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Pumps, Channels, and Transporters: Methods of Functional Analysis
Citation key TF2015_2
Author Thomas Friedrich
Pages 387-410
Year 2015
DOI 10.1002/9781119085126.ch14
Editor Ronald J. Clarke and Mohammed A. A. Khalid
Publisher John Wiley & Sons, Inc
Chapter Cation Uptake Studies with Atomic Absorption Spectrophotometry (AAS)
Abstract This chapter describes a method to quantify the activity of cation-transporting membrane proteins, with a particular focus on electroneutrally operating cation exchange transporters that are inaccessible to conventional voltage or patch clamping. It particularly focuses on two closely related K+-countertransporting P-type ATPases, the net electrogenic Na+,K+-ATPase and the electroneutrally operating H+,K+-ATPase. Upon heterologous expression of these enzymes in Xenopus laevis oocytes, the uptake of monovalent alkali metal cations such as Li+, Rb+, or Cs+ into individual cells can be assayed by atomic absorption spectrophotometry (AAS) with high accuracy, offering in addition the promising perspective of measuring ion fluxes under accurate membrane voltage control. Xenopus laevis is a common laboratory animal because its oocytes are easy to handle as an expression system. The method described here takes advantage of large cell size and that the protein expression level achievable exhibits low cell-to-cell variability.
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