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Author ORCID Identifier

https://orcid.org/0000-0003-1300-4599

AccessType

Campus-Only Access for Five (5) Years

Document Type

dissertation

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

Year Degree Awarded

2021

Month Degree Awarded

February

First Advisor

Jianhan Chen

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

The gating mechanism of transmembrane ion channels is crucial for understanding how these proteins control ion flow across membranes in various physiological processes. Big potassium (BK) channels, in particular, are important in neuronal and muscle functions and are associated with pathogenesis of hypertension, autism, epilepsy, stroke, asthma and other human diseases. BK channels have large single-channel conductance and dual regulation by membrane voltage and intracellular Ca2+. Recent atomistic structures of BK channels failed to identify structural features that could serve as the “gate” to block the ion flow in the closed state. Using atomistic simulations, we showed that gating of BK channels did not seem to require a physical gate. Instead, changes in the pore shape and surface hydrophobicity in the Ca2+-free state allow the pore to readily undergo hydrophobic dewetting transitions, giving rise to a large free energy barrier for K+ permeation. Importantly, the dry pore remains physically open and is readily accessible to quaternary ammonium channel blockers. The hydrophobic gating mechanism is also consistent with scanning mutagenesis studies showing that modulation of pore hydrophobicity is correlated with activation properties.

Knowledge of the gating mechanism provides a basis for understanding the allosteric coupling between the sensor and pore domains. In particular, the only covalent connection between the cytosolic Ca2+ sensing domain and the TM pore and voltage sensing domains is a 15-residue “C-linker”. Referred to as a passive spring, C-linker was largely believed to be an inactive component of the sensing apparatus. To determine the linker’s role in BK activation, we designed a series of linker sequence scrambling mutants to suppress potential complex interplay of specific interactions with the rest of the protein. The results revealed a surprising sensitivity of BK activation to the linker sequence. Combining atomistic simulations and additional mutagenesis experiments, we demonstrated that nonspecific interactions of the linker with membrane alone could directly modulate BK activation. The C-linker thus plays more direct roles in mediating allosteric coupling between BK domains than previously assumed. Our results also suggest that covalent linkers could directly modulate TM protein function and should be considered an integral component of the sensing apparatus. To further quantify the nonspecific interactions of the C-linker with the membrane, we calculated the free energy profiles of pore opening of the WT BK channel and a C-linker mutant. This is a challenging calculation given the size of the system and the complexity of the conformational transition involved. We showed that converged free energy profiles could be calculated and that they successfully recapitulated the effect of the C-linker mutation on the gating properties of the channel. Subsequent force decomposition analysis will allow quantification of various individual interactions of the C-linker residues to the pore open-close equilibrium.

DOI

https://doi.org/10.7275/20879726

Available for download on Sunday, August 01, 2021

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