Measurement of intracellular Ca²⁺ concentration in mammalian myocardium using newly synthesized recombinant aequorin

Abstract

Measurement of cytosolic Ca²⁺ ([Ca²⁺]_i) is one of the important methods for investigating various cellular functions. Aequorin is a Ca²⁺-sensitive photoprotein extracted from jellyfish (native aequorin; N-AQ) and has been widely used for measurement of [Ca²⁺]_i in living cells. Aequorin has been employed for the simultaneous measurement of [Ca²⁺]_i and tension in muscle, particularly in mammalian myocardium. Recently, aequorin can be produced using a genetic engineering method (recombinant aequorin; R-AQ). However, the property of R-AQ and the applicability of R-AQ to the measurement of [Ca²⁺]_i in living cells were not fully investigated. We prepared R-AQ using apoaequorin expressed in Escherichia coli and coelenterazine. R-AQ showed higher sensitivity for Ca²⁺ in the middle range of pCa-light relation. Decay of the aequorin light signal in the solution with higher Ca²⁺ concentration, expressing the consumption rate of aequorin, was almost the same in both N-RQ and R-AQ. The pCa-light relation of N-AQ and R-AQ was similarly shifted to the right by Mg²⁺. The pCa-light relation of N-RQ and R-AQ was hardly influenced by pH change in the range tested (pH 6.5, 7.0 and 7.5). We tried to measure the intracellular Ca²⁺ transient (CaT) with tension in mouse papillary muscle using R-AQ. The measured CaT did not significantly differ form that with N-RQ. These results indicate that R-AQ could be used for the measurement of [Ca²⁺]_i in living cells as in the case of N-AQ. [Jpn J Physiol 55 Suppl:S89 (2005)]