Novel variants in human Aquaporin-4 reduce cellular water permeability

Abstract

Cerebral edema contributes significantly to morbidity and mortality after brain injury and stroke. Aquaporin-4 (AQP4), a water channel expressed in astrocytes, plays a key role in brain water homeostasis. Genetic variants in other aquaporin family members have been associated with disease phenotypes. However, in human AQP4, only one non-synonymous single-nucleotide polymorphism (nsSNP) has been reported, with no characterization of protein function or disease phenotype. We analyzed DNA from an ethnically diverse cohort of 188 individuals to identify novel AQP4 variants. AQP4 variants were constructed by site-directed mutagenesis and expressed in cells. Water permeability assays in the cells were used to measure protein function. We identified 24 variants in AQP4 including four novel nsSNPs (I128T, D184E, I205L and M224T). We did not observe the previously documented M278T in our sample. The nsSNPs found were rare ( approximately 1-2% allele frequency) and heterozygous. Computational analysis predicted reduced function mutations. Protein expression and membrane localization were similar for reference AQP4 and the five AQP4 mutants. Cellular assays confirmed that four variant AQP4 channels reduced normalized water permeability to between 26 and 48% of the reference (P < 0.001), while the M278T mutation increased normalized water permeability (P < 0.001). We identified multiple novel AQP4 SNPs and showed that four nsSNPs reduced water permeability. The previously reported M278T mutation resulted in gain of function. Our experiments provide insight into the function of the AQP4 protein. These nsSNPs may have clinical implications for patients with cerebral edema and related disorders.

Figure 1.

(A) Characteristics of the AQP4 gene. The long isoform of AQP4 is composed of five exons. The 323 amino acid protein includes six transmembrane domains, and its central structure consists of the large Major Intrinsic Protein domain. (B) Schematic secondary structure of AQP4, showing the intra-cellular N-and C-terminal domains, the six transmembrane domains and five loops, the four novel SNPs (squares), and the previously identified M278T (circle). Figure generated by TOPO2 (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl). (C and D) Three-dimensional crystal structure of rat AQP4 (PDB code, 2d57) with 93% identity to human AQP4, showing the four novel nsSNPs (green, orange, yellow, yellow-green) we identified distal to the pore motifs (yellow and blue), (C) looking through the channel and (D) profile view. Figures generated by UCSF Chimera (http://www.sacs.ucsf.edu/TOPO-run/wtopo.pl).

Figure 2.

Haplotypes are shown by allele frequency for each ethnic group. Frequencies reveal population substructure. The African American group is most homogenous, while the Chinese American and Mexican American groups both have two major haplotypes. Variant alleles are highlighted in yellow. Singletons are not shown.

Figure 3.

Linkage disequilibrium plots with variant IDs and position in the gene. D′ < 1 and LOD < 2 white, D′ < 1 and LOD ≥ 2 shades of pink/red, D′ = 1 and LOD ≥ 2 bright red. LD is high for the few variants in Chinese Americans but generally low in other ethnicities. There were few cases of LD among the four nsSNPs. In African Americans, the D184E SNP was in LD with the L164 SNP, and the I128T SNP was in LD with a novel, non-coding singleton. In Mexican Americans, the same linkage was seen for I128T.

Figure 4.

Multiple sequence alignment of AQP4 orthologs spanning identified SNPs (in grey) and showing high evolutionary conservation, suggesting selective pressure. (‘*’, identical; ‘:’, conserved physicochemical substitutions; ‘.’, semi-conserved substitutions.)

Figure 5.

(A) Original light scattering curves of a single recording of CHO cell suspensions, expressing different AQP4 mutants. (B) Schematic of the light scattering experiments. (C) Cell-surface proteins were biotinylated, isolated by strepavidin and blotted with an AQP4 antibody. Western blots of biotinylated cell-surface protein demonstrating consistent membrane protein expression of AQP4 mutants (lanes 4–8). (D) Relative water permeability calculated from the individual t_1/2 normalized to the cell surface AQP4 expression. I128T, D184E, I205L and M224T had lower water permeability relative to reference, reduced to 48.3 ± 11.5%, 36.3 ± 8.1%, 25.6 ± 5.8% and 31 ± 5.6%, respectively (P < 0.001).

Figure 6.

For the M278T SNP not found in our sample set, (A) representative light scattering curve for the CHO cell suspensions transfected with the empty vector, AQP4 and the M278T variant. (B) Western blots quantifying surface protein expression of AQP4 M278T. (C) Relative water permeability calculated from the individual t_1/2 and normalized cell surface AQP4 expression. The M278T variant showed increased relative water permeability (138.1 ± 9.0%, P < 0.001).