AQP4 Extended Region Binds to α-Syntrophin with a Novel Binding Motif

Abstract Aquaporin-4 (AQP4) is an integral membrane protein channel specific for water molecules. It is highly polarized in the endfeet of astrocytes in the central nervous system where it allows water–ion homeostasis. AQP4 has two N-terminal isoforms: M1 (long) and M23 (short). Both isoforms retain their function but differ in their N-terminal lengths, which regulate AQP4 aggregation size. There are also long and short isoforms at the C-terminus. The long form has a 29-amino-acid extension (AQP4ex) that interacts with α-syntrophin, a component of the dystrophin-associated protein complex (DAPC). This interaction anchors AQP4 to the perivascularastrocyte endfeet through the DAPC, thereby maintaining its polarized localization at the blood–brain barrier. This study uses peptides with the sequence of the extended region to demonstrate a strong interaction with α-syntrophin, bound to a glass surface via maleimide-PEG silane chemistry. It was observed that substituting alanine for threonine, four amino acids from the C-terminus, eliminates binding, and an alanine scan shows that the last six amino acids at the C-terminus also play a crucial role. Kinetic studies using time-lapse single-molecule fluorescence imaging were used to determine the Gibbs free energy of activation for both forward and reverse rates (ΔG‡) using an Eyring analysis. Equilibrium constants (Keq) were measured between 15 and 37 °C, and standard-state thermodynamic free energies (ΔG°), enthalpies (ΔH°), and entropies (ΔS°) were determined using a van’t Hoff analysis. It was found that forward rates ranged from 4.5 × 104 to 1.0 × 105 M–1 s–1 depending on peptide length, with ΔG‡forward = 45–46 kJ mol–1. Reverse rates ranged from 11.6 to 21.1 s–1 depending on peptide length, with ΔG‡reverse = 65.2–67.0 kJ mol–1. Using a vant’ Hoff analysis, the overall standard-state free energies were between −19 and −22 kJ mol–1. The work also describes how dynamic single-molecule imaging can be used to determine kinetic and thermodynamic properties.