Theses Doctoral

Functional and Biochemical Characterization of KCNQ1/KCNE1 Subunit Interactions in the Cardiac IKs Potassium Channel

Chan, Priscilla Jay

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of pore-forming alpha subunits (KCNQ1) and accessory beta (KCNE1) subunits. IKs is the slowly activating component of delayed rectifier K+ current in the heart and is a major contributor to the timing of repolarization of the cardiomyocyte membrane potential. Inherited mutations in either IKs channel subunit are associated with cardiac arrhythmia syndromes, including long QT syndrome (LQTS), short QT syndrome (SQTS) and familial atrial fibrillation (FAF). The biophysical properties of IKs channel current are dramatically altered when KCNE1 associates with the KCNQ1 channel. Functional tetrameric channels can be formed by KCNQ1 alone, but co-assembly with KCNE1 is required for the unique kinetics necessary to regulate human cardiac electrical activity as well as for the channel's functional response to the sympathetic nervous system. Specifically, KCNE1 co-assembly results in a depolarizing shift in the voltage dependence of activation, an increase in the single channel conductance, and an increase in current density. IKs channel current is also characterized by slow activation and deactivation kinetics, with little or no inactivation, in contrast to the KCNQ1 homomeric channel, which is characterized by fast activation and deactivation kinetics and clear inactivation. We wanted to understand how KCNE1 modulates the KCNQ1 channel functionally and investigate the structural determinants of this modulation. In Chapter II, we explore the role(s) of KCNE1 in the context of two KCNQ1 atrial fibrillation associated mutations, S140G and V141M. In contrast to published results, we find distinct dependence on the KCNE1 subunit for V141M, but not for S140G. Having determined the importance of KCNE1 for V141M functionally, we continued to explore the role of KCNE1 structurally for this mutation. Using cysteine substitution in both KCNQ1 and KCNE1 subunits, we monitored spontaneous disulfide bond formation and find that V141C crosslinks to KCNE1, while S140C does not. Having established the functional and structural importance of KCNE1 for V141M, we proposed that there could be mutations in KCNE1 that could reverse the consequences of slow deactivation in the V141M mutation. In Chapter III, we engineer amino terminal KCNE1 mutations and demonstrate that this domain is important for controlling deactivation, but not activation, kinetics of the KCNQ1 channel. We find two KCNE1 mutations, L45F and Y46W, which when co-expressed with either V141M or S140G mutations in KCNQ1, help restore the mutant channel back towards a wild-type IKs channel. From these results, we propose that the amino-terminal domain could play an important role in mediating the rate of deactivation in KCNQ1/KCNE1 channels. After testing mutations on KCNE1 that could affect normal channel function, we continued with a project to study mutations on KCNQ1 that would have similar dramatic effects on the channel. In Chapter IV, we mutated KCNQ1 residue S140 to Threonine and found that S140T co-assembled with KCNE1 produced a channel having functional characteristics opposite to that of S140G/KCNE1 channels. In contrast to S140G/KCNE1 channels, where channels tend to stay open due to very slow deactivation kinetics, S140T/KCNE1 channels tend to be stabilized in the closed state and require more depolarized pulses to open channels. In addition, we find that a mutation at position Y46 in KCNE1, when co-expressed with the S140T mutation in KCNQ1, helps restore the mutant channel back towards a wild-type channel. Again, here we provide evidence that the amino terminal end of KCNE1 could play a role in controlling deactivation. In Chapter V, we investigated the importance of where KCNE1 is located in the channel and also how KCNQ1/KCNE1 subunits assemble using a tandem construct, with 1 KCNE1 subunit tethered to 2 KCNQ1 subunits (EQQ). To investigate the significance of KCNE1 location, we explored the functional consequences of having the S140G or V141M mutations in the proximal (closest to KCNE1) or distal (farthest from KCNE1) KCNQ1 subunit. We find that having a mutation in the proximal subunit is subject to modulation by KCNE1, but not the distal subunit. Using crosslinking, we want to confirm proper assembly of the heterotetrameric channel to verify that KCNE1 assembles between S1 from one KCNQ1 subunit and the S6 domain of an opposing KCNQ1 subunit. Taken together, we demonstrate that the proximity between the N-terminus of KCNE1 and the S1 domain of KCNQ1 could play a role in modulating deactivation kinetics of KCNQ1. These findings will be of great importance in understanding normal IKs channel function, which will be essential for maintaining proper heart function.


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More About This Work

Academic Units
Pharmacology and Molecular Signaling
Thesis Advisors
Kass, Robert S.
Ph.D., Columbia University
Published Here
November 11, 2011