Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells

Neuron. 2001 Sep 27;31(6):973-85. doi: 10.1016/s0896-6273(01)00438-x.


Among the most intriguing forms of Ca(2+) channel modulation is the regulation of L-type and P/Q-type channels by intracellular Ca(2+), acting via unconventional channel-calmodulin (CaM) interactions. In particular, overexpressing Ca(2+)-insensitive mutant CaM abolishes Ca(2+)-dependent modulation, hinting that Ca(2+)-free CaM may "preassociate" with these channels to enhance detection of local Ca(2+). Despite the far-reaching consequences of this proposal, in vitro experiments testing for preassociation provide conflicting results. Here, we develop a three filter-cube fluorescence resonance energy transfer method (three-cube FRET) to directly probe for constitutive associations between channel subunits and CaM in single living cells. This FRET assay detects Ca(2+)-independent associations between CaM and the pore-forming alpha(1) subunit of L-type, P/Q-type, and, surprisingly, R-type channels. These results now definitively demonstrate channel-CaM preassociation in resting cells and underscore the potential of three-cube FRET for probing protein-protein interactions.

Publication types

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

MeSH terms

  • Calcium / metabolism*
  • Calcium Channels, L-Type / chemistry
  • Calcium Channels, L-Type / metabolism*
  • Calcium Channels, N-Type / chemistry
  • Calcium Channels, N-Type / metabolism*
  • Calcium Channels, R-Type / chemistry
  • Calcium Channels, R-Type / metabolism*
  • Calmodulin / chemistry
  • Calmodulin / metabolism*
  • Cell Line
  • Energy Transfer
  • Feedback
  • Genes, Reporter
  • Green Fluorescent Proteins
  • Humans
  • Ion Channel Gating
  • Luminescent Proteins / analysis
  • Macromolecular Substances
  • Patch-Clamp Techniques
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / analysis
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / instrumentation
  • Spectrometry, Fluorescence / methods*
  • Transfection


  • Calcium Channels, L-Type
  • Calcium Channels, N-Type
  • Calcium Channels, R-Type
  • Calmodulin
  • Luminescent Proteins
  • Macromolecular Substances
  • Recombinant Fusion Proteins
  • voltage-dependent calcium channel (P-Q type)
  • Green Fluorescent Proteins
  • Calcium