Intracellular pH Measurements In Vivo Using Green Fluorescent Protein Variants

Whether by patch-clamp techniques or the use of fluorescent vital dyes, measurements of transepithelial ion flux in mammals are limited by cell accessibility. Furthermore, redundant functions and complex regulatory mechanisms can mask loss-of-function phenotypes through compensatory mechanisms. In this chapter, we present a technique whereby the optically transparent nematode Caenorhabditis elegans, engineered to express a fluorescent pH indicator protein, can be used to study how intracellular pH (pHi) fluctuates in response to environmental and/or experimental challenge. By using a live whole animal model, systemic, and even behavioral relationships to individual cellular pHi can be inferred. In combination with dye loading of excised or cultured cells, this technique also provides a powerful means of contrasting these relationships to biophysical measurements of ion flux.