Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis

PLoS One. 2012;7(4):e36215. doi: 10.1371/journal.pone.0036215. Epub 2012 Apr 27.

Abstract

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / immunology
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Fluorescent Dyes / chemistry
  • Green Fluorescent Proteins / genetics
  • HEK293 Cells
  • Humans
  • Kidney / metabolism
  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / immunology
  • Membrane Transport Proteins / metabolism
  • Microscopy, Confocal
  • Molecular Imaging / methods*
  • Photons
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Rats
  • Sodium-Bicarbonate Symporters / chemistry
  • Sodium-Bicarbonate Symporters / genetics
  • Sodium-Bicarbonate Symporters / immunology
  • Sodium-Bicarbonate Symporters / metabolism
  • Spectrometry, Fluorescence / methods*

Substances

  • Antibodies
  • Fluorescent Dyes
  • Membrane Transport Proteins
  • SLC4A4 protein, human
  • Sodium-Bicarbonate Symporters
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins