Title: Mechanistic studies of ion binding and inactivation in the potassium channel KcsA by solid state NMR
Description: The prototypical prokaryotic channel, KcsA, is model system for mammalian potassium channels. This thesis describes solid-state NMR studies of the conformational dynamics involved in ion binding and channel inactivation. Our studies are conducted on the full-length wildtype channel incorporated into native-like lipid bilayers. We focus on the selectivity filter, which is a key conserved region across all potassium channels and is responsible for the selective passage of potassium ions across cellular membranes. KcsA is known to alter the structure of its pore as a function of the ambient potassium level; at high potassium the selectivity filter adopts a conductive state with high ion occupancy and at low potassium it collapses into a non conductive state with reduced ion occupancy. We report solid-state NMR resonance assignments for ~ 200 15N and 13C atoms in KcsA using two and three dimensional correlation spectroscopy. Using the assignments we characterize the conductive and collapsed states of the selectivity filter and show that the transition between the states occurs at a potassium concentration of 1-15 micromolar.

We interpret the shape of the binding curves in terms of the complex equilibria involved in the structural collapse of the filter. Our results describe the detailed structural and kinetic landscape of the selectivity filter as it collapses at low potassium. To gain more mechanistic insight into the physiological role of the collapsed state, we conduct studies of an inactivation resistant mutant of KcsA, E71A. C-type inactivation is the potassium and voltage dependent closure of the outer mouth of the channel and is a clinically important process in mammalian potassium channels. We show that the collapsed state is similar to the inactivated state in terms of structure and chemical shifts, which establishes that inactivation processes can be studied by lowering the ambient potassium level. We also use site-specific chemical shift tensor measurements, to show that glutamic acid 71 is protonated at neutral pH and therefore must have an abnormally high pKa (<7.5). Our studies clarify a previous inconsistency in the literature regarding the pKa of E71 and propose a new model for the role of E71 in channel inactivation.

The results have led to insights into the molecular factors that stabilize the collapsed or inactivated state of the channel, and firmly establish KcsA as a model membrane protein system for future studies of structure and dynamics by solid state NMR.