Stopped-flow Light Scattering Analysis of Red Blood Cell Glycerol Permeability

Background

The movement of water and solutes across biological and artificial membranes can be analyzed using different approaches. Assessing the rate with which the volume of any sealed particle lined by a membrane changes following the osmotically-driven movement of water represents an indirect approach widely used to measure biophysically the water and solute permeability of the particle specimen.

The Stopped-Flow Light Scattering (SFLS) method was devised to analyze the kinetics of rapid enzymatic reactions accompanied by significant changes of the molecular weight and/or shape of the reacting substrates (Riesner and Buenemann, 1973). SFLS soon proved to be an implementable, reliable and reproducible spectroscopic technique with a number of advantages and only few limitations. While the original method remains extensively used to study the chemical kinetics of fast reactions in solution the conventional SFLS apparatus has been modified to allow the measurement of the coefficient of solute and osmotic water permeability (P_s and P_f, respectively; cm/s) of both biological and artificial membranes (Terwilliger and Solomon, 1981; Mlekoday et al., 1983; van Heeswijk and van Os, 1986). SFLS is largely employed to assess the P_s and P_f of whole cells (Yang and Verkman, 2002; Maggio et al., 2018), organelles, extracellular vesicles (Calamita et al., 2005 and 2006; Ivanova et al., 2008; Soria et al., 2010), and vesicles of any kind of biological membrane (Shi et al., 1990; Calamita et al., 2012; Gena et al., 2017; Miyazawa et al., 2018). The method is also used to measure the permeability of artificial membranes such as liposomes (Zeidel et al., 1992 and 1994; Harris et al., 1994; Agre et al., 1999; Müller-Lucks et al., 2013) and polymersomes (Erbakan et al., 2014; Loo et al., 2017). SFLS is also employed to evaluate the Arrhenius activation energy (E_a; kcal/mol) of water and solute transport based on the temperature dependence with which molecules cross the membrane (Agre et al., 1999; Yang and Verkman, 2002; Mathai et al., 2001; Mouro-Chanteloup et al., 2010).

Here, we describe a protocol of SFLS to measure the glycerol permeability of isolated human RBCs in presence or absence of three isoform-specific blockers of AQP3, AQP7 and AQP9 (Jelen et al., 2011; Sonntag et al., 2019), three aquaglyceroporin channels conducting glycerol, water and some other molecules. AQP3, AQP7 and AQP9 are variously expressed in the body playing roles both in health and disease (Yang et al., 2001; Hara-Chikuma and Verkman, 2006) and triggering strong pharmacological interest (Calamita et al., 2018). RBC glycerol permeability is measured after their exposure to different concentrations of the three blockers (vs. control RBCs treated with the vehicle alone). Useful information is acquired regarding the efficiency, selectivity and potency of the compounds in blocking the Aquaglyceroporin-facilitated glycerol transport as well as their preclinical sustainability (solubility, toxicity) for drug developments.