Characterization of Calcium-dependent Phosphatase Activity of Calcineurin in Cultured Cortical Neurons

Nanteetip Limpeanchob

Authors

Nanteetip Limpeanchob Department of Pharmacy Practice, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok 65000, Thailand.

Keywords:

calcineurin, phosphopeptide, calcium

Abstract

Calcineurin (CaN) is a serine/threonine phosphatase enzyme that is expressed in many types of tissues such as immune cells, muscle cells and neurons. It plays important roles in regulating immunological responses in lymphocytes cells. In neurons, CaN is involved in synaptic plasticity associated with learning and memory formation. It is known that the phosphatase activity of CaN required small increase in intracellular Ca²⁺concentration due to its high affinity to Ca²⁺ ions. Various types of CaN inhibitors are widely used to indirectly determine CaN activity in vivo and in intact individual neurons. In in vitro experiments, phosphatase activity of CaN from cell lysate can be directly measured by using CaN substrates. Most of these in vitro assays were conducted in buffer solutions containing Ca²⁺ at the physiological level of extracellular fluid (mM). Due to high concentration of Ca²⁺, this condition might not be the optimal assay condition for measuring CaN activity. Thus, the objective of this study was to investigate Ca²⁺-dependent pattern of CaN activity in cell lysate of cultured cortical neurons. Phosphatase activity of CaN was determined by using RII phosphopeptide substrate, a specific substrate for CaN. The results from this study showed that CaN was highest activated in the presence of low concentrations of free Ca²⁺ (80-100 nM) in the reaction. In contrast, high level of Ca²⁺ appeared to reduce CaN activity in vitro. This study also showed non-specific dephosphorylation of RII phosphopeptide by other phosphatases which was probably due to high concentration of substrate used in the assay. Therefore, titrating free Ca²⁺ and substrate concentrations seems to be an important process for CaN activity measurement in test tubes. In addition, this study suggests that in vitro CaN activity requires low amount of Ca²⁺ as well as previous in vivo studies.

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