scholarly journals Cryo-electron microscopy structures and progress toward a dynamic understanding of KATP channels

2018 ◽  
Vol 150 (5) ◽  
pp. 653-669 ◽  
Author(s):  
Michael C. Puljung
Keyword(s):  
Electron Microscopy ◽  
Ligand Binding ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Cell Metabolism ◽  
Katp Channels ◽  
K Channel ◽  
Sulfonylurea Receptor ◽  
Binding Domains ◽  
Cryo Electron Microscopy

Adenosine triphosphate (ATP)–sensitive K+ (KATP) channels are molecular sensors of cell metabolism. These hetero-octameric channels, comprising four inward rectifier K+ channel subunits (Kir6.1 or Kir6.2) and four sulfonylurea receptor (SUR1 or SUR2A/B) subunits, detect metabolic changes via three classes of intracellular adenine nucleotide (ATP/ADP) binding site. One site, located on the Kir subunit, causes inhibition of the channel when ATP or ADP is bound. The other two sites, located on the SUR subunit, excite the channel when bound to Mg nucleotides. In pancreatic β cells, an increase in extracellular glucose causes a change in oxidative metabolism and thus turnover of adenine nucleotides in the cytoplasm. This leads to the closure of KATP channels, which depolarizes the plasma membrane and permits Ca2+ influx and insulin secretion. Many of the molecular details regarding the assembly of the KATP complex, and how changes in nucleotide concentrations affect gating, have recently been uncovered by several single-particle cryo-electron microscopy structures of the pancreatic KATP channel (Kir6.2/SUR1) at near-atomic resolution. Here, the author discusses the detailed picture of excitatory and inhibitory ligand binding to KATP that these structures present and suggests a possible mechanism by which channel activation may proceed from the ligand-binding domains of SUR to the channel pore.

Characterization of ATP-sensitive potassium channels in freshly dissociated rabbit aortic endothelial cells

1997 ◽  
Vol 272 (5) ◽  
pp. H2507-H2511 ◽  
Author(s):  
C. Katnik ◽  
D. J. Adams
Keyword(s):  
Endothelial Cells ◽  
Single Channel ◽  
Muscle Tone ◽  
Cell Metabolism ◽  
Katp Channels ◽  
K Channel ◽  
Membrane Hyperpolarization ◽  
Aortic Endothelial Cells ◽  
Intracellular Atp ◽  
Aortic Endothelial

ATP-sensitive potassium (KATP) channels represent a class of K+ channel regulated by intracellular ATP and serve to transduce changes in cell metabolism into changes in membrane potential. The presence of an KATP conductance has recently been demonstrated in freshly dissociated endothelial cells from rabbit arteries. In the present study, the single-channel activity underlying the KATP conductance in rabbit aortic endothelial cells was examined. Unitary currents were evoked in response to lowering intracellular ATP concentration or application of the K(+)-channel activator levcromakalim and were inhibited by the sulfonylurea drug glibenclamide. Exposure of the cytoplasmic face of an inside-out membrane patch to a solution containing 0.1 mM ATP produced single-channel events with unitary conductances of approximately 150 and approximately 25 pS that were inhibited by either 6 mM ATP or 10 microM glibenclamide. A small conductance channel was also activated in cell-attached patches by bath-applied levcromakalim (25 microM). Activation of endothelial cell KATP channels, and subsequent membrane hyperpolarization, may contribute to endothelium-dependent regulation of vascular smooth muscle tone in response to changes in levels of intracellular metabolites.

scholarly journals Regulation of Cloned Atp–Sensitive K Channels by Phosphorylation, Mgadp, and Phosphatidylinositol Bisphosphate (Pip2)

2000 ◽  
Vol 116 (3) ◽  
pp. 391-410 ◽  
Author(s):  
Bernard Ribalet ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Prolonged Exposure ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Adenine Nucleotides ◽  
Katp Channels ◽  
Hek293 Cells ◽  
Slow Phase ◽  
Functional Coupling ◽  
Sulfonylurea Receptor ◽  
Phosphatidylinositol Bisphosphate

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6.2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of KATP channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP2), and phosphorylation. Upon excision of inside-out patches into a Ca2+- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6.2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca2+ accelerated this process, suggesting a role for PIP2 hydrolysis mediated by a Ca2+-dependent phospholipase C. PIP2 could reactivate channel activity after a brief exposure to Ca2+, but not after prolonged exposure. However, in both cases, PIP2 reversed the increase in ATP sensitivity, indicating that PIP2 lowers the ATP sensitivity by increasing Po as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca2+ facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5–15 min, increased ATP sensitivity and loss of reactivation by PIP2 and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP2 responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP2 and phosphorylation.

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 Differential mechanisms of Cantú syndrome–associated gain of function mutations in the ABCC9 (SUR2) subunit of the KATP channel

2015 ◽  
Vol 146 (6) ◽  
pp. 527-540 ◽  
Author(s):  
Paige E. Cooper ◽  
Monica Sala-Rabanal ◽  
Sun Joo Lee ◽  
Colin G. Nichols
Keyword(s):  
Cell Metabolism ◽  
Katp Channels ◽  
Sulfonylurea Receptor ◽  
Membrane Excitability ◽  
Gain Of Function ◽  
Equivalent Position ◽  
Functional Consequences ◽  
Atp Inhibition ◽  
Patch Clamp Electrophysiology ◽  
Inwardly Rectifying

Cantú syndrome (CS) is a rare disease characterized by congenital hypertrichosis, distinct facial features, osteochondrodysplasia, and cardiac defects. Recent genetic analysis has revealed that the majority of CS patients carry a missense mutation in ABCC9, which codes for the sulfonylurea receptor SUR2. SUR2 subunits couple with Kir6.x, inwardly rectifying potassium pore-forming subunits, to form adenosine triphosphate (ATP)-sensitive potassium (KATP) channels, which link cell metabolism to membrane excitability in a variety of tissues including vascular smooth muscle, skeletal muscle, and the heart. The functional consequences of multiple uncharacterized CS mutations remain unclear. Here, we have focused on determining the functional consequences of three documented human CS-associated ABCC9 mutations: human P432L, A478V, and C1043Y. The mutations were engineered in the equivalent position in rat SUR2A (P429L, A475V, and C1039Y), and each was coexpressed with mouse Kir6.2. Using macroscopic rubidium (86Rb+) efflux assays, we show that KATP channels formed with P429L, A475V, or C1039Y mutants enhance KATP activity compared with wild-type (WT) channels. We used inside-out patch-clamp electrophysiology to measure channel sensitivity to ATP inhibition and to MgADP activation. For P429L and A475V mutants, sensitivity to ATP inhibition was comparable to WT channels, but activation by MgADP was significantly greater. C1039Y-dependent channels were significantly less sensitive to inhibition by ATP or by glibenclamide, but MgADP activation was comparable to WT. The results indicate that these three CS mutations all lead to overactive KATP channels, but at least two mechanisms underlie the observed gain of function: decreased ATP inhibition and enhanced MgADP activation.

scholarly journals Architecture and subunit arrangement of native AMPA receptors elucidated by cryo-EM

Science ◽  
2019 ◽  
Vol 364 (6438) ◽  
pp. 355-362 ◽  
Author(s):  
Yan Zhao ◽  
Shanshuang Chen ◽  
Adam C. Swensen ◽  
Wei-Jun Qian ◽  
Eric Gouaux
Keyword(s):  
Molecular Structure ◽  
Electron Microscopy ◽  
Signal Transduction ◽  
Ligand Binding ◽  
Ampa Receptor ◽  
Ampa Receptors ◽  
Mammalian Brain ◽  
Receptor Complexes ◽  
Cryo Electron Microscopy ◽  
Chemical Synapses

Glutamate-gated AMPA receptors mediate the fast component of excitatory signal transduction at chemical synapses throughout all regions of the mammalian brain. AMPA receptors are tetrameric assemblies composed of four subunits, GluA1–GluA4. Despite decades of study, the subunit composition, subunit arrangement, and molecular structure of native AMPA receptors remain unknown. Here we elucidate the structures of 10 distinct native AMPA receptor complexes by single-particle cryo–electron microscopy (cryo-EM). We find that receptor subunits are arranged nonstochastically, with the GluA2 subunit preferentially occupying the B and D positions of the tetramer and with triheteromeric assemblies comprising a major population of native AMPA receptors. Cryo-EM maps define the structure for S2-M4 linkers between the ligand-binding and transmembrane domains, suggesting how neurotransmitter binding is coupled to ion channel gating.

scholarly journals Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels

2012 ◽  
Vol 140 (2) ◽  
pp. 175-187 ◽  
Author(s):  
Emily B. Pratt ◽  
Qing Zhou ◽  
Joel W. Gay ◽  
Show-Ling Shyng
Keyword(s):  
Cell Metabolism ◽  
Katp Channels ◽  
Dynamic Parameter ◽  
Sulfonylurea Receptor ◽  
Wild Type ◽  
Membrane Excitability ◽  
Close Proximity ◽  
Secretion Inhibition ◽  
Sulfonylurea Receptor 1 ◽  
Hallmark Feature

The ATP-sensitive potassium (KATP) channel consisting of the inward rectifier Kir6.2 and SUR1 (sulfonylurea receptor 1) couples cell metabolism to membrane excitability and regulates insulin secretion. Inhibition by intracellular ATP is a hallmark feature of the channel. ATP sensitivity is conferred by Kir6.2 but enhanced by SUR1. The mechanism by which SUR1 increases channel ATP sensitivity is not understood. In this study, we report molecular interactions between SUR1 and Kir6.2 that markedly alter channel ATP sensitivity. Channels bearing an E203K mutation in SUR1 and a Q52E in Kir6.2 exhibit ATP sensitivity ∼100-fold higher than wild-type channels. Cross-linking of E203C in SUR1 and Q52C in Kir6.2 locks the channel in a closed state and is reversible by reducing agents, demonstrating close proximity of the two residues. Our results reveal that ATP sensitivity in KATP channels is a dynamic parameter dictated by interactions between SUR1 and Kir6.2.

scholarly journals Molecular structure of human KATP in complex with ATP and ADP

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Kenneth Pak Kin Lee ◽  
Jue Chen ◽  
Roderick MacKinnon
Keyword(s):  
Regulatory Element ◽  
Katp Channels ◽  
Inward Rectifier ◽  
K Channel ◽  
Excitable Cells ◽  
Binding Domains ◽  
Inhibitory Site ◽  
Atp Inhibition ◽  
Adenosine Nucleotides

In many excitable cells, KATP channels respond to intracellular adenosine nucleotides: ATP inhibits while ADP activates. We present two structures of the human pancreatic KATP channel, containing the ABC transporter SUR1 and the inward-rectifier K+ channel Kir6.2, in the presence of Mg2+ and nucleotides. These structures, referred to as quatrefoil and propeller forms, were determined by single-particle cryo-EM at 3.9 Å and 5.6 Å, respectively. In both forms, ATP occupies the inhibitory site in Kir6.2. The nucleotide-binding domains of SUR1 are dimerized with Mg2+-ATP in the degenerate site and Mg2+-ADP in the consensus site. A lasso extension forms an interface between SUR1 and Kir6.2 adjacent to the ATP site in the propeller form and is disrupted in the quatrefoil form. These structures support the role of SUR1 as an ADP sensor and highlight the lasso extension as a key regulatory element in ADP’s ability to override ATP inhibition.

scholarly journals Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel

Nature ◽  
2015 ◽  
Vol 527 (7577) ◽  
pp. 198-203 ◽  
Author(s):  
Richard K. Hite ◽  
Peng Yuan ◽  
Zongli Li ◽  
Yichun Hsuing ◽  
Thomas Walz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
K Channel ◽  
Cryo Electron Microscopy
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