Electroporation loading and flash photolysis to investigate intra-and intercellular Ca2+ signaling

Abstract

Many cellular functions are driven by variations in the intracellular Ca2+ concentration ([Ca2+]i), which may appear as a single-event transient [Ca2+]i elevation, repetitive [Ca2+]i increases known as Ca2+ oscillations, or [Ca2+]i increases propagating in the cytoplasm as Ca2+ waves. Additionally, [Ca2+]i changes can be communicated between cells as intercellular Ca2+waves (ICWs). ICWs are mediated by two possible mechanisms acting in parallel: one involving gap junctions that form channels directly linking the cytoplasm of adjacent cells and one involving a paracrine messenger, in most cases ATP, that is released into the extracellular space, leading to [Ca2+]i changes in neighboring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP3) that triggers Ca2+ release from Ca2+ stores is crucial in these two ICW propagation scenarios, and is also a potent trigger to initiate ICWs. Loading inactive, “caged” IP3 into cells followed by photolytic “uncaging” with UV light, thereby liberating IP3, is a well-established method to trigger [Ca2+]i changes in single cells that is also effective in initiating ICWs. We here describe a method to load cells with caged IP3 by local electroporation of monolayer cell cultures and to apply flash photolysis to increase intracellular IP3 and induce [Ca2+]i changes, or initiate ICWs. Moreover, the electroporation method allows loading of membrane-impermeable agents that interfere with IP3 and Ca2+ signaling.