Diabetes mellitus is characterized by hyperglycemia, oxidative stress and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl. MGO can readily interact with proteins, lipids and DNA, and cause an imbalance of the cellular antioxidant system leading to carbonyl stress. The effects of MGO can be devastating if the targeted molecules are responsible for the maintenance of membrane potentials and ionic homeostasis. Here we show that MGO disrupts the vascular isoform of ATP-sensitive K+ (KATP) channels by acting on the mRNAs of Kir6.1 and SUR2B subunits thereby regulating vascular tone. Our results show that the 3’ untranslated region (UTR) of Kir6.1 mRNA and the coding region of SUR2B mRNA are targeted by MGO causing a disruption of vascular KATP channels. The destabilization of the mRNAs of KATP channel can in turn affect K+ homeostasis of vascular smooth muscles as well as vascular responses to circulating vasodilators and vasoconstrictors.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

Contractility of vascular smooth muscles (VSMs) in resistance arteries determines systemic blood pressure and blood supplies to local tissues, in which ATP sensitive K+ (KATP) channels play a role. The KATP channels that couple metabolic state to cellular activity are activated by multiple hormonal vasodilators and inhibited by vasoconstrictors. To understand the molecular mechanisms for the channel regulation by vasodilators, we studied the effects of β-adrenergic receptors on Kir6.1/SUR2B in HEK cells. Stimulation of β-adrenergic receptors activated the channels, which relied on the GS-protein, adenylyl cyclase, cAMP and PKA system. Using mutational analysis, we scanned all the putative PKA sites on Kir6.1 and SUR2B subunits and identified two residues (Ser1351 and Ser1387) in SUR2B critical for channel activation. In vitro phosphorylation experiments confirmed that Ser1387 but not Ser1351 was phosphorylated in isolated SUR2B peptides. Molecular modeling and molecular dynamics simulations reveal that phosphorylation at Ser1387 causes interdomain movements in SUR2B subunit. Blockage of the movements by engineering a disulfide bond across NBD2 and TMD1 eliminated the PKA-dependent channel activation. We also studied the molecular basis for the inhibition of vascular KATP channels by PKC. In the HEK expression system, we found that the Kir6.1/SUR2B channel but not the Kir6.2/SUR2B was drastically inhibited by PKC stimulation. We constructed Kir6.1/Kir6.2 chimeras and identified two critical protein domains for the Kir6.1 channel inhibition by PKC. The distal C-terminus was the direct target of PKC where multiple phosphorylation sites were identified. These phosphorylation sites were located in a short sequence with stereotypical sequence repeats. Mutation of any decreased the effects of PKC. Joint mutation of all of them prevented the channel inhibition by PKC. The proximal N-terminus is also involved in PKC effects without phosphorylation sites, suggesting it may play a role in channel gating. Thus, this thesis provides experimental evidence for the vascular KATP channel modulation by PKA and PKC. Phosphorylation of the Kir6.1 and SUR2B subunits by PKC and PKA produce inhibition and activation of the vascular KATP channel, respectively, which appears to be one of the molecular bases contributing to vascular tone regulation by both vasoconstricting and vasodilating hormones and neurotransmitters.

Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

The KATP channels play an important role in the membrane excitability and vascular tone regulation. Previous studies indicate that the function of KATP channels is disrupted in oxidative stress seen in a variety of cardiovascular diseases, while the underlying mechanism remains unclear. Here, we demonstrate S-glutathionylation to be a modulation mechanism underlying the oxidant-mediated vascular KATP channel inhibition, the molecular basis for the channel inhibition and the alleviation of the channel inhibition by vasoactive intestinal peptide (VIP). We found that an exposure of isolated mesenteric rings to H2O2 impaired the KATP channel-mediated vascular dilation. In whole-cell recordings and inside-out patches, micromolar H2O2 or diamide caused a strong inhibition of the vascular KATP channel (Kir6.1/SUR2B) in the presence, but not in the absence, of glutathione (GSH), indicating S-glutathionylation. By co-expressions of Kir6.1 or Kir6.2 with SUR2B subunits, we found that the oxidant sensitivity of the KATP channel relied on the Kir6.1 subunit. Systematic mutational analysis revealed three cysteine residues (Cys43, Cys120 and Cys176) to be important. Among them, Cys176 was prominent, contributing to >80% oxidant sensitivity. Biochemical pull-down assay with biotinylated glutathione ethyl ester (BioGEE) showed that mutations of Cys176 impaired the oxidant-induced incorporation of GSH to the Kir6.1 subunit. Simulation modeling of Kir6.1 S-glutathionylation revealed that after incorporation to residue 176, the GSH moiety occupied a space between slide helix and two transmembrane helices. This prevented the necessary conformational change of the inner helix for channel gating, and retained the channel in its closed state. VIP is a potent vasodilator, and is shown to have protective role against oxidative stress. We found that the channel was strongly augmented by VIP and the channel activation relied on PKA phosphorylation. These results therefore indicate that 1) the vascular KATP channel is strongly inhibited in oxidative stress, 2) S-glutathionylation underlies the oxidant-mediated KATP channel inhibition, 3) Cys176 in the Kir6.1 subunit is the major site for S-glutathionylation, and 4) the Kir6.1/SUR2B channel is activated in a PKA-dependent manner by VIP that has been previously shown to alleviate oxidative stress.