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

Usher, Samuel; Ashcroft, Frances M.; Puljung, Michael C.

doi:10.7554/elife.52775

Cited by 21 publications

(31 citation statements)

References 66 publications

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“…If bleaching correction does not introduce any additional artefacts, the results should be comparable 11 . As some non-specific binding of TNP-ATP to naive plasma membranes from untransfected HEK-293T cells was observed, it is recommended to quantify FRET as a reduction in the donor (ANAP) fluorescence 10 , 11 .…”

Section: Representative Resultsmentioning

confidence: 99%

“…Importantly, this technique can be directly applied to small amounts of protein in a cellular environment under conditions that preserve protein function. Using our binding method in conjunction with direct, electrophysiological readout of ion channel currents allows us to obtain rich insights into the molecular underpinnings of channel gating 11 .…”

Section: Discussionmentioning

confidence: 99%

“…We quantify binding as a decrement in the donor fluorescence, as this signal is specific to the labeled channel. However, we recommend performing additional control experiments for each agonist/receptor pair, for example mutating the nucleotide binding site if known, to verify that the change in donor fluorescence is really the result of direct binding to the labeled receptor 11 . Finally, working with constructs that contain a fluorescent protein tag in addition to the ANAP label is recommended.…”

Section: Discussionmentioning

confidence: 99%

“…In the second method, nucleotide binding to ANAP-labeled K ATP channels is measured in a membrane patch under voltage clamp, allowing for the simultaneous measurement of ionic currents and fluorescence. By combining these two experimental approaches, changes in binding can be directly correlated with changes in channel function 11 . Typical results, potential pitfalls, and data analysis are discussed.…”

Section: Introductionmentioning

confidence: 99%

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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Usher

Ashcroft

Puljung

2021

JoVE

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We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment.This method combines expression of proteins tagged with the fluorescent noncanonical amino acid ANAP, and FRET between ANAP and fluorescent (trinitrophenyl) nucleotide derivatives. We present examples of nucleotide binding to ANAP-tagged K ATP ion channels measured in unroofed plasma membranes and excised, insideout membrane patches under voltage clamp. The latter allows for simultaneous measurements of ligand binding and channel current, a direct readout of protein function. Data treatment and analysis are discussed extensively, along with potential pitfalls and artefacts. This method provides rich mechanistic insights into the liganddependent gating of K ATP channels and can readily be adapted to the study of other nucleotide-regulated proteins or any receptor for which a suitable fluorescent ligand can be identified.

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Section: Representative Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Usher

Ashcroft

Puljung

2021

JoVE

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“…2019; Usher et al . 2020). These studies made use of a double negative eukaryotic release factor that was developed to increase the efficiency of ncAA incorporation and thus fluorescence signals (Schmied et al .…”

Section: Genetic Modification Of Proteinsmentioning

confidence: 99%

The current chemical biology tool box for studying ion channels

Braun

Sheikh

Pless

2020

The Journal of Physiology

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Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be Nina Braun studied biochemistry at the University of Bayreuth and Stockholm University before discovering her interest in ion channels and non-canonical amino acids while working on her Masters thesis with Stephan A. Pless at the University of Copenhagen. She completed her PhD in the Pless lab and is currently working on high-throughput assessment of non-canonical amino acid incorporation using photocrosslinkers in acid-sensing ion channels, with the goal of studying protein-protein interactions. Zeshan P. Sheikh completed his MSc degree at the University of Copenhagen, where he discovered his passion for ion channels, mainly focusing on recombinant expression and purification. He embarked on a PhD in the Pless lab pursuing his interest in ion channel biophysics. Here, he gained particular interest in the application of chemical biological techniques for modifying ion channels. His current research priority is applying split inteins to modify acid-sensing ion channels to gain insights into their structural dynamics.

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ANAP: A versatile, fluorescent probe of ion channel gating and regulation

Puljung

2021

Methods in Enzymology

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

Cited by 21 publications

References 66 publications

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

The current chemical biology tool box for studying ion channels

ANAP: A versatile, fluorescent probe of ion channel gating and regulation

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