Design and mechanistic insight into ultrafast calcium indicators for monitoring intracellular calcium dynamics.

ABSTRACT: Calmodulin-based genetically encoded fluorescent calcium indicators (GCaMP-s) are powerful tools of imaging calcium dynamics from cells to freely moving animals. High affinity indicators with slow kinetics however distort the temporal profile of calcium transients. Here we report the development of reduced affinity ultrafast variants of GCaMP6s and GCaMP6f. We hypothesized that GCaMP-s have a common kinetic mechanism with a rate-limiting process in the interaction of the RS20 peptide and calcium-calmodulin. Therefore we targeted specific residues in the binding interface by rational design generating improved indicators with GCaMP6f_u displaying fluorescence rise and decay times (t_1/2) of 1 and 3 ms (37 °C) in vitro, 9 and 22-fold faster than GCaMP6f respectively. In H

SUBMITTER: Helassa N 

PROVIDER: S-EPMC5138832 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature