Study of human Aquaporin-4 extended isoform by heterologous expression in Xenopus laevis oocytes and molecular dynamics.

VITALI, VICTORIA; CAPURRO, CLAUDIA; CASAL, JUAN JOSÉ; NETTI, VANINA; MOLINA PONCE, TOMÁS; ALLEVA, KARINA

Mar del Plata

Congreso; Reunión Conjunta SAIC SAI&FAIC SAFIS 2022; 2022

Aquaporin-4 (AQP4) is expressed at the plasma membrane as 2 isoforms,M1-AQP4 of 323 amino acids (aa) and M23-AQP4 of 301 aa.Recently, a new AQP4 isoform with a 29 aa C-terminal (Ct) extension(AQP4ex, 352 aa) generated by translational readthrough (TRT)was described, which has not been fully characterized yet. Thus,the aim of our work was to study the properties of the Ct extensionby heterologous expression in Xenopus laevis oocytes and moleculardynamics (MD) simulations. Human M1-AQP4 and M1-AQP4ex(Biomatik) were subcloned into T7T-derived vector and expressed inoocytes to measure osmotic permeability coefficient (Pf) in responseto a hypotonic shock. For MD simulations, the Ct end was modeledand linked to human AQP4 crystalized structure (3GD8) to build M1-AQP4 and M1-AQP4ex homotetramers by Chimera. Then, a 100 nsMD simulation was run in GROMACS for both isoforms embeddedin a bilayer of lipid POPC molecules and solvated with TIP3P as asolvent model. M1ex was built with S324 in the stop codon skippedby TRT in both systems. Our results indicate that M1 and M1exwere expressed at the plasma membrane and functional for watertransport, presenting Pf values different from non-inyected oocytes.Both isoforms presented an increase in Pf at both acidic (5.8) andalkaline (7.4) intracellular pH, being this increase higher in alkalineconditions. Homology modeling of the extended Ct showed that itis a random coil. MD simulations evidenced that M1ex has a largerroot mean square deviation compared to M1, indicating that M1exwould be less compact and stable. The distance from H201 to R216,representative of the selectivity filter (M1: 7.7 ± 4.2Å vs. M1ex: 8.3± 2.1 Å, ns), is similar in both isoforms. Our preliminary results indicatethat the Ct extension may not be involved in regulation of waterpermeability of M1ex. However, this is the first report evaluating Pfvalues of human M1-AQP4ex with S324 and demonstrating its modulationby acidic/alkaline pH.