Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Zhe Lu

Abstract

TrkAH and MthK are bacterial ligand-gated K+ channels with no sequence similarity and remarkably different structural and functional properties. TrkAH has a two-pore module with non-zero basal open probability that can be up- or downregulated in response to cytosolic ATP or ADP, respectively. In MthK, the open probability of a single pore is increased from zero to one by cytosolic Ca2+. Due to their different pore and selectivity filter architectures, the two channels are predicted to have distinct gating mechanisms. The only common feature between TrkAH and MthK is that in each system the effect of ligand on the open probability is exerted through a cytosolic regulatory module, composed of eight structurally conserved Regulator of Conductance for K+ (RCK) motifs, harboring the ligand binding sites. We hypothesized that, despite their pronounced differences, TrkAH and MthK have similar gating energetics, dictated by the primary involvement of their RCK motifs in ligand recognition and pore regulation. To test this hypothesis, we studied the energetics of ligand-induced conformational changes of individual RCK motifs in each regulatory module by implementing a recently reported single-molecule fluorescence-based technique, in which the conformational state of a target protein is deduced from the measured polarization of a bifunctional rhodamine fluorophore attached to a suitable reporter a-helix. From the RCK energetics in each regulatory module we derived an analytic model, which quantitatively predicts the experimentally observed ligand dependence of each channel’s open probability. Our findings demonstrate that, in each regulatory module type, the structurally conserved RCK motif visits one of three states with comparable probabilities, related by similar equilibrium constants, thus exhibiting essentially the same intrinsic energetics in Trk as in MthK. Furthermore, the gating energetics of each system are governed by a unifying principle, whereby the channel’s opening is conditioned upon a particular combination of RCK subunit states and ligand binding affects the probability of observing this combination by regulating the degree of inter-subunit coupling.

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