Chemical-Biological Investigation of KCNQ1/KCNE K⁺ Channel Complexes: A Dissertation

Abstract

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.