Whole Endosome Recording of Vesicular Neurotransmitter Transporter Currents

Abstract

The analysis of organellar membrane transporters presents many technical problems. In general, their activity depends on a H⁺ electrochemical driving force (Δμ_H+). However, transport itself influences the expression of Δμ_H+ in standard radiotracer flux assays, making it difficult to disentangle the role of the chemical component ΔpH and the membrane potential Δψ. Whole endosome recording in voltage clamp circumvents many of these problems, controlling ionic conditions as well as membrane potential inside and outside the organelle . This approach has been used primarily to study the properties of endolysosomal channels, which generate substantial currents (Saito et al., J Biol Chem 282(37):27327–27333, 2007; Cang et al., Nat Chem Biol 10(6):463–469, 2014; Cang et al., Cell 152(4):778–790, 2013; Chen et al., Nat Protoc 12(8):1639–1658, 2017; Samie et al., Dev Cell 26(5):511–524, 2013; Wang et al., Cell 151(2):372–383, 2012). Electrogenic transport produces much smaller currents, but we have recently reported the detection of transport currents and an uncoupled Cl⁻ conductance associated with the vesicular glutamate transporters (VGLUTs) that fill synaptic vesicles with glutamate (Chang et al., eLife 7:e34896, 2018). In this protocol, we will focus on the measurement of transport currents on enlarged endosomes of heterologous mammalian cells.