Date of Award
Doctor of Philosophy (PhD)
Ion channels require proper ion selectivity and regulated gating in order to perform their cellular functions. Bacterial ion channels serve as excellent model systems to study structure/function relationships concerning the fundamental processes of ion selectivity and gating. NaK, a non-selective cation channel from Bacillus cereus, has the conserved pore structure of K+ channels. However, its non-canonical selectivity filter structure leads to non-selectivity between Na+ and K+. Full selectivity is restored with two mutations that lead to the restoration of a Kcsa-like selectivity filter structure. Many mutations can be made to the selectivity filter of NaK without loss of protein stability or function, and this structural stability makes it an excellent model system to study the molecular mechanism of ion selectivity. Experimental measurements on the dynamics and stability of the selectivity filter and how this relates to ion selective have not been performed in this system. The data presented in this thesis establishes that solution NMR dynamic studies of NaK that will lead to a better understanding of how backbone dynamics tune ion selectivity.
The results presented here also reveal an unexpected allostery between the selectivity filter and inner gate of NaK. Allosteric coupling between the selectivity filter and inner gate links gating at the selectivity filter (C-type inactivation) to the state of the inner gate, thus regulating the cycle of channel gating. My work demonstrates that this coupling is an intrinsic structural and dynamic property of NaK and is mechanistically distinct from the well-studied KcsA model. Allostery in channel gating is also important for transmitting signals from regulatory domains to the channel pore. I investigated this coupling of the amphipathic regulatory helix of NaK to the inner gate and found that interaction is mediated through inter- and intra-molecular side chain interactions between M0 and the pore domain. When these interactions are disrupted, the channel reverts to a state similar to what is seen without the regulatory helix.
Chair and Committee
Gaya Amarasinghe, Christopher Lingle, Colin Nichols, Paul Schlesinger
Brettmann, Joshua B., "Ion Selectivity and Allostery in the NaK Channel" (2015). Arts & Sciences Electronic Theses and Dissertations. 637.