Fast GCaMPs for improved tracking of neuronal activity

Nat Commun. 2013;4:2170. doi: 10.1038/ncomms3170.

Abstract

The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca(2+)-sensitive range. Here we identify three performance-limiting features of GCaMP3, a popular genetically encodable calcium indicator protein. First, we find that affinity is regulated by the calmodulin domain's Ca(2+)-chelating residues. Second, we find that off-responses to Ca(2+) are rate-limited by dissociation of the RS20 domain from calmodulin's hydrophobic pocket. Third, we find that on-responses are limited by fast binding to the N-lobe at high Ca(2+) and by slow binding to the C-lobe at lower Ca(2+). We develop Fast-GCaMPs, which have up to 20-fold accelerated off-responses and show that they have a 200-fold range of K(D), allowing coexpression of multiple variants to span an expanded range of Ca(2+) concentrations. Finally, we show that Fast-GCaMPs track natural song in Drosophila auditory neurons and generate rapid responses in mammalian neurons, supporting the utility of our approach.

Publication types

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

MeSH terms

  • Acoustic Stimulation
  • Amino Acid Sequence
  • Animals
  • Auditory Perception / physiology
  • Binding Sites
  • Calcium / metabolism*
  • Calmodulin / chemistry*
  • Calmodulin / genetics
  • Calmodulin / metabolism
  • Drosophila melanogaster / cytology
  • Drosophila melanogaster / physiology*
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Kinetics
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Neurons / cytology
  • Neurons / physiology*
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Time Factors

Substances

  • Calmodulin
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Calcium