Improved calcium sensor GCaMP-X overcomes the calcium channel perturbations induced by the calmodulin in GCaMP

Nat Commun. 2018 Apr 17;9(1):1504. doi: 10.1038/s41467-018-03719-6.

Abstract

GCaMP, one popular type of genetically-encoded Ca2+ indicator, has been associated with various side-effects. Here we unveil the intrinsic problem prevailing over different versions and applications, showing that GCaMP containing CaM (calmodulin) interferes with both gating and signaling of L-type calcium channels (CaV1). GCaMP acts as an impaired apoCaM and Ca2+/CaM, both critical to CaV1, which disrupts Ca2+ dynamics and gene expression. We then design and implement GCaMP-X, by incorporating an extra apoCaM-binding motif, effectively protecting CaV1-dependent excitation-transcription coupling from perturbations. GCaMP-X resolves the problems of detrimental nuclear accumulation, acute and chronic Ca2+ dysregulation, and aberrant transcription signaling and cell morphogenesis, while still demonstrating excellent Ca2+-sensing characteristics partly inherited from GCaMP. In summary, CaM/CaV1 gating and signaling mechanisms are elucidated for GCaMP side-effects, while allowing the development of GCaMP-X to appropriately monitor cytosolic, submembrane or nuclear Ca2+, which is also expected to guide the future design of CaM-based molecular tools.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • Biosensing Techniques / methods
  • Calcium / chemistry
  • Calcium / metabolism*
  • Calcium Channels, L-Type / metabolism
  • Calcium Signaling*
  • Calmodulin / chemistry
  • Calmodulin / genetics
  • Calmodulin / metabolism*
  • Fluorescent Dyes / chemistry*
  • Genetic Engineering / methods
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • HEK293 Cells
  • Humans
  • Intravital Microscopy / methods
  • Ion Channel Gating
  • Mice
  • Mice, Inbred ICR
  • Molecular Imaging / methods*
  • Neurons
  • Patch-Clamp Techniques
  • Primary Cell Culture
  • Protein Binding

Substances

  • Calcium Channels, L-Type
  • Calmodulin
  • Fluorescent Dyes
  • Green Fluorescent Proteins
  • Calcium