ATP suppresses the K(+) current responses to FSH and adenosine in the follicular cells of Xenopus oocyte

The application of either follicle-stimulating hormone (FSH) or adenosine (Ade) induces a K(+)-current response in the follicular cells surrounding a Xenopus oocyte under a voltage clamp. These K(+)-current responses are reported to be produced by an increase in intracellular cAMP. A previous application of ATP to the same cells markedly depressed the K(+)-current responses to FSH and Ade. Furthermore, a 2 min application of phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C (PKC), significantly depressed the K(+)-current responses to FSH and Ade, but it had no significant effect on the Cl(-)-current response to ATP. An application of either ATP or PDBu also depressed the K(+)-current response induced by intracellularly applied cAMP. In contrast to the effect of PDBu, the application of 1-octanol, an inhibitor of gap junction channel, significantly depressed both the Ade- and ATP-induced responses, indicating that the acting site of 1-octanol is different from that of PKC. The results suggest that the depressing effect of ATP on the FSH- and Ade-induced K(+)-current responses might be mediated by PKC activation and that the site of PKC action might be downstream of the cAMP production involved in the K(+) channel opening.     

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.     

Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells

We have monitored whole-cell and single channel ATP-sensitive K+ (KATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell KATP conductance (Ki = 6 microM) and initiated action potential firing. The K+ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K+ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited KATP channel activity with a Ki of 17 microM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect KATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated KATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding KATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express KATP channels with the nucleotide-, sulphonylurea- and K+ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.     

Developmental expression and functional significance of Kir channel subunits in ureteric bud and nephron epithelia

Kir channel subunit expression during development of the rat collecting-duct epithelium was quantified by RT-PCR of primary monolayer cultures. mRNAs of the vascular-type K(ATP) (K(NDP)) channel-forming subunits Kir6.1/SUR2 were highly expressed in early ureteric bud generations (embryonic day E14) and downregulated thereafter, while Kir1.1b (ROMK2) mRNA increased fourfold during cortical collecting duct (CCD) maturation. As assessed by immunohistochemistry, Kir6.1 protein was abundant in the apical and basolateral plasma membranes of early ureteric buds and trunks (E15 to postnatal day P1), downregulated thereafter and not detectable in CCD and outer medullary collecting ducts (OMCD) (P7). During nephron development, Kir6.1 protein was expressed ubiquitously on plasma membranes of early nephron stages from mesenchymal condensations to S-shaped bodies. After fusion of nephron and CCD, Kir6.1 protein was restricted to the apical membrane of proximal tubule. The Kir6/SUR2 channel opener, pinacidil (100 microM/2 days), increased tubulogenesis in organ culture by a factor of 3. Cell proliferation of human embryonic kidney cells (HEK 293) which endogenously express Kir6.1/SUR2 mRNA was stimulated by pinacidil in a dose-dependent manner, an effect that was partially abolished by glibenclamide (3 microM). In summary, Kir6.1/SUR2 channel subunits are highly expressed during early development of ureteric bud and nephron epithelia where Kir6.1/SUR2 activity regulates cell proliferation.     

The regulation of rotenone-induced inflammatory factor production by ATP-sensitive potassium channel expressed in BV-2 cells

Our previous studies have demonstrated that activating ATP-sensitive potassium channel (K(ATP) channel), not only improved Parkinsonian behavior and neurochemical symptoms, but also reduced iNOS activity and mRNA levels in striatum and nigra of rotenone rat model of Parkinson's disease (PD). In this study, it was first shown that the subunits of K(ATP) channels are expressed in BV-2 cells, and then it was investigated whether K(ATP) channel was involved in regulating inflammatory factor production from BV-2 cells activated by rotenone. It was found that K(ATP) channel was expressed in BV-2 cell and formed by the combination of Kir 6.1 and SUR 2A/2B. K(ATP) channel openers (KCOs) including pinacidil, diazoxide and iptakalim (Ipt) exerted beneficial effects on rotenone-induced morphological alterations of BV-2 cells, decreased tumor necrosis factor alpha (TNF-alpha) production and the expression and activity of inducible isoform of nitric oxide synthase (iNOS). Either glibenclamide or 5-hydroxydecanoate acid (a selective mitochondrial K(ATP) channel blocker) could abolish the effects of KCOs, suggesting that K(ATP) channels, especially mitochondrial ATP-sensitive potassium channels (mitoK(ATP) channels), played a crucial role in preventing the activation of BV-2 cells, and subsequently the production of a variety of proinflammatory factors. Therefore, activation of K(ATP) channel might be a new therapeutic strategy for treating neuroinflammatory and neurodegenerative disorders.     

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.     

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.     

Differential distribution of Kir4.1 in spinal cord astrocytes suggests regional differences in K+ homeostasis

Neuronal activity in the spinal cord results in extracellular potassium accumulation that is significantly higher in the dorsal horn than in the ventral horn. This is suggestive of differences in K(+) clearance, widely thought to involve diffusional K(+) uptake by astrocytes. We previously identified the inward rectifying K(+) channel Kir4.1 as the major K(+) conductance in spinal cord astrocytes in situ and hence hypothesized that different expression levels of Kir4.1 may account for the observed differences in potassium dynamics in spinal cord. Our results with immunohistochemical staining demonstrated highest Kir4.1 channel expression in the ventral horn and very low levels of Kir4.1 in the apex of the dorsal horn. Western blots from tissue of these two regions similarly confirmed much lower levels of Kir4.1 in the apex of the dorsal horn. Whole cell patch-clamp recordings from astrocytes in rat spinal cord slices also showed a difference in inwardly rectifying currents in these two regions. However, no statistical difference in either fast-inactivating (Ka) or delayed rectifying potassium currents (Kd) was observed, suggesting these differences were specific to Kir currents. Importantly, when astrocytes in each region were challenged with high [K(+)](o), astrocytes from the dorsal horn showed significantly smaller (60%) K(+) uptake currents than astrocytes from the ventral horn. Taken together, these data support the conclusion that regional differences in astrocytic expression of Kir4.1 channels result in marked changes in potassium clearance rates in these two regions of the spinal cord.     

Cirrhosis decreases vasoconstrictor response to electrical field stimulation in rat mesenteric artery: role of calcitonin gene-related peptide

Our study determines alterations in the vasoconstrictor response elicited by electric field stimulation (EFS) in mesenteric arteries from cirrhotic rats treated with CCl(4), and how calcitonin gene-related peptide (CGRP) participates in this response. Vasoconstriction induced by EFS was analysed in the absence and presence of the CGRP receptor antagonist CGRP(8-37) in arterial segments from control and cirrhotic rats. The vasodilator response to exogenous CGRP was tested in both groups of rats, and the interference of the guanylate cyclase inhibitor ODQ or the K(ATP) channel blocker glibenclamide was analysed only in segments from cirrhotic rats. The vasodilator response to the K(ATP) channel opener pinacidil and to 8-bromo-cyclic GMP was tested. The K(ATP) currents were recorded using the patch-clamp technique. Expression of receptor activity-modifying protein 1 (RAMP1), calcitonin receptor-like receptor, Kir 6.1 and sulfonylurea receptor 2B (SUR2B) was also analysed. Release of CGRP and cGMP was measured. The EFS-elicited vasoconstriction was less in segments from cirrhotic rats. The presence of CGRP(8-37) increased the EFS-induced response only in segments from cirrhotic rats. The CGRP-induced vasodilatation was greater in segments from cirrhotic rats, and was inhibited by ODQ or glibenclamide. Both pinacidil and 8-bromo-cyclic GMP induced a stronger vasodilator response in segments from cirrhotic rats. Pinacidil induced greater K(ATP) currents in cirrhotic myocytes. Expression of RAMP1, calcitonin receptor-like receptor, Kir 6.1 and SUR2B was not modified by liver cirrhosis. Liver cirrhosis increased CGRP release, but did not modify cGMP formation. The decreased vasoconstrictor response to EFS in cirrhosis is mediated by increased vasodilator response to CGRP, as well as increased K(ATP) channel gating. This effect of CGRP may play a role in the splanchnic vasodilatation present in liver cirrhosis.     

ATP-sensitive K+ channels in rat colonic epithelium

ATP-sensitive K⁺ (K_ATP) channels couple the metabolic state of a cell to its electrical activity. They consist of a hetero-octameric complex with pore-forming Kir6.x (Kir6.1, Kir6.2) and regulatory sulfonylurea receptor (SUR) subunits. Functional data indicate that K_ATP channels contribute to epithelial K⁺ currents at colonic epithelia. However, their molecular identity and their properties are largely unknown. Therefore, changes in short-circuit current (I _sc) induced by the K_ATP channel opener pinacidil (5 10^?4?mol?l^?1) were measured in Ussing chambers under control conditions and in the presence of different blockers of K_ATP channels. The channel subunits expressed by the colonic epithelium were identified by immunohistochemistry and by RT-PCR. The K⁺ channel opener, when administered at the serosal side, induced an increase in I _sc consistent with the induction of transepithelial Cl^? secretion after activation of basolateral K⁺ channels, whereas mucosal administration of pinacidil resulted in a negative I _sc. The increase in I _sc evoked by serosal pinacidil was inhibited by serosal administration of glibenclamide (5 10^?4?mol l^?1) and gliclazide (10^?6?mol l^?1), but was resistant even against a high concentration (10^?2?mol l^?1) of tolbutamide. In contrast, none of these inhibitors (administered at the mucosal side) reduced significantly the negative I _sc induced by mucosal pinacidil. Instead, pinacidil inhibited Cl^? currents across apical Cl^? channels in basolaterally depolarized epithelia indicating that the negative I _sc induced by mucosal pinacidil is due to a transient inhibition of Cl^? secretion. In mRNA prepared from isolated colonic crypts, messenger RNA for both pore-forming subunits, Kir6.1 and Kir6.2, and two regulatory subunits (SUR1and SUR2B) was found. Expression within the colonic epithelium was confirmed for these subunits by immunohistochemistry. In consequence, K_ATP channels are present in the basolateral membrane of the colonic epithelium; their exact subunit composition, however, has still to be revealed.     

CO2-dependent opening of an inwardly rectifying K+ channel

CO(2) chemosensing is a vital function for the maintenance of life that helps to control acid-base balance. Most studies have reported that CO(2) is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO(2) under conditions of constant extracellular pH. As PCO(2) increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of -70?mV, which lay between the K(+) and Cl(-) equilibrium potentials. This reversal potential was shifted by +61?mV following a tenfold increase in extracellular [K(+)] but was insensitive to variations of extracellular [Cl(-)]. The single-channel conductance increased with extracellular [K(+)]. We propose that this channel is a member of the Kir family. In addition to this K(+) channel, we found that many of the excised patches also contained a conductance carried via a Cl(-)-selective channel. This CO(2)-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO(2)-dependent inhibition during hypercapnia.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Physiological and pharmacological characteristics of quisqualic acid-induced K(+)-current response in the ganglion cells of Aplysia

The extracellular application of either quisqualic acid (QA) or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces an outward current in identified neurons of Aplysia ganglion under voltage clamp. The time course of the QA-induced response is significantly slower than that induced by FMRFamide. The reversal potential for both responses was -92 mV and was shifted 17 mV in a positive direction for a twofold increase in the extracellular K(+) concentration. The QA-induced response was markedly depressed in the presence of Ba(2+), a blocker of inward rectifier K(+)-channel, whereas TEA, a Ca(2+)-activated K(+)-channel (BK(Ca)) blocker, or 4-AP, a transient K(+) (A)-channel blocker, had no effect on the response. The QA-induced K(+)-current was significantly suppressed by CNQX and GYKI52466, antagonists of non-NMDA receptors. However, the application of either kainate or AMPA, agonists for non-NMDA receptors, produced no type of response in the same neurons. The QA-induced K(+)-current response was not depressed at all by an intracellular injection of either guanosine 5'-O-(2-thiodiphosphate) (GDP-betaS) or guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS), but the FMRFamide-induced response was markedly blocked by both GDP-betaS and GTP-gammaS in the same cell. Furthermore, the QA- and FMRFamide-induced K(+)-current responses were both decreased markedly when the temperature was lowered to 15 degrees C, from 23 degrees C. These results suggested that the QA-induced K(+)-current response is produced by an activation of a novel type of QA-receptor and that this response is not produced by an activation of the G protein.     

Different Localization of ATP Sensitive K+ Channel Subunits in Rat Testis

ATP sensitive K⁺ (K_ATP) channels are important linkage of cell membrane excitability to its cellular bioenergetic state. These channels are composed of pore‐forming subunits and regulatory subunits. The present study focused on the cellular expressions and localizations of these subunits in rat testis. RT‐PCR analysis showed that rat testis contained five K_ATP channel subunits, Kir6.1, Kir6.2, SUR1, SUR2A and SUR2B. Immunoblot assay showed that proteins of Kir6.1, Kir6.2, SUR2A and SUR2B were expressed in rat testis. Immunohistochemistry revealed these K_ATP channel subunits were positive in different localizations of spermatogenic cells, Sertoli cells and Leydig cells, which implies these subunits playing important roles in spermatogenesis. Co‐localization of Kir6.2 with SUR2B was determined in acrosome or head cap of spermatids by double immunofluorescence analysis by indicating K_ATP channel might be formed by Kir6.2 and SUR2B in acrosome of spermatids. Different localizations of the K_ATP channel subunits in the cell membrane and membranous organelles of spermatogenic cells and Sertoli cells indicated the complex and multiple functions of K_ATP channels in rat testis. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Mechanism of action of a sulphonylurea receptor SUR1 mutation (F132L) that causes DEND syndrome

Activating mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 and SUR1 are a common cause of neonatal diabetes. Here, we analyse the molecular mechanism of action of the heterozygous mutation F132L, which lies in the first set of transmembrane helices (TMD0) of SUR1. This mutation causes severe developmental delay, epilepsy and permanent neonatal diabetes (DEND syndrome). We show that the F132L mutation reduces the ATP sensitivity of K(ATP) channels indirectly, by altering the intrinsic gating of the channel. Thus, the open probability is markedly increased when Kir6.2 is co-expressed with mutant TMD0 alone or with mutant SUR1. The F132L mutation disrupts the physical interaction between Kir6.2 and TMD0, but does not alter the plasmalemma channel density. Our results explain how a mutation in an accessory subunit can produce enhanced activity of the K(ATP) channel pore (formed by Kir6.2). They also provide further evidence that interactions between TMD0 of SUR1 and Kir6.2 are critical for K(ATP) channel gating and identify a residue crucial for this interaction at both physical and functional levels.     

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells

 To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5′-triphosphate (ATP)-sensitive K⁺ (K_ATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal K_ATP currents (at 0 mV) induced by metabolic inhibition were 48.9±2.8 pA/pF in control (n=48), 34.3±3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5±3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na⁺ current and inward-rectifying K⁺ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of K_ATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional K_ATP channels. Furthermore, in four out of seven control cells tested, outward K⁺ currents were stimulated by diazoxide, which is a potent K⁺ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional K_ATP channels. The larger population of K_ATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2. Received: 29 May 1998 / Received after revision: 8 September 1998 / Accepted: 13 October 1998