Biochemical Characterizations of Nicastrin, Barium Blockage of Potassium Channels and Calcium Blockage of Cyclic Nucleotide-Gated Channels

This dissertation is divided into three independent chapters dedicated to research work in biochemical characterizations of nicastrin, barium blockage of potassium channels and calcium blockage of cyclic nucleotide-gated channels. The first chapter describes studies on an intramembrane protease called γ-secretase. This protease complex is known to generate the pathogenic peptide species in Alzheimer's disease, a neurodegenerative disease affecting billions around the globe. In particular, the study focused on understanding one subunit of the protease complex, the substrate receptor nicastrin. While the ultimate goal of crystallizing nicastrin was not achieved, the purification processes have revealed that glycosylation is important for folding or trafficking of the protein and disulfide bonds are crucial to maintain the tertiary structure of nicastrin. The second chapter focuses on a classic blocking phenomenon in potassium channels known as barium blocking. Previous studies on barium blocking have fueled our understanding on the ion conduction pore of potassium channels. A model of blockage has been proposed but has yet to be proven. This study recapitulated properties of barium blocking in the potassium channel NaK2K and provided structural evidence for the model of barium block. The crystal structure of NaK2K in the presence of sodium and barium showed a barium-binding pattern different from that of a structure in the presence of potassium and barium. The difference in binding explains the different blocking behaviors in the presence and absence of low concentrations of external potassium. The third chapter details a study on the weak calcium block of monovalent ion current in Drosophila cyclic nucleotide-gated (CNG) channel. Calcium blockage in the CNG channel arises from preferential but slow conduction of calcium. This blocking phenomenon is observed in all CNG channels, but to various extents. In this study, a threonine immediately outside of the selectivity filter was found to modulate the calcium block of Drosophila CNG channels, likely by causing the channel to adopt a selectivity filter structure different from canonical CNG channels. This threonine is unique to the CNG channels of Drosophila and a few insects, whereas most other CNG channels have a proline at the equivalent position. These findings have allowed us to identify a structure-function relationship for calcium blockage in Drosophila CNG channels.