scholarly journals Mechanism of pharmacochaperoning in KATP channels revealed by cryo-EM

2019 ◽  
Author(s):  
Gregory M. Martin ◽  
Min Woo Sung ◽  
Zhongying Yang ◽  
Laura M. Innes ◽  
Balamurugan Kandasamy ◽  
...  
Keyword(s):  
Katp Channels ◽  
Binding Pocket ◽  
Surface Expression ◽  
Drug Binding ◽  
Cell Surface Expression ◽  
Common Mechanism ◽  
Congenital Hyperinsulinism ◽  
Sulfonylurea Receptor ◽  
N Terminus ◽  
Channel Expression

AbstractATP-sensitive potassium (KATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic β-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATPinhibitors have been shown to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of KATPchannels bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within the SUR1 ABC core, allowing it to act as a firm “handle” for the assembly of metastable mutant SUR1-Kir6.2 complexes.

scholarly journals Mechanism of pharmacochaperoning in a mammalian KATP channel revealed by cryo-EM

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Gregory M Martin ◽  
Min Woo Sung ◽  
Zhongying Yang ◽  
Laura M Innes ◽  
Balamurugan Kandasamy ◽  
...  
Keyword(s):  
Katp Channel ◽  
Katp Channels ◽  
Binding Pocket ◽  
Surface Expression ◽  
Drug Binding ◽  
Cell Surface Expression ◽  
Common Mechanism ◽  
Congenital Hyperinsulinism ◽  
Sulfonylurea Receptor ◽  
N Terminus

ATP-sensitive potassium (KATP) channels composed of a pore-forming Kir6.2 potassium channel and a regulatory ABC transporter sulfonylurea receptor 1 (SUR1) regulate insulin secretion in pancreatic β-cells to maintain glucose homeostasis. Mutations that impair channel folding or assembly prevent cell surface expression and cause congenital hyperinsulinism. Structurally diverse KATP inhibitors are known to act as pharmacochaperones to correct mutant channel expression, but the mechanism is unknown. Here, we compare cryoEM structures of a mammalian KATP channel bound to pharmacochaperones glibenclamide, repaglinide, and carbamazepine. We found all three drugs bind within a common pocket in SUR1. Further, we found the N-terminus of Kir6.2 inserted within the central cavity of the SUR1 ABC core, adjacent the drug binding pocket. The findings reveal a common mechanism by which diverse compounds stabilize the Kir6.2 N-terminus within SUR1’s ABC core, allowing it to act as a firm ‘handle’ for the assembly of metastable mutant SUR1-Kir6.2 complexes.

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 Anti-diabetic drug binding site in KATP channels revealed by Cryo-EM

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

AbstractSulfonylureas 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 the channel bound to a high-affinity sulfonylurea drug glibenclamide and ATP at 3.8Å resolution, which reveals in 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 The PAR2 signal peptide prevents premature receptor cleavage and activation

2019 ◽  
Author(s):  
Belinda Liu ◽  
Grace Lee ◽  
Jiejun Wu ◽  
Janise Deming ◽  
Chester Kuei ◽  
...  
Keyword(s):  
Cell Surface ◽  
Signal Peptide ◽  
Synthetic Peptide ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Peptide Ligand ◽  
Protease Activated Receptors ◽  
N Terminus

AbstractUnlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that these is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.

scholarly journals N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Hao Wang ◽  
Shuo Li ◽  
Juejin Wang ◽  
Shenghan Chen ◽  
Xue-Long Sun ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
General Mechanism ◽  
Common Mechanism ◽  
Physiological Processes ◽  
Glycosylation Sites ◽  
Underlying Mechanisms

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

S-acylation regulates Kv1.5 channel surface expression

2007 ◽  
Vol 293 (1) ◽  
pp. C152-C161 ◽  
Author(s):  
Lian Zhang ◽  
Karyn Foster ◽  
Qiuju Li ◽  
Jeffrey R. Martens
Keyword(s):  
Cell Surface ◽  
Time Course ◽  
Electrical Response ◽  
Proteasome Inhibitors ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Channel Protein ◽  
Kv Channels ◽  
Channel Expression ◽  
Intracellular Compartments

The number of ion channels expressed on the cell surface shapes the complex electrical response of excitable cells. An imbalance in the ratio of inward and outward conducting channels is unfavorable and often detrimental. For example, over- or underexpression of voltage-gated K+ (Kv) channels can be cytotoxic and in some cases lead to disease. In this study, we demonstrated a novel role for S-acylation in Kv1.5 cell surface expression. In transfected fibroblasts, biochemical evidence showed that Kv1.5 is posttranslationally modified on both the NH2 and COOH termini via hydroxylamine-sensitive thioester bonds. Pharmacological inhibition of S-acylation, but not myristoylation, significantly decreased Kv1.5 expression and resulted in accumulation of channel protein in intracellular compartments and targeting for degradation. Channel protein degradation was rescued by treatment with proteasome inhibitors. Time course experiments revealed that S-acylation occurred in the biosynthetic pathway of nascent channel protein and showed that newly synthesized Kv1.5 protein, but not protein expressed on the cell surface, is sensitive to inhibitors of thioacylation. Sensitivity to inhibitors of S-acylation was governed by COOH-terminal, but not NH2-terminal, cysteines. Surprisingly, although intracellular cysteines were required for S-acylation, mutation of these residues resulted in an increase in Kv1.5 cell surface channel expression, suggesting that screening of free cysteines by fatty acylation is an important regulatory step in the quality control pathway. Together, these results show that S-acylation can regulate steady-state expression of Kv1.5.

scholarly journals Characterization of a Nonclathrin Endocytic Pathway: Membrane Cargo and Lipid Requirements

2004 ◽  
Vol 15 (8) ◽  
pp. 3542-3552 ◽  
Author(s):  
Naava Naslavsky ◽  
Roberto Weigert ◽  
Julie G. Donaldson
Keyword(s):  
Adaptor Proteins ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
Endocytic Pathway ◽  
Cell Surface Expression ◽  
Common Mechanism ◽  
Cargo Proteins ◽  
Clathrin Adaptor ◽  
Free Membrane ◽  
Glycosylphosphatidyl Inositol

Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.

scholarly journals Listeria monocytogenesTriggers the Cell Surface Expression of Gp96 Protein and Interacts with Its N Terminus to Support Cellular Infection

2012 ◽  
Vol 287 (51) ◽  
pp. 43083-43093 ◽  
Author(s):  
Mariana Martins ◽  
Rafael Custódio ◽  
Ana Camejo ◽  
Maria Teresa Almeida ◽  
Didier Cabanes ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
N Terminus

scholarly journals Pharmacologically diverse antidepressant drugs disrupt the interaction of BDNF receptor TRKB and the endocytic adaptor AP-2

2019 ◽  
Author(s):  
Senem Merve Fred ◽  
Liina Laukkanen ◽  
Cecilia A Brunello ◽  
Liisa Vesa ◽  
Helka Goos ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Antidepressant Drugs ◽  
Adaptor Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Common Mechanism ◽  
Chemical Structures ◽  
Tropomyosin Related Kinase B

AbstractAntidepressant drugs activate TRKB (tropomyosin-related kinase B), however it remains unclear whether these compounds employ a common mechanism for achieving this effect. We found by using mass spectrometry that the interaction of several proteins with TRKB was disrupted in the hippocampus of fluoxetine-treated animals (single intraperitoneal injection), including members of the AP-2 complex (adaptor protein complex-2) involved in vesicular endocytosis. The interaction of TRKB with the cargo-docking mu subunit of the AP-2 complex (AP2M) was disrupted by both acute and repeated fluoxetine treatment. However, while the coupling between full length TRKB and AP2M was disrupted by fluoxetine, the interaction between AP2M and the TRKB C-terminal peptide was resistant to this drug, indicating that the binding site targeted by fluoxetine must lie outside of the TRKB:AP2M interface. In addition to fluoxetine, other pharmacologically diverse antidepressants imipramine, rolipram, phenelzine, ketamine, and the ketamine metabolite 2R,6R-hydroxynorketamine (RR-HNK) also decreased the interaction between TRKB:AP2M in vitro, as measured by ELISA. Silencing the expression of AP2M in MG87.TRKB cell line led to increased surface positioning of TRKB and to a higher response to BDNF (brain-derived neurotrophic factor), observed as the levels of active TRKB. Moreover, animals haploinsufficient for the Ap2m1 gene displayed increased levels of active TRKB in vivo, as well as an enhanced cell surface expression of the receptor in cultured hippocampal neurons.Taken together, our data suggests that disruption of the TRKB:AP2M interaction is an effect shared by several antidepressants with diverse chemical structures and canonical modes of action.

Sign in / Sign up

Export Citation Format

Share Document