Abstract
Purpose: :
Aquaporin-4 (AQP4) is a major water channel of retinal Müller cells, and the nonpigmented layer of the ciliary body, a major site of aqueous humor secretion in the eye. Knockout studies suggest a role for AQP4 in the regulation of intraocular pressure (IOP). The shorter splice variant of AQP4 (M23) forms orthogonal array particle aggregates (OAPs) in the membrane, while the longer M1 isoform inhibits this higher-order organization of M23, leading to a significant reduction of the single-channel intrinsic water permeability of AQP4. However, the exact physiological mechanisms that alter AQP4 higher-order organization and function remain unclear. In depth studies on the pathophysiology and regulation of human AQP4 in the eye and other organs have been hampered by the lack of a cell line that expresses human AQP4 in vitro. Therefore, we set out to create CHO cells stably expressing human AQP4 M1 and M23 splice variants.
Methods: :
The cDNA encoding human C-terminal cmyc/his tagged M1- or untagged M23-AQP4 in a pcDNA 3.1 expression vector was propagated in bacteria and utilized to transfect CHO cells with the aid of Lipofectin or electroporation. Following selection with geneticin or hygromycin, subclones were isolated and harvested by incubation of cultures with 10 mM EDTA in PBS (EDTA-PBS). Cells grown on coverslips, fixed with 4% paraformaldehyde and incubated with anti AQP4 antibodies were employed for immunohistochemical characterization. Stopped-flow light scattering was used to examine AQP4 function and freeze fracture electron microscopy to inspect the higher-order organization of AQP4 in the plasma membrane.
Results: :
Immunostaining of the transfected cells showed M1- and M23-AQP4 at the cell plasma membrane often in a punctate pattern. We confirmed that human M23-AQP4 (but not M1 AQP4) forms orthogonal arrays by freeze-fracture electron microscopy. Cells suspended in EDTA-PBS (300 mOsm NaCl) were rapidly mixed in the stopped-flow apparatus with an equal amount of phosphate-buffered 1200 mOsm NaCl to produce a 450 mOsm inwardly directed osmotic gradient. At 10 °C, AQP4-expressing cells shrunk 9.7 (M23) or 8.9 (M1) -fold faster than nontransfected CHO cells.
Conclusions: :
The results indicate that functional expression of human AQP4 isoforms has been achieved in CHO cell lines and demonstrate their utility in revealing different characteristics of the M1 and M23 splice variants, as well as potential physiologically relevant interactions between these AQP4 isoforms.
Keywords: protein structure/function • protein modifications-post translational • aqueous