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

GCaMP, one popular type of genetically-encoded Ca(2+) 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 (Ca(V)1). GCaMP acts as an impaired apoCaM and Ca(2+)/CaM, both critical to Ca(V)1, which disrupts Ca(2+) dynamics and gene expression. We then design and implement GCaMP-X, by incorporating an extra apoCaM-binding motif, effectively protecting Ca(V)1-dependent excitation–transcription coupling from perturbations. GCaMP-X resolves the problems of detrimental nuclear accumulation, acute and chronic Ca(2+) dysregulation, and aberrant transcription signaling and cell morphogenesis, while still demonstrating excellent Ca(2+)-sensing characteristics partly inherited from GCaMP. In summary, CaM/Ca(V)1 gating and signaling mechanisms are elucidated for GCaMP side-effects, while allowing the development of GCaMP-X to appropriately monitor cytosolic, submembrane or nuclear Ca(2+), which is also expected to guide the future design of CaM-based molecular tools.