S-palmitoylation dependent self assembly of human aquaporin-4 orthogonal arrays of particles in biomimetic membranes

Aquaporin-4 (AQP4) is a water channel protein located in the plasma membrane of astrocytes in the central nervous system (CNS) and helps to regulate water-ion homeostasis. Isoforms of AQP4 further order themselves into aggregates known as orthogonal arrays of particles (OAPs). AQP4 exists in two main isoforms, M1 and M23, with M23 being the isoform that favors stabilization by aggregation into OAPs. It has been hypothesized that the aggregation size is regulated by the amount of M1 isoform present in the membrane. In turn, the palmitoylation state of two cysteine residues on the N-terminal tail of the M1 isoform greatly affects its mobility and ability to inhibit OAP formation. In this study, M1 was chemically cleaved and palmitoylated in vitro, and inserted in biomimetic lipid bilayers to study kinetics and aggregation behavior using single-protein fluorescence tracking. This study showed that when M1 was palmitoylated, it displays fast diffusion through the membrane with short lived protein-protein interactions and no evidence of aggregation. The depalmitoylated form of M1 showed evidence of extensive aggregation and long-lived protein-protein interactions. This study is the first of its kind to investigate human AQP4 in vitro and to extract the kinetic rate constants for protein-protein interactions as well as estimates of their equilibrium constants.