Conformational changes of the Ca(2+) regulatory site of the Na(+)-Ca(2+) exchanger detected by FRET

Biophys J. 2004 Aug;87(2):899-906. doi: 10.1529/biophysj.104.043471.

Abstract

The Na(+)-Ca(2+) exchanger is a plasma membrane protein expressed at high levels in cardiomyocytes. It extrudes 1 Ca(2+) for 3 Na(+) ions entering the cell, regulating intracellular Ca(2+) levels and thereby contractility. Na(+)-Ca(2+) exchanger activity is regulated by intracellular Ca(2+), which binds to a region (amino acids 371-508) within the large cytoplasmic loop between transmembrane segments 5 and 6. Regulatory Ca(2+) activates the exchanger and removes Na(+)-dependent inactivation. The physiological role of intracellular Ca(2+) regulation of the exchanger is not yet established. Yellow (YFP) and cyan (CFP) fluorescent proteins were linked to the NH(2)- and CO(2)H-termini of the exchanger Ca(2+) binding domain (CBD) to generate a construct (YFP-CBD-CFP) capable of responding to changes in intracellular Ca(2+) concentrations by FRET efficiency measurements. The two fluorophores linked to the CBD are sufficiently close to generate FRET. FRET efficiency was reduced with increasing Ca(2+) concentrations. Titrations of Ca(2+) concentration versus FRET efficiency indicate a K(D) for Ca(2+) of approximately 140 nM, which increased to approximately 400 nM in the presence of 1 mM Mg(2+). Expression of YFP-CBD-CFP in myocytes, generated changes in FRET associated with contraction, suggesting that NCX is regulated by Ca(2+) on a beat-to-beat basis during excitation-contraction coupling.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Animals, Newborn
  • Binding Sites
  • Calcium / chemistry*
  • Calcium / metabolism*
  • Cell Membrane / chemistry
  • Cell Membrane / metabolism
  • Cells, Cultured
  • Fluorescence Resonance Energy Transfer / methods*
  • Myocardial Contraction / physiology*
  • Myocytes, Cardiac / chemistry
  • Myocytes, Cardiac / physiology*
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Rats
  • Sodium-Calcium Exchanger / chemistry*
  • Sodium-Calcium Exchanger / metabolism*

Substances

  • Sodium-Calcium Exchanger
  • Calcium