Indicator-dependent differences in detection of local intracellular Ca2+ release events evoked by voltage-gated Ca2+ entry in pancreatic β-cells

Abstract

Genetically encoded Ca²⁺ indicators have become widely used in cell signalling studies as they offer advantages over cell-loaded dye indicators in enabling specific cellular or subcellular targeting. Comparing responses from dye and protein-based indicators may provide information about indicator properties and cell physiology, but side-by-side recordings in cells are scarce. In this study, we compared cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) changes in insulin-secreting β-cells recorded with commonly used dyes and indicators based on circularly permuted fluorescent proteins. Total internal reflection fluorescence (TIRF) imaging of K⁺ depolarization-triggered submembrane [Ca²⁺]_i increases showed that the dyes Fluo-4 and Fluo-5F mainly reported stable [Ca²⁺]_i elevations, whereas the proteins R-GECO1 and GCaMP5G more often reported distinct [Ca²⁺]_i spikes from an elevated level. [Ca²⁺]_i spiking occurred also in glucose-stimulated cells. The spikes reflected Ca²⁺ release from the endoplasmic reticulum, triggered by autocrine activation of purinergic receptors after exocytotic release of ATP and/or ADP, and the spikes were consequently prevented by SERCA inhibition or P2Y₁-receptor antagonism. Widefield imaging, which monitors the entire cytoplasm, increased the spike detection by the Ca²⁺ dyes. The indicator-dependent response patterns were unrelated to Ca²⁺ binding affinity, buffering and mobility, and probably reflects the much slower dissociation kinetics of protein compared to dye indicators. Ca²⁺ dyes thus report signalling within the submembrane space excited by TIRF illumination, whereas the protein indicators also catch Ca²⁺ events originating outside this volume. The study highlights that voltage-dependent Ca²⁺ entry in β-cells is tightly linked to local intracellular Ca²⁺ release mediated via an autocrine route that may be more important than previously reported direct Ca²⁺ effects on phospholipase C or on intracellular channels mediating calcium-induced calcium release.

Keywords: Ca(2+) oscillations; Fluo-4; Fluo-5F; Insulin secretion; P2Y(1) receptor; R-GECO1; TIRF microscopy.