A glutathione probe finds caffeine as a CaSR PAM
Photoaffinity GSH analog DAZ-G labels the CaSR amino-acid site and shows caffeine is a positive allosteric modulator.
Source
Glutathione-Based Photoaffinity Probe Identifies Caffeine as a Positive Allosteric Modulator of the Calcium-Sensing Receptor
Study at a glance
- Design
- Animal / in-vitro — DAZ-G photoaffinity labeling of CaSR in HEK293 cells plus intracellular Ca2+ flux
- N
- Competition and Ca2+ assays across caffeine concentrations rather than a single N
- Population
- HEK293 cells expressing calcium-sensing receptor
- Outcome
- Caffeine is a positive allosteric modulator of CaSR, enhancing DAZ-G labeling at low micromolar concentrations when extracellular Ca2+ is present
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A glutathione probe finds caffeine as a CaSR PAM
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What new CaSR ligand did DAZ-G reveal?
finding that caffeine is a potent PAM for CaSR
Where was human CaSR expressed for labeling?
Human CaSR was ectopically expressed in HEK293 cells
What they did
Authors synthesized DAZ-G (diazirine/alkyne GSH), photocrosslinked it to CaSR in HEK293 cells, competed with known ligands, and read intracellular Ca2+ flux to test whether caffeine activates the receptor.
What they found
DAZ-G binds the amino-acid binding site and is a potent PAM. Caffeine at 0–1 μM enhances labeling via the calcium-binding site, while higher concentrations compete at the amino-acid site; caffeine raises Ca2+ only when extracellular Ca2+ is present.
The limits
What it doesn't show
The probe is currently a Western-blot tool, not a high-throughput screen, and caffeine’s in vivo CaSR contribution versus adenosine receptors is not measured here.
Key terms
- CaSR
- Calcium-sensing receptor, a class C GPCR that sets parathyroid hormone and Ca2+ homeostasis.
- PAM
- Positive allosteric modulator that boosts receptor activity in the presence of an agonist.
- DAZ-G
- Glutathione analog with diazirine and alkyne handles for photoaffinity labeling and click detection.
- ABS
- Amino-acid binding site in the CaSR extracellular domain.
- CBS
- Cation (Ca2+) binding site; caffeine appears to engage it at low micromolar levels.
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DAZ-G is derived from:
Common questions
What is DAZ-G built from?
Glutathione with diazirine and alkyne at Gly and Cys.
What unexpected ligand did it find?
Caffeine as a CaSR positive allosteric modulator.
Where does DAZ-G bind?
Primarily the amino-acid binding site (ABS).
Does caffeine work without Ca2+?
No—it is a PAM that needs extracellular Ca2+.
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