Aquaporin 4 (AQP4) is a tetrameric water channel protein with a pore in each monomer. AQP4 is the most abundant water channel protein in the brain, highly expressed in astrocytes. AQP4 knockout mice are protected against cytotoxic brain oedema. A cytotoxic brain oedema is where the blood brain barrier remains intact and the oedema is thought to form as a result of deterioration of cellular metabolism. There is no generally accepted current therapy for cytotoxic oedema, in part due to a lack of understanding surrounding the cellular signaling events. Work from our laboratory on a novel trigger for sub-cellular redistribution of the homologous AQP1 kidney protein has allowed us to investigate this phenomena in AQP4. GFP tagged aquaporin constructs were created by inserting the Vector-N75 in the PcDNA-DEST47, by swapping out the ccdB gene which kills E.coli. The Vector-N75 doesn’t contain a stop codon and is instead followed by attb attachment sites then the GFP tag sequence. The cellular relocalisation of GFP-tagged AQP4 was exposed to a hypotonic extracellular environment transfected into a live Human Embryonic Kidney (HEK293) cell line using confocal microscopy. Relative membrane expression (RME) of the AQPGFP was measured by comparing the fluorescence intensity profiles of GFP tagged proteins across lines drawn using the image analysis software which bisect the membrane and cytosol of cells exposed to isotonic and hypotonic extracellular environments. These lines were drawn across the membrane, cytosol and opposite membrane, without bisecting the nucleus. These profile intensities were calculated using the software ImageJ. Cell volume was estimated by converting the image, in ImageJ, to a binary form, then, using the analyse particle function the cell was scanned to find the edge, an outline drawn and the area calculated. AQP4 rapidly and reversibly translocated to the cell surface in response to hypotonicity increasing RME from 29.3% ± 6.4% to 54.9% ± 6.6% (p<0.05; N=3). The cellular signaling required for this translocation response was investigated by exhibiting cells to different activation inhibitors. PKAi, Cytochalasin D, and extracellular calcium-free media were found to fully prevent the translocation and functional swelling response of HEK293 cells transfected with AQP4 (p [sic] Motif and conservation analysis was used to identify potential PKA activation sites. These sites were substituted from serine to both alanine and aspartic acid using site-directed mutagenesis (SDM). One, highly conserved serine residue of transmembrane region 1 (TM1) at position 52, when mutated to aspartic acid (S52D) lost the hypotonicity-induced translocation and cell volume increase (p<0.01; N=3 in all cases). The alanine substitution (S52A) was unaffected. Molecular modeling of AQP4 and the mutant suggest a potential hydrostatic interaction with nearby cysteine and serine residues of TM2. Consequently the S52 residue was mutated to leucine, which has a similar size but no charge to form the potential hydrostatic interactions with neighboring residues. S52L also removed the translocation and functional swelling in response to hypotonicity, suggesting steric hindrance as the most likely factor inhibiting the translocation process rather than the interaction with neighboring residues. This study demonstrates that a cellular signaling response to a change in tonicity of the cellular environment leads to AQP4 translocation to and from the cell surface. This involves the influx of extracellular calcium, the activation of PKA and cytoskeletal reorganization.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:658971 |
| Date | January 2014 |
| Creators | Taylor, Luke H. J. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/71046/ |
Page generated in 0.0018 seconds