Determination of Ion Channel and Pump Fluxes for Chinese Hamster Ovary Cells using Rubidium Uptake Measurement

Abstract

Cells expend a significant portion of their energy and have considerable apparatus to control the flow ions across the cell membrane. Ions concentrations have also been demonstrated to be important to processes such as apoptosis and implicated in monitoring cells metabolism for biopharmaceutical production. Accurate quantitative modelling of ion transport is challenging since the densities of ion channels and pumps are known for relatively few cells. In this work the ion channels and pumps fluxes for CHO cells were estimated using Rubidium uptake measurements combined with Ouabain inhibition of pumps.Attached CHO cells were incubated in a buffer containing 5.4 mM Rb and in some cases Ouabain was also added to the buffer in different concentrations over a period of two hours to inhibit the Na+-K+ ATPase pump to study passive and active ion fluxes across the membrane.Using Inductively Coupled Plasma Optical Emission Spectrometry, the Rb content of the lysed cells after two hours of incubation was 10.6 mg/L, which is a result of Rb transfer through the channels and pumps. From this, the Rb flux through the active and passive pathways is estimated to be 8.5×10−12 mol.cm−2.s−1.The Rb entry through the diffusion pathway was estimated using Ouabain to inhibit the Na+-K+ ATPase activity. With 5 mM concentration of Ouabain the inhibition was maximized and the Rb uptake is reduced by 80% compared to the control. From this result the Rb flux through the channels is estimated to be 2.8×10−12 mol.cm−2.s−1.The obtained results were used in temporal ion concentration simulations using methodology of Hernandez 2000. These simulations are in agreement with the work where ion pumps were inhibited via ATP production inhibition in Mitochondria using Oligomycin (B. Saboktakin Rizi, Biomicrofluidics, 2014).