Background

Activation of G protein coupled receptors triggers production in hundreds or thousands of second messenger molecules. Once stimulated, G_q protein coupled receptors activate phospholipases C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). DAG remains membrane bound and IP₃ is released into the cytosol where it binds to a specific IP₃ receptor (calcium channel) in the endoplasmic reticulum (ER). This causes an increase of intracellular calcium concentration that modulates the activation of calcium binding proteins and Protein Kinase C (PKC). Therefore, given the importance of calcium in biological systems, many techniques/methods to analyze and measure Ca²⁺ levels have been established.

To determine the intracellular Ca²⁺ concentration, fluorescent indicators are particularly useful. Ca²⁺ indicators are available with variations in affinity, brightness or spectral characteristics. Fura-2-acetoxymethyl ester (fura-2 AM), is a membrane-permeable, non-invasive derivative of the ratiometric calcium indicator fura-2. Fura-2 AM crosses cell membranes and once inside the cell, the cellular esterases remove the acetoxymethyl group. Ca²⁺-bound fura-2 AM has its excitation maximum at 335 nm, Ca²⁺-free fura-2 AM has its excitation maximum at 363 nm. In both states, the emission maximum is about 510 nm. The typical excitation wavelengths used are 340 nm and 380 nm for Ca²⁺-bound and Ca²⁺-free fura-2 AM, respectively. The ratios 510 nm/340 nm and 510 nm/380 nm are directly related to the amount of intracellular Ca²⁺ (Figure 1). Traditionally, changes in intracellular Ca²⁺ mobilization/concentration were measured using fluorescent calcium indicators and a fluorescent confocal microscope, known as calcium imaging. This protocol explains how to use the Tecan Infinite M200^® microplate reader equipped with an injector to measure intracellular Ca²⁺ concentration. With the aid of an injector equipped microplate reader, researchers may be able to screen new agonist for G_q protein couple receptors and evaluate how the intracellular calcium mobilization is affected by mutant receptors. This protocol can be adapted to measure agonist mediated intracellular calcium mobilization in several cellular/receptor/agonist models using the appropriate growth conditions.


Figure 1. The human P2Y2 receptor mobilizes intracellular Ca²⁺ efficiently when expressed in 1321N1 human astrocytoma cells. Representative traces of intracellular calcium responses to 100 μM ATP in 1321N1 cells transfected with hHA-P2Y₂R (solid squares) and control non-transfected 1321N1 cells (solid circles). Data represent the mean ± SEM of readings from 4 independent experiments.