Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells

1995 ◽  
Vol 269 (5) ◽  
pp. H1625-H1633 ◽  
Author(s):  
W. A. Coetzee ◽  
T. Y. Nakamura ◽  
J. F. Faivre
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Channel Activity ◽  
Nitrobenzoic Acid ◽  
Ventricular Cells ◽  
Channel Opening ◽  
Sh Group ◽  
Constant Potential ◽  
High Degree

ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of ATP-sensitive K+ channels by ATP and nucleotide diphosphate in rabbit portal vein

1994 ◽  
Vol 266 (5) ◽  
pp. H1687-H1698 ◽  
Author(s):  
M. Kamouchi ◽  
K. Kitamura
Keyword(s):  
Portal Vein ◽  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
K Channel ◽  
Channel Activity ◽  
Rabbit Portal Vein ◽  
Internal Side ◽  
The Right ◽  
Single Channel Currents

The modulation of ATP-sensitive K+ (KATP)-channel activity was investigated by recording single-channel currents in isolated smooth muscle cells from rabbit portal vein. K(+)-channel openers (KCOs; pinacidil, lemakalim, and nicorandil) induced burstlike openings of single KATP channels in the cell-attached configuration. After patch excision, KATP channels showed "run-down" phenomenon in the presence of KCOs, but subsequent application of Mg-ATP (1 mM) restored KATP-channel activity. Removal of Mg-ATP resulted in transient augmentation of KATP currents, which eventually decayed out. Nucleotide diphosphates (NDPs; GDP, ADP, UDP, IDP, and CDP) also induced channel reopening in the presence of KCOs, which was markedly enhanced by addition of Mg2+ in millimolar concentrations at the internal side of the membrane. The dose-response relation between ATP and the UDP-induced KATP-channel activity was shifted to the right in the presence of Mg2+ (2 mM). These results suggest that intracellular ATP, NDPs, and Mg2+ regulate the channel state of KATP channels (operative and inoperative states) and that KCOs open KATP channels only in the operative state.

scholarly journals Ligand-insensitive State of Cardiac ATP-sensitive K+ Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 381-394 ◽  
Author(s):  
Alexey E. Alekseev ◽  
Peter A. Brady ◽  
Andre Terzic
Keyword(s):  
Kinetic Model ◽  
Katp Channel ◽  
Katp Channels ◽  
Cardiac Cell ◽  
Channel Activity ◽  
K Channels ◽  
Channel Interaction ◽  
Channel Opening ◽  
State Kinetic ◽  
Allosteric Model

The mechanism by which ATP-sensitive K+ (KATP) channels open in the presence of inhibitory concentrations of ATP remains unknown. Herein, using a four-state kinetic model, we found that the nucleotide diphosphate UDP directed cardiac KATP channels to operate within intraburst transitions. These transitions are not targeted by ATP, nor the structurally unrelated sulfonylurea glyburide, which inhibit channel opening by acting on interburst transitions. Therefore, the channel remained insensitive to ATP and glyburide in the presence of UDP. “Rundown” of channel activity decreased the efficacy with which UDP could direct and maintain the channel to operate within intraburst transitions. Under this condition, the channel was sensitive to inhibition by ATP and glyburide despite the presence of UDP. This behavior of the KATP channel could be accounted for by an allosteric model of ligand-channel interaction. Thus, the response of cardiac KATP channels towards inhibitory ligands is determined by the relative lifetime the channel spends in a ligand-sensitive versus -insensitive state. Interconversion between these two conformational states represents a novel basis for KATP channel opening in the presence of inhibitory concentrations of ATP in a cardiac cell.

Cardiac ATP-sensitive K+ channels: regulation by intracellular nucleotides and K+ channel-opening drugs

1995 ◽  
Vol 269 (3) ◽  
pp. C525-C545 ◽  
Author(s):  
A. Terzic ◽  
A. Jahangir ◽  
Y. Kurachi
Keyword(s):  
Katp Channel ◽  
Molecular Mechanisms ◽  
Enzymatic Reaction ◽  
Katp Channels ◽  
Cardiac Cells ◽  
K Channel ◽  
Channel Activity ◽  
Pharmacological Agents ◽  
Channel Opening ◽  
Main Regulator

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells where they regulate cardiac function during cellular metabolic impairment. KATP channels have been implicated in the shortening of the action potential duration and the cellular loss of K+ that occurs during metabolic inhibition. KATP channels have been associated with the cardioprotective mechanism of ischemia-related preconditioning. Intracellular ATP (ATPi) is the main regulator of KATP channels. ATPi has two functions: 1) to close the channel (ligand function) and 2) in the presence of Mg2+, to maintain the activity of KATP channels (presumably through an enzymatic reaction). KATP channel activity is modulated by intracellular nucleoside diphosphates that antagonize the ATPi-induced inhibition of channel opening or induce KATP channels to open. How nucleotides will affect KATP channels depends on the state of the channel. K+ channel-opening drugs are pharmacological agents that enhance KATP channel activity through different mechanisms and have great potential in the management of cardiovascular conditions. KATP channel activity is also modulated by neurohormones. Adenosine, through the activation of a GTP-binding protein, antagonizes the ATPi-induced channel closure. Understanding the molecular mechanisms that underlie KATP channel regulation should prove essential to further define the function of KATP channels and to elucidate the pharmacological regulation of this channel protein. Since the molecular structure of the KATP channel has now become available, it is anticipated that major progress in the KATP channel field will be achieved.

Diadenosine Tetraphosphate-Gating of Recombinant Pancreatic ATP-Sensitive K+ Channels

2001 ◽  
Vol 21 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Sofija Jovanovic ◽  
Aleksandar Jovanovic
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Single Channels ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Concentration Dependent Manner ◽  
Diadenosine Tetraphosphate ◽  
Channel Opening

Diadenosine tetraphosphate (Ap4A) has been recently discovered in the pancreatic γ cells where targets ATP-sensitive K+ (KATP) channels, depolarizes the cell membrane and induces insulin secretion. However, whether Ap4A inhibit pancreatic KATP channels by targeting protein channel complex itself was unknown. Therefore, we coexpressed pancreatic KATP channel subunits, Kir6.2 and SUR1, in COS-7 cells and examined the effect of Ap4A on the single channel behavior using the inside-out configuration of the patch-clamp technique. Ap4A inhibited channel opening in a concentration-dependent manner. Analysis of single channels demonstrated that Ap4A did not change intraburst kinetic behavior of KATP channels, but rather decreased burst duration and increased between-burst duration. It is concluded that Ap4A antagonizes KATP channel opening by targeting channel subunits themselves and by keeping channels longer in closed interburst states.

KATP channel opening does not contribute significantly to the vasodilatory effect of SH-group-containing ACE inhibitors

1996 ◽  
Vol 11 (4) ◽  
pp. 192-196 ◽  
Author(s):  
H. Köppel ◽  
S. Holzmann ◽  
W. Klein ◽  
E. Horn ◽  
S. Horn ◽  
...  
Keyword(s):  
Ace Inhibitors ◽  
Katp Channel ◽  
Vasodilatory Effect ◽  
Channel Opening ◽  
Sh Group

Blockade of Adenosine Triphosphate–sensitive Potassium Channels by Thiamylal in Rat Ventricular Myocytes

2000 ◽  
Vol 92 (4) ◽  
pp. 1154-1159 ◽  
Author(s):  
Yasuo Tsutsumi ◽  
Shuzo Oshita ◽  
Hiroshi Kitahata ◽  
Yasuhiro Kuroda ◽  
Takashi Kawano ◽  
...  
Keyword(s):  
Adenosine Triphosphate ◽  
Katp Channel ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Katp Channels ◽  
Dependent Manner ◽  
Open Probability ◽  
Rat Ventricular Myocytes ◽  
Inside Out ◽  
Cell Attached Configuration

Background The adenosine triphosphate (ATP)-sensitive potassium (KATP) channels protect myocytes during ischemia and reperfusion. This study investigated the effects of thiamylal on the activities of KATP channels in isolated rat ventricular myocytes during simulated ischemia. Methods Male Wistar rats were anesthetized with ether. Single, quiescent ventricular myocytes were dispersed enzymatically. Membrane currents were recorded using patch-clamp techniques. In the cell-attached configuration, KATP channel currents were assessed before and during activation of these channels by 2,4-dinitrophenol and after administration of 25, 50, and 100 mg/l thiamylal. The open probability was determined from current-amplitude histograms. In the inside-out configuration, the current-voltage relation was obtained before and after the application of thiamylal (50 mg/1). Results In the cell-attached configuration, 2,4-dinitrophenol caused frequent channel opening. 2,4-Dinitrophenol-induced channel activities were reduced significantly by glibenclamide, suggesting that the channels studied were KATP channels. Open probability of KATP channels was reduced by thiamylal in a concentration-dependent manner. KATP channels could be activated in the inside-out configuration because of the absence of ATP. Thiamylal inhibited KATP channel activity without changing the single-channel conductance. Conclusions The results obtained in this study indicate that thiamylal inhibits KATP channel activities in cell-attached and inside-out patches, suggesting a direct action of this drug on these channels.

scholarly journals Regulation of Cloned Atp-Sensitive K Channels by Adenine Nucleotides and Sulfonylureas

2001 ◽  
Vol 118 (4) ◽  
pp. 391-406 ◽  
Author(s):  
Scott A. John ◽  
James N. Weiss ◽  
Bernard Ribalet
Keyword(s):  
Katp Channel ◽  
Adenine Nucleotides ◽  
Katp Channels ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
K Channels ◽  
Channel Regulation ◽  
Channel Opening ◽  
Insight Into ◽  
Positively Charged

KATP channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 “PIP2-insensitive” mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the KATP channel. In contrast, the R301C and R314C mutants, which are also “PIP2-insensitive,” remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the KATP channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since KATP channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

scholarly journals Mutations within the P-Loop of Kir6.2 Modulate the Intraburst Kinetics of the Atp-Sensitive Potassium Channel

2001 ◽  
Vol 118 (4) ◽  
pp. 341-353 ◽  
Author(s):  
Peter Proks ◽  
Charlotte E. Capener ◽  
Phillippa Jones ◽  
Frances M. Ashcroft
Keyword(s):  
Katp Channel ◽  
Single Channel ◽  
Katp Channels ◽  
Burst Duration ◽  
Open Probability ◽  
Closed Intervals ◽  
Open Time ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of

The ATP-sensitive potassium (KATP) channel exhibits spontaneous bursts of rapid openings, which are separated by long closed intervals. Previous studies have shown that mutations at the internal mouth of the pore-forming (Kir6.2) subunit of this channel affect the burst duration and the long interburst closings, but do not alter the fast intraburst kinetics. In this study, we have investigated the nature of the intraburst kinetics by using recombinant Kir6.2/SUR1 KATP channels heterologously expressed in Xenopus oocytes. Single-channel currents were studied in inside-out membrane patches. Mutations within the pore loop of Kir6.2 (V127T, G135F, and M137C) dramatically affected the mean open time (τo) and the short closed time (τC1) within a burst, and the number of openings per burst, but did not alter the burst duration, the interburst closed time, or the channel open probability. Thus, the V127T and M137C mutations produced longer τo, shorter τC1, and fewer openings per burst, whereas the G135F mutation had the opposite effect. All three mutations also reduced the single-channel conductance: from 70 pS for the wild-type channel to 62 pS (G135F), 50 pS (M137C), and 38 pS (V127T). These results are consistent with the idea that the KATP channel possesses a gate that governs the intraburst kinetics, which lies close to the selectivity filter. This gate appears to be able to operate independently of that which regulates the long interburst closings.

scholarly journals Anti-diabetic drug binding site in a mammalian KATP channel revealed by Cryo-EM

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Gregory M Martin ◽  
Balamurugan Kandasamy ◽  
Frank DiMaio ◽  
Craig Yoshioka ◽  
Show-Ling Shyng
Keyword(s):  
Binding Sites ◽  
Katp Channel ◽  
Katp Channels ◽  
Binding Pocket ◽  
Drug Binding ◽  
Channel Activity ◽  
Nucleotide Binding Domain ◽  
High Affinity ◽  
Drug Binding Site ◽  
First Time

Sulfonylureas are anti-diabetic medications that act by inhibiting pancreatic KATP channels composed of SUR1 and Kir6.2. The mechanism by which these drugs interact with and inhibit the channel has been extensively investigated, yet it remains unclear where the drug binding pocket resides. Here, we present a cryo-EM structure of a hamster SUR1/rat Kir6.2 channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.63 Å resolution, which reveals unprecedented details of the ATP and glibenclamide binding sites. Importantly, the structure shows for the first time that glibenclamide is lodged in the transmembrane bundle of the SUR1-ABC core connected to the first nucleotide binding domain near the inner leaflet of the lipid bilayer. Mutation of residues predicted to interact with glibenclamide in our model led to reduced sensitivity to glibenclamide. Our structure provides novel mechanistic insights of how sulfonylureas and ATP interact with the KATP channel complex to inhibit channel activity.

scholarly journals A Gastropod Toxin Selectively Slows Early Transitions in the Shaker K Channel's Activation Pathway

2004 ◽  
Vol 123 (6) ◽  
pp. 685-696 ◽  
Author(s):  
Jon T. Sack ◽  
Richard W. Aldrich ◽  
William F. Gilly
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Gating Currents ◽  
K Channels ◽  
Voltage Sensors ◽  
Essentially Normal ◽  
Channel Opening ◽  
Single Channel Activity ◽  
Kinetics Of

A toxin from a marine gastropod's defensive mucus, a disulfide-linked dimer of 6-bromo-2-mercaptotryptamine (BrMT), was found to inhibit voltage-gated potassium channels by a novel mechanism. Voltage-clamp experiments with Shaker K channels reveal that externally applied BrMT slows channel opening but not closing. BrMT slows K channel activation in a graded fashion: channels activate progressively slower as the concentration of BrMT is increased. Analysis of single-channel activity indicates that once a channel opens, the unitary conductance and bursting behavior are essentially normal in BrMT. Paralleling its effects against channel opening, BrMT greatly slows the kinetics of ON, but not OFF, gating currents. BrMT was found to slow early activation transitions but not the final opening transition of the Shaker ILT mutant, and can be used to pharmacologically distinguish early from late gating steps. This novel toxin thus inhibits activation of Shaker K channels by specifically slowing early movement of their voltage sensors, thereby hindering channel opening. A model of BrMT action is developed that suggests BrMT rapidly binds to and stabilizes resting channel conformations.

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