Ca²⁺ indicator constructed using fluorescence resonance energy transfer and its properties

Abstract

Ca²⁺ regulates numerous cellular functions. The intracellular Ca²⁺ concentration of free Ca²⁺ have been measured by using synthetic fluorescent chelators such as FURA2. The chelators are easily imaged, but they are hard to be targeted to specific intracellular locations, and they gradually leak out of the cell. We report here properties of a fluorescence resonance energy transfer (FRET)-based calcium indicator protein. We designed a tandem fusion of cyan- and yellow- fluorescent protein and N-terminal icosapeptide (palmitoylation signal) of growth-associated protein-43 (GAP43). This fusion protein associates to cell membrane by palmitoylation signal. Two fluorescent protein was combined with the calpain cleavage site of fodrin. Calpain is known as a calcium-dependent protease widely distributed in mammalian cells. The construction of this calpain sensitive fusion protein is the same as Vanderklish et. al reported except for N-terminal signal. They reported that the fusion protein was cleaved by calpain and marked synaptic activity, but our fusion protein was not cleaved by calpain, surprisingly. Purkinje cells from rat cerebellum were transiently infected with virus encoded this protein using Sindbis virus system, and then fluorescent image analysis was performed. The fluorescence signal from this protein was reversible and was repeatedly changed when intracellular Ca²⁺ concentration was altered. Basically the same results were acquired in vivo infection. These properties of this protein is useful to be a Ca²⁺ indicator. [Jpn J Physiol 55 Suppl:S204 (2005)]