Measurement of Free Ca²⁺ Concentration in the Lumen of Neuronal Endoplasmic Reticulum

INTRODUCTION

This protocol describes a technique for the simultaneous monitoring of free calcium concentrations in the cytosol ([Ca²⁺]_i) and within the lumen of the endoplasmic reticulum (ER) ([Ca²⁺]_L) of cultured/freshly isolated dorsal root ganglia (DRG) neurons. The method uses two synthetic fluorescent Ca²⁺ probes (fluo-3 and mag-fura-2) in combination with fluorescence microscopy and a whole-cell patch-clamp technique. This approach has been used successfully in acutely isolated/cultured DRG neurons, in Purkinje neurons, acutely isolated from cerebellar slices, and in cultured astrocytes. In this protocol, isolated neurons are first loaded with the membrane-permeant, low-affinity Ca²⁺ indicator, mag-fura-2, which preferentially, though not exclusively, accumulates in the ER. Cells are then loaded with the membrane-impermeant, high-affinity calcium indicator fluo-3 using the whole-cell patch-clamp configuration. This second loading removes the majority of cytosolic mag-fura-2, replacing it with fluo-3. Mag-fura-2 and fluo-3 signals can be separated by virtue of their distinct excitation properties (340 nm and 380 nm for mag-fura-2, and 488 nm for fluo-3). An equation is provided to determine [Ca²⁺]_L values using the 340/380 nm ratio.