新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

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.     

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.     

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

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

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.     

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.     

Random assembly of SUR subunits in K(ATP) channel complexes

Sulfonylurea receptors (SURs) associate with Kir6.x subunits to form tetradimeric K(ATP) channel complexes. SUR1 and SUR2 confer differential channel sensitivities to nucleotides and pharmacological agents, and are expressed in specific, but overlapping, tissues. This raises the question of whether these different SUR subtypes can assemble in the same channel complex and generate channels with hybrid properties. To test this, we engineered dimeric constructs of wild type or N160D mutant Kir6.2 fused to SUR1 or SUR2A. Dimeric fusions formed functional, ATP-sensitive, channels. Coexpression of weakly rectifying SUR1-Kir6.2 (WTF-1) with strongly rectifying SUR1-Kir6.2[N160D] (NDF-1) in COSm6 cells results in mixed subunit complexes that exhibit unique rectification properties. Coexpression of NDF-1 and SUR2A-Kir6.2 (WTF-2) results in similar complex rectification, reflecting the presence of SUR1- and SUR2A-containing dimers in the same channel. The data demonstrate clearly that SUR1 and SUR2A subunits associate randomly, and suggest that heteromeric channels will occur in native tissues.     

Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: Possible role in glucose sensing

ATP‐sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose‐sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT‐1 and GLUT‐3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia‐like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT‐1 and GLUT‐3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT‐1 and GLUT‐3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes. J. Cell. Biochem. 114: 1879–1889, 2013. © 2013 Wiley Periodicals, Inc.     

ATP modulation of ATP-sensitive potassium channel ATP sensitivity varies with the type of SUR subunit

ATP-sensitive potassium (K(ATP)) channels comprise Kir and SUR subunits. Using recombinant K(ATP) channels expressed in Xenopus oocytes, we observed that MgATP (100 microm) block of Kir6.2/SUR2A currents gradually declined with time, whereas inhibition of Kir6.2/SUR1 or Kir6.2DeltaC36 currents did not change. The decline in Kir6.2/SUR2A ATP sensitivity was not observed in Mg(2+) free solution and was blocked by the phosphatidylinositol (PI) 3-kinase inhibitors LY 294002 (10 microm) and wortmannin (100 microm), and by neomycin (100 microm). These results suggest that a MgATP-dependent synthesis of membrane phospholipids produces a secondary decrease in the ATP sensitivity of Kir6.2/SUR2A. Direct application of the phospholipids PI 4,5-bisphosphate and PI 3,4,5-trisphosphate in the presence of 100 microm MgATP activated all three types of channel, but the response was faster for Kir6.2/SUR2A. Chimeric studies indicate that the different responses of Kir6.2/SUR2A and Kir6.2/SUR1 are mediated by the first six transmembrane domains of SUR. The MgATP-dependent loss of ATP sensitivity of Kir6.2/SUR2A was enhanced by the actin filament disrupter cytochalasin and blocked by phalloidin (which stabilizes the cytoskeleton). Phalloidin did not block the effect of PI 3,4,5-trisphosphate. This suggests that MgATP may cause disruption of the cytoskeleton, leading to enhanced membrane phospholipid levels (or better targeting to the K(ATP) channel) and thus to decreased channel ATP sensitivity.     

Structure-function relationships in the beta-cell K(ATP) channel

The ATP-sensitive potassium (K(ATP)) channel plays a key role in controlling beta-cell membrane potential and insulin secretion. The channels are composed of two subunits, Kir6.2, which forms the channel pore, and SUR1, which contains binding sites for nucleotides and sulphonylureas and acts as a channel regulator. Our current studies are aimed at delineating the molecular interactions involved in assembly and ligand binding by K(ATP) channel proteins. We have employed a complementation approach in which SUR1 half-molecules are co-expressed in insect cells using a baculovirus system. Together with data from truncated SUR1 molecules and a fusion protein in which SUR1 is linked to Kir6.2, we have interpreted our findings in terms of a model for the structure of the K(ATP) channel. The main features of the model are: (i) the C-terminal end of SUR1 is close to the N-terminus of Kir6.2; (ii) the two nucleotide binding domains (NBDs) of SUR1--NBD1 and NBD2--are in proximity; (iii) transmembrane helix 12 of SUR1 is orientated in such a way that it can make contact with Kir6.2; (iv) formation of the glibenclamide binding site requires that the two cytosolic loops (CLs) CL3 and CL8 are located close to each other; (v) there are homomeric interactions between the NBD1 domains of neighbouring subunits. We suggest that binding of glibenclamide leads to conformational changes in CL3 and CL8 leading to rearrangement of transmembrane helices. These effects are transmitted to Kir6.2 to result in channel closure.     

Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter

K(ATP) channels are large heteromultimeric complexes containing four subunits from the inwardly rectifying K+ channel family (Kir6.2) and four regulatory sulphonylurea receptor subunits from the ATP-binding cassette (ABC) transporter family (SUR1 and SUR2A/B). The molecular basis for interactions between these two unrelated protein families is poorly understood. Using novel trafficking-based interaction assays, coimmunoprecipitation, and current measurements, we show that the first transmembrane segment (M1) and the N terminus of Kir6.2 are involved in K(ATP) assembly and gating. Additionally, the transmembrane domains, but not the nucleotide-binding domains, of SUR1 are required for interaction with Kir6.2. The identification of specific transmembrane interactions involved in K(ATP) assembly may provide a clue as to how ABC proteins that transport hydrophobic substrates evolved to regulate other membrane proteins.     

Investigation of the molecular assembly of beta-cell K(ATP) channels

We have investigated the protein interactions involved in the assembly of pancreatic beta-cell ATP-sensitive potassium channels. The channels are a heterooligomeric complex of pore-forming Kir6.2 subunits and sulfonylurea receptor (SUR1) subunits. SUR1 belongs to the ATP binding cassette (ABC) family of proteins and has two nucleotide binding domains (NBD1 and NBD2) and 17 putative transmembrane (TM) sequences. Previously we showed that co-expression in a baculovirus expression system of two parts of SUR1 divided at Pro1042 between TM12 and 13 leads to restoration of glibenclamide binding activity, whereas expression of either individual N- or C-terminal domain alone gave no glibenclamide binding activity [M.V. Mikhailov and S.J.H. Ashcroft (2000) J. Biol. Chem. 275, 3360-3364]. Here we show that the two half-molecules formed by division of SUR1 between NBD1 and TM12 or between TM13 and 14 also self-assemble to give glibenclamide binding activity. However, deletion of NBD1 from the N-part of SUR1 abolished SUR1 assembly, indicating a critical role for NBD1 in SUR1 assembly. We found that differences in glibenclamide binding activity obtained after co-expression of different half-molecules are attributable to different amounts of binding sites, but the binding affinities remained nearly the same. Simultaneous expression of Kir6.2 resulted in enhanced glibenclamide binding activity only when the N-half of SUR1 included TM12. We conclude that TM12 and 13 are not essential for SUR1 assembly whereas TM12 takes part in SUR1 Kir6.2 interaction. This interaction is specific for Kir 6.2 because no enhancement of glibenclamide binding was observed when half-molecules were expressed together with Kir4.1. We propose a model of K(ATP) channel organisation based on these data.     

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits

ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia.     

KATP通道基因在大鼠组织中的表达

为检测大鼠肺动脉平滑肌及支气管平滑肌中KATP．通道亚基的表达情况,应用RT-PCR技术．从原代培养的Wistar大鼠肺动脉平滑肌及支气管平滑肌细胞第3—5代提取总RNA,逆转录．并进行PCR扩增鉴定．发现肺动脉平滑肌细胞有Kir6.1、SUR1和SUR2B的表达,其中SUR1表达量较弱,支气管平滑肌细胞有Kir6.1和SUR2B的表达。     

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.     

PKA-mediated phosphorylation of the human K(ATP) channel: separate roles of Kir6.2 and SUR1 subunit phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.     

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.