Fast GCaMPs for improved tracking of neuronal activity

The use of genetically encodable calcium indicator proteins to monitor neuronal activity is hampered by slow response times and a narrow Ca²⁺-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 Ca²⁺-chelating residues. Second, we find that off-responses to Ca²⁺ 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 Ca²⁺ and by slow binding to the C-lobe at lower Ca²⁺. 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 Ca²⁺ 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.