It has been shown that oxidative stress and activation of the c-Jun N-terminal kinase (JNK) pathway induce the nucleocytoplasmic translocation of the pancreatic transcription factor PDX-1, which leads to pancreatic -cell dysfunction. In this study, we have shown that the forkhead transcription factor Foxo1/FKHR plays a role as a mediator between the JNK pathway and PDX-1. Under oxidative stress conditions, Foxo1 changed its intracellular localization from the cytoplasm to the nucleus in the pancreatic -cell line HIT-T15. The overexpression of JNK also induced the nuclear localization of Foxo1, but in contrast, suppression of JNK reduced the oxidative stress-induced nuclear localization of Foxo1, suggesting the involvement of the JNK pathway in Foxo1 translocation. In addition, oxidative stress or activation of the JNK pathway decreased the activity of Akt in HIT cells, leading to the decreased phosphorylation of Foxo1 following nuclear localization. Furthermore, adenovirus-mediated Foxo1 overexpression reduced the nuclear expression of PDX-1, whereas repression of Foxo1 by Foxo1-specific small interfering RNA retained the nuclear expression of PDX-1 under oxidative stress conditions. Taken together, Foxo1 is involved in the nucleocytoplasmic translocation of PDX-1 by oxidative stress and the JNK pathway.
The ATP-sensitive K(+) (K(ATP)) channels are composed of the pore-forming K(+) channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle-type K(ATP) channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K(ATP) channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K(ATP) channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K(ATP) channels containing different SURs.
SUMMARY1. The ATP-regulated potassium channel (KiATP) was investigated with respect to modulation by intracellular pH (pHi) by using the inside-out membrane patch clamp technique in ventricular cells isolated from the heart of the guinea-pig. Channels which had been closed by internal ATP (0 3-3 mM) were dose-dependently activated by decreasing the pHi over the range of pH 7-6-6-0. However, the channel was conversely inhibited when the pHi was further decreased below 6-0. Inwardly rectifying K+ channels were also decreased in activity when pHi fell from 7-2 to 6-0.2. The channel activation was also observed with constant concentration of free Ca2+ (1 nM) and Mg2+ (1 mM) in the bathing solution, suggesting that a change in divalent cation concentration is not involved in channel modulation by pHi.3. When the dose-response relations of the channel activity for ATP concentrations at different pHi were examined, the channel activity obtained at 1 /tM ATP was increased by decreasing pH from 7-2 to 6-4. The half-maximal inhibition for ATP concentration at pH 7-2 and 6-4 was 20 and 40 ,(M, respectively, and the Hill coefficient was 2-5 in both curves.4. In the absence of ATP, internal H+ was able to reactivate run-down channels but it had less effect on the channel as long as the activity was maintained at a higher level. The increase in the channel activity by H+ was facilitated with a proceeding of the run-down. However, after the channel was completely inactivated by a long exposure of the membrane patch to ATP-free solution, a reduction of pH could not activate the channel.5. The decrease of pH from 7-2 to 6-4 reduced single channel conductance from 89-0 to 77-7 pS in the absence of Mg2+, whereas it reduced the conductance only at the negative membrane potentials in the presence of 2 mm Mg2+.6. Mean open and closed times within the burst-like openings of the channel remained unaffected during the change in pHi.7. We conclude that the cardiac KATP channel is modulated by a change in the intracellular pH. The channel modulation consisted of the increase in the channel * To whom offprints should be requested. MS 9964T. KOYANO AND OTHERS activity and a decrease in the permeability. The former effect was due to the decrease in the sensitivity of the channel to ATP and the reactivation of the channel which is during the process of run-down in activity.
Abstract-ATP-sensitive K ϩ (K ATP ) channels are composed of sulfonylurea receptors (SURs) and inwardly rectifying Kir6.2-channels. The C-terminal 42 amino acid residues (C42) of SURs are responsible for ADP-induced differential activation of K ATP channels in SUR-subtypes. By examining ADP-effect on K ATP channels containing various chimeras of SUR2A and SUR2B, we identified a segment of 7 residues at central portion of C42 critical for this phenomenon. A 3-D structure model of the region containing the second nucleotide-binding domain (NBD2) of SUR and C42 was developed based on the structure of HisP, a nucleotide-binding protein forming the bacterial Histidine transporter complex. In the model, the polar and charged residues in the critical segment located within a distance that allows their electrostatic interaction with Arg1344 at the Walker-A loop of NBD2. Therefore, the interaction might be involved in the control of ADP-induced differential activation of SUR2-subtype K ATP channels. A TP-sensitive K ϩ (K ATP ) channels are inhibited by intracellular ATP and activated by nucleoside diphosphates and thus provide a link between the metabolic state and cellular excitability in various organs, including pancreas, heart, vasculature, and brain. 1 These channels are associated with such cellular functions as insulin secretion, cardiac preconditioning, vasodilatation, and neurotransmitter release. K ATP channels are heterooctamers composed of an ATPbinding cassette protein, known as the sulfonylurea receptor (SUR), and an inwardly rectifying K ϩ channel (Kir) subunit, Kir6.0. 2,3 Three kinds of SUR, SUR1, SUR2A, and SUR2B, have been isolated and their roles in formation of functional K ϩ channels with Kir6.0 subunits have been extensively examined. [2][3][4][5][6] It is now widely accepted that SUR1, SUR2A, and SUR2B represent pancreatic, cardiac, and vascular smooth muscle types of SUR, respectively. When expressed with Kir6.2, all 3 subtypes of SUR form K ATP channels that exhibit the same single-channel characteristics; weak inwardrectification and a unitary conductance of Ϸ80 pS in the inward-direction with 150 mmol/L extracellular K ϩ . They, however, show distinct sensitivities to different vasorelaxant K ϩ channel opener compounds (KCOs), intracellular MgADP, and sulfonylurea drug derivatives. [7][8][9][10][11] The different pharmacological sensitivities depend on the SUR-subtype. SUR has been assigned to the ATP-binding-cassette (ABC) superfamily, as is assumed to possess 17 transmembrane segments and 2 nucleotide-binding domains (NBDs) with Walker-A and -B consensus motifs for binding intracellular nucleotides. 12,13 However, structural elements of SURs and their functional roles responsible for their different features have not been fully determined.The majority of ABC proteins are active transporters, utilizing the energy of ATP hydrolysis to pump solutes and small substances across the membrane. SUR on the other hand regulates the behavior of Kir6.0 channel pores, and thus, the role of the 2 NBDs dif...
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