Biophysical and structural characterization on a subgroup of prokaryotic voltage-gated ion channels
Biofysische en structurele karakterisatie van een subgroep van prokaryotische voltage-gereguleerde ionenkanalen
Kesters, Divya; S0169766
This project comprises biophysical and structural characterization of three types of voltage-gated ion channels, namely cyclic nucleotide-modulated channels (CNG and HCN channels), voltage-gated sodium channels (Nav) and voltage-gated calcium channels (Cav). Due to low abundance of the mammalian Cav, Nav, CNG and HCN channels from natural sources and their complex assembly and purification from recombinant expression systems, we focused on biophysical and structural characterization of some prokaryotic counterparts. The prokaryotic Cav, Nav, CNG and HCN channels that have been studied in this project all share a similar core transmembrane structure that consists of a homotetrameric subunit assembly. Each of these subunits consists of six transmembrane spanning domains termed S1 to S6, and a re-entrant pore-loop that determines the cation selectivity of these channels. In addition, CNG and HCN channels contain a C-terminal intracellular cyclic nucleotide binding domain (CNBD), which enables cyclic nucleotide induced channel modulation of CNG and HCN channels. The starting point for the first project was the bacterial voltage-gated sodium channel, termed NaChBac, which shares biophysical properties both with eukaryotic Nav and Cav channels. Even though currently available structures of bacterial Nav channels reveal these channels at atomic resolution, additional structures of full length Nav channels in the resting state, open state and drug-bound conformations are still highly desirable. In our search for ligands that confine Nav channels in oneof these conformational states we identified cinnamaldehyde as a compound which stabilizes NaChBac in the inactivated state. This observation opens up perspectives to employ cinnamaldehyde as a molecular tool to aidcrystallization of bacterial Nav channels in an inactivated conformation structure to elucidate the conformational changes that contribute to channel inactivation. Additionally, we used a multidisciplinary approach to identify new NaChBac homologs that are suitable for large-scale expression and purification of stable and monodisperse target proteins suitable for future X-ray crystallography projects on drug binding in Cav or Nav channels. In this study, we identified two proteins which might be valuable tools for those future structural studies on Nav and Cav channels. The second part of this PhD projects has focused on another family of ion channels, namely CNG and HCN channels. Whereas CNG channels are relatively insensitive to voltage and activated by cyclic nucleotides, HCN channels are activated by hyperpolarization and modulated by cyclic nucleotides. Currently available structures of CNBDs reveal little to no conformational changes between the ligand-bound and unbound form. In this study, we determined the X-ray crystal structure of the CNBD both in the presence of an agonist and an antagonist. Of interest, we observed two distinct conformational states of the CNBD. More specifically, our results show the CNBD structure in a possible activated and resting conformation in the presence of the agonist and antagonist, respectively. These results provide new insights into the conformational changesof the CNBD and thereby contribute to understanding the underlying mechanism of CNG or HCN channel modulation via its CNBD.