Project Summary
Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide.
Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, functions as a
water channel and as an adhesive protein. Defects in AQP0 can produce cataract, as
well as have adverse effects on lens development. Despite its critical role in lens
physiology, the functions of AQP0 are not fully understood. Our proposed research
seeks to advance our understanding of how AQP0 water permeability (Pf), the exquisite
control of which is required to maintain lens clarity, is regulated by Ca2+ and protons,
whose concentrations depend on the AQP0 location within the lens. The proposed
studies also seek to identify the amino acid residues that are crucial for Pf regulation and
for the adhesive function of AQP0, through protein-protein and/or protein-membrane
interactions, and to determine the effects of genetic modifications of AQP0 on lens
physiology and development. To these ends, we will employ a tightly coupled, multi-
disciplinary approach, unique within the field of aquaporin research, which employs
techniques ranging in scale from the atomic/molecular to the cellular and organismal
level. Specifically, in Aim 1 we propose to use in vitro Xenopus oocyte permeability
measurements on a panel of mammalian and fish AQP0 mutants to assess the
contribution of particular residues to AQP0 Pf and its regulation, along with adhesion
assays on lens fiber cells from zebrafish containing wild-type and mutant Aqp0s. These
experimental approaches will be complemented in Aim 1 with in silico multi-µs molecular
dynamics simulations, validated by comparison with experimental Pf measurements, to
elucidate mechanistic aspects of the influence of Ca2+ via calmodulin binding, pH,
including the effects of a variety of strategically chosen mutations on the Pf of
mammalian and fish AQP0s. The computer simulations proposed in Aim 1 will also
address the relative role of protein-protein and protein-membrane interactions in the
adhesive function of AQP0. Aim 2 will use genetically modified zebrafish to determine
how AQP0 contributes to the structure and function of the lens and its development in
vivo. Our work will improve understanding of lens physiology and the molecular
mechanisms of water channel gating and its regulation by Ca2+ and pH, and uncover
fundamental principles that could inform the future development of therapeutic strategies
for delaying or eliminating cataract formation.