Receptor-independent modulation of reconstituted Galpha(i) protein mediated by liposomes

Mol Biosyst. 2009 Apr;5(4):356-67. doi: 10.1039/b815042g. Epub 2009 Jan 29.

Abstract

A cationic amphiphile, BC5 (N-pentadecylpiperidin-4-amine), was recently designed and tested for its ability to directly stimulate the activity of recombinant Galpha inhibitory subunits. However, amphiphilic drugs can self-associate and bind to plasma membranes, causing undesired side effects. In this contribution, we report on the incorporation of BC5 in 1,2-dipalmytoyl-sn-glycerophosphocoline (DPPC) liposomes and on the characterization of the mixed DPPC/BC5 systems at various lipid/drug mole ratios by means of dynamic light scattering, differential scanning calorimetry and fluorescence spectroscopy. The myristoylated Galpha(i) subunit (Galpha-mir) was reconstituted in 1,2-dimiristoyl-sn-glycerophosphocoline (DMPC) bilayers, as a mimic of the drug target. We compare several reconstitution procedures in liposomes and present for the first time a complete characterization of a Galpha subunit reconstitution in model membranes in terms of protein activity as a function of the reconstitution protocol. The incorporation of the drug in DPPC bilayers resulted in enhanced Gi-modulating efficiency (evaluated in terms of binding to GTPgammaS (guanosine-5'-(gamma-thio)-triphosphate)). A correlation of the physico-chemical features and binding activity of protein-containing membrane model is proposed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1,2-Dipalmitoylphosphatidylcholine / chemistry
  • 1,2-Dipalmitoylphosphatidylcholine / metabolism
  • Binding Sites
  • Calorimetry, Differential Scanning
  • Circular Dichroism
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism
  • Liposomes / metabolism*
  • Piperidines / chemistry
  • Piperidines / metabolism
  • Spectrometry, Fluorescence
  • Surface-Active Agents / chemistry
  • Surface-Active Agents / metabolism

Substances

  • Liposomes
  • N-pentadecylpiperidin-4-amine
  • Piperidines
  • Surface-Active Agents
  • 1,2-Dipalmitoylphosphatidylcholine
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • GTP-Binding Protein alpha Subunits, Gi-Go