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Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets
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Photoaffinity labeling the propofol binding site in GLIC.

David C Chiara1, Jonathan F Gill, Qiang Chen

  • 1Department of Neurobiology, Harvard Medical School , Boston, Massachusetts 02115, United States.

Biochemistry
|December 18, 2013
PubMed
Summary

Researchers identified propofol binding sites in Gloeobacter violaceus ion channel (GLIC) using a novel photoreactive analog. This reveals propofol

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Area of Science:

  • Neuroscience and Pharmacology
  • Structural Biology
  • Ion Channel Research

Background:

  • Propofol, a general anesthetic, primarily acts by potentiating GABA type A receptors (GABAAR).
  • Propofol also inhibits cation-selective pentameric ligand-gated ion channels (pLGICs), such as Gloeobacter violaceus ion channel (GLIC).
  • Previous crystal structures showed propofol within an intrasubunit pocket of GLIC at acidic pH.

Purpose of the Study:

  • To determine propofol binding sites in GLIC in solution.
  • To utilize a novel photoreactive propofol analog, AziPm, for photolabeling studies.
  • To elucidate the mechanism of propofol inhibition on GLIC.

Main Methods:

  • Functional characterization of GLIC inhibition by propofol and AziPm in Xenopus oocytes.
  • Photolabeling of detergent-solubilized GLIC with radiolabeled AziPm.
  • Protein microsequencing to identify photolabeled residues.

Main Results:

  • Propofol and AziPm inhibited GLIC currents with IC50 values of 20 and 50 μM, respectively.
  • Photolabeling identified three key residues (Met-205, Tyr-254, Asn-307) in the GLIC transmembrane domain.
  • These residues are located in proximity to the intrasubunit pocket identified in crystal structures.

Conclusions:

  • Propofol and AziPm bind competitively to a site within the GLIC transmembrane domain in solution.
  • The identified binding site is consistent with the location observed in crystal structures.
  • This study provides insights into the molecular interactions of propofol with GLIC.