A novel Ca super(2) super(+) indicator protein using FRET and calpain-sensitive linker

Here, we report the properties of a FRET-based calcium indicator protein. We constructed a tandem fusion protein, named F2C, of ECFP and EYFP combined with calpain-sensitive sequences of alpha -spectrin, with N-terminal palmitoylation signal of GAP-43. It was previously reported that calpain cleaved a similar ECFP-EYFP fusion protein linked by a calpain-sensitive sequence of alpha -spectrin (fodrin). Unexpectedly, F2C was not cleaved by calpain, but demonstrated properties of a Ca super(2) super(+) indicator when transiently infected in Purkinje cells of rat primary cerebellar culture or in the brainstem neurons infected in vivo using Sindbis virus encoding F2C. The emission ratio of 480nm/535nm was repeatedly increased when the intracellular Ca super(2) super(+) concentration ([Ca super(2) super(+)] sub(i)) was raised. F2C had a Ca super(2) super(+) sensitivity with an apparent dissociation constant (K sub(d) for Ca super(2) super(+)) of 150nM, and demonstrated kinetics that paralleled Fura-2 when [Ca super(2) super(+)] sub(i) was measured simultaneously. These properties of F2C are useful to be a Ca super(2) super(+) indicator.