Discovering the Active Site on Aquaporin 0 for Calmodulin
Aquaporin 0 is the most abundant protein of the ocular lens fiber membrane in mammals. A seven segment transmembrane protein that forms a water channel, Aquaporin 0 is crucial in maintaining healthy osmotic pressure in the lens. As humans grow old their ocular lenses lose flexibility and are unable to bend and focus the eye to produce a sharp image which leads to the need for a cataract surgery to replace their lenses. Due to their prevalence, cataract surgeries account for the greatest aggregate consumption of money of all surgical procedures. It is known that when released, calmodulin binds to at least the C-terminal tail of Aquaporin 0 and restricts the movement of water through the protein’s central channel. This function allows the brain to regulate lens flexibility by regulating calmodulin levels. It is unknown, but highly suspected, that calmodulin also binds to an intracellular loop of Aquaporin 0. It is thought that degradation of binding between Aquaporin 0 and calmodulin could be the cause of hampered lens flexibility that accompanies aging. If the binding site is found it is possible that drugs could be developed to help maintain an efficient bind and release between these proteins reducing the need for cataract surgeries. I have isolated aquaporin 0 from bovine lenses through dissection, homogenization, and anion exchange chromatography and confirmed its identity with a Western Blot. My next steps are to use protein cross linking and Hydrogen Deuterium Exchange (HDX) to image the proteins both separately and bound to find the active site.