Evaluation of Connexin Hemichannel Activity In Vivo

Connexin hemichannels (Cx HCs) are hexameric structures at the cell plasma membrane, whose function as membrane transport proteins allows for the passive flow of small hydrophilic molecules and ions (≤1 kDa) between the cytosol and the extracellular environment. Activation of Cx HCs is highly dependent on pathological conditions. HC activity provokes changes in the microenvironment, inducing the dissemination of signaling molecules in both an autocrine and paracrine manner. Given the elicitation of a variety of signaling pathways, and assortment of Cx species and dispersion throughout the body, Cx HCs have been implicated in a range of processes such as cell proliferation, differentiation, cell death, and tissue modeling and remodeling. While studying the expression and localization of Cx HCs can be done using traditional laboratory techniques, such as immunoblot analysis, measuring the functionality/activity of the HCs requires a more explicit methodology and is essential for determining Cx-mediated physiological changes. The study of Cx HC function/activity has focused mainly on in vitro measurements through electrophysiological characterization or, more commonly, using HC-permeable dye uptake studies. Here, we describe the use of dye uptake to measure Cx HC activity in vivo using mechanically stimulated osteocytic Cx43 HCs with Evans blue dye as our model.