K_ATP) channels in MSC and upon in vitro differentiation. K_ATP channels are present in many cells and regulate a variety of cellular functions by coupling cell metabolism with membrane potential. Kir6.1, Kir6.2 and SUR2A were expressed in undifferentiated MSC, whereas SUR2B and SUR1 were not detected on cDNA and protein level. Upon adipogenic differentiation Kir6.1 and SUR2A showed a significant reduction of the amount of mRNA by 84% and 95%, respectively, whereas Kir6.2 expression was unchanged. Osteogenic differentiation strongly up-regulated Kir6.2 mRNA (28-fold) whereas Kir6.1 and SUR2A showed no significant change in expression. Quantitative Western blot analysis and immunofluorescence staining confirmed the elevated expression of Kir6.2 upon osteogenic differentiation. Taken together, expression changes of K_ATP channels may contribute to in vitro differentiation of MSC and represent changes in the metabolic state of the developing tissue.     

Regulation of the ATP-sensitive potassium channel subunit, Kir6.2, by a Ca2+-dependent protein kinase C

The activity of ATP-sensitive potassium (K(ATP)) channels is governed by the concentration of intracellular ATP and ADP and is thus responsive to the metabolic status of the cell. Phosphorylation of K(ATP) channels by protein kinase A (PKA) or protein kinase C (PKC) results in the modulation of channel activity and is particularly important in regulating smooth muscle tone. At the molecular level the smooth muscle channel is composed of a sulfonylurea subunit (SUR2B) and a pore-forming subunit Kir6.1 and/or Kir6.2. Previously, Kir6.1/SUR2B channels have been shown to be inhibited by PKC, and Kir6.2/SUR2B channels have been shown to be activated or have no response to PKC. In this study we have examined the modulation of channel complexes formed of the inward rectifier subunit, Kir6.2, and the sulfonylurea subunit, SUR2B. Using a combination of biochemical and electrophysiological techniques we show that this complex can be inhibited by protein kinase C in a Ca(2+)-dependent manner and that this inhibition is likely to be as a result of internalization. We identify a residue in the distal C terminus of Kir6.2 (Ser-372) whose phosphorylation leads to down-regulation of the channel complex. This inhibitory effect is distinct from activation which is seen with low levels of channel activity.     

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.     

Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6.2-SUR1 interactions

ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.2 and regulatory SUR1 subunits, play a critical role in regulating insulin secretion. Binding of ATP to Kir6.2 inhibits, whereas interaction of MgATP with SUR1 activates, K(ATP) channels. We tested the functional effects of two Kir6.2 mutations (Y330C, F333I) that cause permanent neonatal diabetes mellitus, by heterologous expression in Xenopus oocytes. Both mutations reduced ATP inhibition and increased whole-cell currents, which in pancreatic beta-cells is expected to reduce insulin secretion and precipitate diabetes. The Y330C mutation reduced ATP inhibition both directly, by impairing ATP binding (and/or transduction), and indirectly, by stabilizing the intrinsic open state of the channel. The F333I mutation altered ATP binding/transduction directly. Both mutations also altered Kir6.2/SUR1 interactions, enhancing the stimulatory effect of MgATP (which is mediated via SUR1). This effect was particularly dramatic for the Kir6.2-F333I mutation, and was abolished by SUR1 mutations that prevent MgATP binding/hydrolysis. Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind/hydrolyse MgATP to open the mutant K(ATP) channel.