Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Biochemistry. 2002 Oct 8;41(40):12253-62. doi: 10.1021/bi0200099.


Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calorimetry, Differential Scanning
  • Fluorescence Polarization
  • Liposomes / chemistry
  • Membrane Lipids / chemistry
  • Phosphatidic Acids / chemistry*
  • Protein Structure, Tertiary
  • Receptors, Cholinergic / chemistry*
  • Spectroscopy, Fourier Transform Infrared
  • Temperature
  • Torpedo


  • Liposomes
  • Membrane Lipids
  • Phosphatidic Acids
  • Receptors, Cholinergic