STUDY OF HUMAN AQUAPORIN-4 EXTENDED ISOFORM BY HETEROLOGOUS EXPRESSION IN XENOPUS LAEVIS OOCYTES AND MOLECULAR DYNAMICS

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

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAI & FAIC SAFIS 2022; 2022

quaporin-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 extension by heterologous expression in Xenopus laevis oocytes and molecular dynamics (MD) simulations. Human M1-AQP4 and M1-AQP4ex (Biomatik) were subcloned into T7T-derived vector and expressed in oocytes to measure osmotic permeability coefficient (Pf) in response to a hypotonic shock. For MD simulations, the Ct end was modeled and linked to human AQP4 crystalized structure (3GD8) to build M1- AQP4 and M1-AQP4ex homotetramers by Chimera. Then, a 100 ns MD simulation was run in GROMACS for both isoforms embedded in a bilayer of lipid POPC molecules and solvated with TIP3P as a solvent model. M1ex was built with S324 in the stop codon skipped by TRT in both systems. Our results indicate that M1 and M1ex were expressed at the plasma membrane and functional for water transport, presenting Pf values different from non-inyected oocytes. Both isoforms presented an increase in Pf at both acidic (5.8) and alkaline (7.4) intracellular pH, being this increase higher in alkaline conditions. Homology modeling of the extended Ct showed that it is a random coil. MD simulations evidenced that M1ex has a larger root mean square deviation compared to M1, indicating that M1ex would 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 indicate that the Ct extension may not be involved in regulation of water permeability of M1ex. However, this is the first report evaluating Pf values of human M1-AQP4ex with S324 and demonstrating its modulation by acidic/alkaline pH.