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

• Published in: Nature communications Vol. 9; no. 1; pp. 1504 - 18
• Main Authors: Yang, Yaxiong, Liu, Nan, He, Yuanyuan, Liu, Yuxia, Ge, Lin, Zou, Linzhi, Song, Sen, Xiong, Wei, Liu, Xiaodong
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 17.04.2018; Nature Publishing Group; Nature Portfolio
• Subjects: 14/1; 14/19; 14/34; 14/35; 14/63; 42/109; 631/1647/245/2225; 631/378/2586; 631/45/269/1146; 631/80/86/1999; 631/80/86/2372; 9/10; 9/74; 96/95; Animals; Biosensing Techniques - methods; Calcium; Calcium - chemistry; Calcium - metabolism; Calcium channels; Calcium channels (L-type); Calcium channels (voltage-gated); Calcium Channels, L-Type - metabolism; Calcium imaging; Calcium ions; Calcium Signaling; Calcium signalling; Calcium-binding protein; Calmodulin; Calmodulin - chemistry; Calmodulin - genetics; Calmodulin - metabolism; Channel gating; Coupling (molecular); Fluorescent Dyes - chemistry; Gene expression; Genetic Engineering - methods; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; HEK293 Cells; Humanities and Social Sciences; Humans; Intravital Microscopy - methods; Ion Channel Gating; Mice; Mice, Inbred ICR; Molecular Imaging - methods; Morphogenesis; multidisciplinary; Neurons; Patch-Clamp Techniques; Primary Cell Culture; Protein Binding; Science; Science (multidisciplinary); Side effects; Transcription
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-018-03719-6

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. The popular genetically-encoded Ca 2+ indicator, GCaMP, has several side-effects. Here the authors show that GCaMP containing CaM interferes with gating and signaling of L-type calcium channels, which disrupts Ca 2+ dynamics and gene expression, and develop GCaMP-X to overcome these limitations.