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Control of the aquaporin-4 channel water permeability by structural dynamics of aromatic/arginine selectivity filter residues Philip Kitchen, and Alex C Conner Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01053 • Publication Date (Web): 29 Oct 2015 Downloaded from http://pubs.acs.org on November 3, 2015
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Biochemistry
Control of the aquaporin-4 channel water permeability by structural dynamics of aromatic/arginine selectivity filter residues *
Philip Kitchen and §Alex C Conner
* §
Molecular Organisation and Assembly in Cells Doctoral Training Centre, University of Warwick, Coventry CV4 7AL, UK. Institute of Clinical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
KEYWORDS Aquaporin, Selectivity, Osmosis, Water transport Supporting Information Placeholder ABSTRACT The aquaporins (AQPs) are a family of integral membrane proteins that control cellular water flow. Gating of the water channel by conformational changes induced by phosphorylation or protein-protein interactions is an established regulatory mechanism for AQPs. Recent in silico and crystallographic analyses of the structural biology of AQPs suggest that the rate of water flow can also be controlled by small movements of single amino acid sidechains lining the water pore. Here we use measurements of the membrane water permeability of mammalian cells expressing AQP4 mutants to provide the first in vitro evidence in support of this hypothesis.
Rapid modulation of AQP-mediated cell membrane water permeability in response to external stimuli is a vital regulatory mechanism that underlies a variety of physiological processes in mammals and plants(1, 2). There are two ways in which this could be achieved: by changing the number of AQP molecules in the membrane via exo- or endocytosis, or by directly blocking or activating AQP molecules via a conformational change that opens or closes the pore. There are examples of each of these approaches to AQP regulation being taken in nature, the best-studied examples of which are the arginine-8 vasopressin (AVP) mediated trafficking of AQP2 to the apical membrane of kidney collecting duct cells(3), and the phosphorylation-mediated gating of the spinach AQP SoPIP2, involving a large scale motion of an intracellular loop which stabilizes a closed conformation of the aqueous pore(4). A conceptual model of AQP gating in which the channel is either open or closed and can be ‘switched off’ by stimuli is probably overly simplistic and indeed, researchers in the field of G proteincoupled receptor (GPCR) structural biology have long since abandoned conceptual models in which receptors are thought of as ‘on’ or ‘off’, in favour of models in which receptors are thought of as continually visiting various conformations, each with unique ligand affinities and signalling capacities. In this framework, rather than ‘switching on’ a receptor, ligands are thought of as stabilizing (increasing the probability to visit) a particular conformation with a particular signalling capacity. In silico work on the bacterial AQPZ(5), human AQP5(6), AQP1(7), and human AQPs 1 and 4(8) suggest that a similar model may be appropriate for AQPs and that particularly important local conformational changes may
involve the pore-lining residues within the aromatic/arginine (ar/R) selectivity filter region. Here we show that specific site-directed mutations to the selectivity filter region of human AQP4 can quantitatively alter the permeability of the channel independently of surface expression. We made alanine substitution mutants of the three AQP4 residues whose sidechains form the ar/R selectivity filter, F77A, H201A and R216A. The histidine is typically replaced by a glycine in the GLP subfamily so we also made a H201G mutant. After finding that the H201A and F77A mutations had no effect, we combined them into the H201A/F77A and H201G/F77A double mutants. Finally, we made the H201E mutant to try to form a salt bridge between the glutamate residue and R216. Mutants were generated by site-directed mutagenesis as previously described(9). The M1 isoform of AQP4 with a C-terminal GFP tag and all selectivity filter mutants of this construct were stably transfected into type I MDCK cells (ATCC CRL-2935) to measure water permeability using a plate-reader based calcein fluorescence quenching method adapted from Fenton et al(10). Expression of AQP4 mutants in stable clones was checked visually using the GFP tag and SDSPAGE/Western blotting against AQP4. Endogenous AQP4 was not detected in MDCK cells in Western blots. After 5 seconds of reading at 50 ms intervals in isotonic culture media, an equal volume of 600 mM mannitol in culture media was injected to give a final mannitol concentration of 300 mM and an osmotic gradient of 300 mOsm/kg H2O and fluorescence was measured for a further 50 s. Normalized fluorescence timeseries were converted to volume timeseries. Cell shrinkage rates were backgroundsubtracted (using an untransfected MDCK control to correct for any basal water permeability of the cells) and normalised to surface expression (measured using a cell surface biotinylation-based ELISA as previously described(9)) to give relative single channel water permeability. For most mutants, the variations in the shrinkage rates of individual clones (figure 1B) were consistent with the variation in surface expression (figure 2A). After normalization none of the mutants except H201E and R216A had significantly different water permeability to wild-type AQP4. The water permeability of H201E was reduced to 63±8% of wild-type (n=4, p=0.002) and the water permeability of R216A was increased to 147±3% of wild-type (n=4, p=0.005); this is shown in figures 1 and 2. Hub et al previously reported that, in molecular dynamics simulations, fluctuations of R216 of AQP4 could switch the channel
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Biochemistry
nel, based on the idea that the R216A mutation forces the channel to permanently occupy the open state. Previous molecular dynamics simulations suggested an approximately 40-fold difference in permeability between the open and closed states of AQP4(8). Using the following equation:
(B) MDCK
0.96 0.94 0.92 0.9 0.88
M
0.86
-5
5
15
25
35
45
Figure 1. (A) Representative calcein fluorescence quenching curves of control MDCK cells and our stable MDCK-AQP4 cell line upon addition of 300 mM mannitol. (B) Cell shrinkage rates of MDCK cells stably transfected with AQP4 mutants. Relative fluorescence data was converted to relative volume and exponential decay functions were fitted.
(B)
1 0.8 0.6 0.4 0.2 0
1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0
*
*
AQ P4 F7 7 H2 A 01 H A 20 1G H 20 1E F7 R 2 7A 16 A F7 /H2 7A 01 A /H 20 1G
1.2
QP F7 4 7 H2 A 01 H A 20 1 H G 20 F7 R 1 E 7A 21 6 F7 /H A 7A 201 /H A 20 1G
Normalized surface expression
1.4
Normalised single channel water permeability
(A)
Figure 2. (A) Surface expression of AQP4 mutants in stable MDCK clones, normalized to wild-type AQP4. (B) Normalized single channel permeability calculated by subtracting the untransfected MDCK background (see figure 1B) and normalizing to surface expression. * represents p
