Purification, Characterization, and Synthesis of an Inward-Rectifier K+Channel Inhibitor from Scorpion Venom†

Biochemistry ◽  
1997 ◽  
Vol 36 (23) ◽  
pp. 6936-6940 ◽  
Author(s):  
Zhe Lu ◽  
Roderick MacKinnon
Keyword(s):  
Scorpion Venom ◽  
Inward Rectifier ◽  
K Channel

scholarly journals Nucleotide inhibition of the pancreatic ATP-sensitive K+ channel explored with patch-clamp fluorometry

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Samuel G Usher ◽  
Frances M Ashcroft ◽  
Michael C Puljung
Keyword(s):  
Nucleotide Binding ◽  
Inward Rectifier ◽  
K Channel ◽  
Data Sets ◽  
Channel Activity ◽  
Unknown Mechanism ◽  
Apparent Affinity ◽  
Nucleotide Inhibition ◽  
Channel Currents ◽  
Combined Data

Pancreatic ATP-sensitive K+ channels (KATP) comprise four inward rectifier subunits (Kir6.2), each associated with a sulphonylurea receptor (SUR1). ATP/ADP binding to Kir6.2 shuts KATP. Mg-nucleotide binding to SUR1 stimulates KATP. In the absence of Mg2+, SUR1 increases the apparent affinity for nucleotide inhibition at Kir6.2 by an unknown mechanism. We simultaneously measured channel currents and nucleotide binding to Kir6.2. Fits to combined data sets suggest that KATP closes with only one nucleotide molecule bound. A Kir6.2 mutation (C166S) that increases channel activity did not affect nucleotide binding, but greatly perturbed the ability of bound nucleotide to inhibit KATP. Mutations at position K205 in SUR1 affected both nucleotide affinity and the ability of bound nucleotide to inhibit KATP. This suggests a dual role for SUR1 in KATP inhibition, both in directly contributing to nucleotide binding and in stabilising the nucleotide-bound closed state.

scholarly journals Functional expression of a vertebrate inwardly rectifying K+ channel in yeast.

1995 ◽  
Vol 6 (9) ◽  
pp. 1231-1240 ◽  
Author(s):  
W Tang ◽  
A Ruknudin ◽  
W P Yang ◽  
S Y Shaw ◽  
A Knickerbocker ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Xenopus Oocytes ◽  
Genetic System ◽  
Functional Expression ◽  
Inward Rectifier ◽  
K Channel ◽  
Coding Region ◽  
Low Potassium ◽  
K Current ◽  
Inwardly Rectifying

We describe the expression of gpIRK1, an inwardly rectifying K+ channel obtained from guinea pig cardiac cDNA. gpIRK1 is a homologue of the mouse IRK1 channel identified in macrophage cells. Expression of gpIRK1 in Xenopus oocytes produces inwardly rectifying K+ current, similar to the cardiac inward rectifier current IK1. This current is blocked by external Ba2+ and Cs+. Plasmids containing the gpIRK1 coding region under the transcriptional control of constitutive (PGK) or inducible (GAL) promoters were constructed for expression in Saccharomyces cerevisiae. Several observations suggest that gpIRK1 forms functional ion channels when expressed in yeast. gpIRK1 complements a trk1 delta trk2 delta strain, which is defective in potassium uptake. Expression of gpIRK1 in this mutant restores growth on low potassium media. Growth dependent on gpIRK1 is inhibited by external Cs+. The strain expressing gpIRK1 provides a versatile genetic system for studying the assembly and composition of inwardly rectifying K+ channels.

scholarly journals Estrogen ameliorates microglial activation by inhibiting the Kir2.1 inward-rectifier K+ channel

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Shih-Ying Wu ◽  
Yun-Wen Chen ◽  
Sheng-Feng Tsai ◽  
Sheng-Nan Wu ◽  
Yao-Hsiang Shih ◽  
...  
Keyword(s):  
Microglial Activation ◽  
Inward Rectifier ◽  
K Channel

Comparative expression of the inward rectifier K+ channel GIRK2 in the cerebellum of normal and weaver mutant mice

1997 ◽  
Vol 753 (1) ◽  
pp. 8-17 ◽  
Author(s):  
Inger Lauritzen ◽  
Jan De Weille ◽  
Christine Adelbrecht ◽  
Florian Lesage ◽  
Gustavo Murer ◽  
...  
Keyword(s):  
Inward Rectifier ◽  
Mutant Mice ◽  
K Channel

scholarly journals High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit

2019 ◽  
Vol 5 (7) ◽  
pp. eaax0495 ◽  
Author(s):  
A. Sumino ◽  
T. Sumikama ◽  
T. Uchihashi ◽  
S. Oiki
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Scorpion Venom ◽  
State Model ◽  
K Channel ◽  
Induced Fit ◽  
Force Microscopy ◽  
Toxin Binding ◽  
Docking Model ◽  
Antigen Antibody

Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

scholarly journals Codon Harmonization of a Kir3.1-KirBac1.3 Chimera for Structural Study Optimization

Biomolecules ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 430
Author(s):  
Evan Van Aalst ◽  
Maryam Yekefallah ◽  
Anil K. Mehta ◽  
Isaac Eason ◽  
Benjamin Wylie
Keyword(s):  
Membrane Proteins ◽  
G Protein ◽  
Recombinant Expression ◽  
Protein Product ◽  
Inward Rectifier ◽  
Correlation Spectroscopy ◽  
Protein Yield ◽  
K Channel ◽  
Functional Assay ◽  
Nmr Studies

The expression of functional, folded, and isotopically enriched membrane proteins is an enduring bottleneck for nuclear magnetic resonance (NMR) studies. Indeed, historically, protein yield optimization has been insufficient to allow NMR analysis of many complex Eukaryotic membrane proteins. However, recent work has found that manipulation of plasmid codons improves the odds of successful NMR-friendly protein production. In the last decade, numerous studies showed that matching codon usage patterns in recombinant gene sequences to those in the native sequence is positively correlated with increased protein yield. This phenomenon, dubbed codon harmonization, may be a powerful tool in optimizing recombinant expression of difficult-to-produce membrane proteins for structural studies. Here, we apply this technique to an inward rectifier K+ Channel (Kir) 3.1-KirBac1.3 chimera. Kir3.1 falls within the G protein-coupled inward rectifier K+ (GIRK) channel family, thus NMR studies may inform on the nuances of GIRK gating action in the presence and absence of its G Protein, lipid, and small molecule ligands. In our hands, harmonized plasmids increase protein yield nearly two-fold compared to the traditional ‘fully codon optimized’ construct. We then employ a fluorescence-based functional assay and solid-state NMR correlation spectroscopy to show the final protein product is folded and functional.

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