Isoform M23 of human aquaporin 4 (AQP4) was over-expressed in Xenopus laevis (X. laevis) oocytes from in vitro transcribed cRNA and then imaged by Atomic Force Microscopy (AFM) in a physiological-like environment. Oocytes were injected with AQP4-cRNA and, three days later, volumetric analysis experiments, giving AQP4-induced cell swelling as estimated from the rate of cell volume change produced by an osmotic gradient (1) verified successful AQP4 expression and its incorporation into the plasma membrane. Plasma membrane patches were purified by ultracentrifugation on discontinuous sucrose gradient (2) and scanned by AFM. Biochemical analyses, namely Western Blotting and Lowry assay, proved that the purified membrane samples have an high content of AQP4.
Tapping mode AFM topography images collected on the isolated plasma membrane patches showed the characteristic square arrangement of AQP4 tetramers, with a centre-to-centre distance of 6 nm. This well recognizable spatial organization was seen only in cRNA-injected oocytes. AFM data, namely the angles and spacing between tetramer subunits as well as the size of individual tetramers, are in agreement with both TEM analyses reported in literature (3) and the current model of AQP4 (4, 5).
These findings show the potential of AFM technique of studying cloned membrane proteins for both their surface topography and structure-function relationship in native eukaryotic membranes without the need for crystallization. In particular, the present study opens appealing perspectives in the AFM investigation of heterologous membrane proteins of relevant biomedical interest cloned in a very efficient expression system such as the X. laevis oocytes.
(1)M. Santacroce et al., Comparative Biochemistry and Physiology A, (2010), 156, 509-517.
(2) F. Orsini et al., Methods, (2010), 51, 106-113.
(3) C.S. Furman et al.,PNAS, (2003), 100, 13609-13614.
(4) J.D. Ho et al., PNAS, (2009), 106, 7437-7442.
(5)Y. Cui et al., Biochemical and Biophysical Research Communications, (2011), 412, 654–659