Date

2016

Department or Program

Biological Chemistry

Primary Wellesley Thesis Advisor

Lousie Darling

Additional Advisor(s)

Don Elmore

Additional Advisor

Vanja Klepac-Ceraj

Additional Advisor

Alex Diesl

Abstract

hERG and KvLQT1 are voltage-gated potassium channel protein α-subunits encoded for by KCNH2 and KCNQ1, respectively, that play a critical role in regulating the human heartbeat. hERG and KvLQT1 respectively produce the rapidly activating (IKr) and slowly activating (IKs) components of the cardiac repolarization current, which plays a critical role in regulating the action potential duration in cardiomyocytes. Both hERG and KvLQT1 channels have been shown to associate with the β-subunit, minK. While the expression of minK is not necessary for the production of IKr, the association of minK with KvLQT1 is essential for the full production of IKs. Previously performed biochemical assays and acceptor photobleach FRET (apFRET) experiments indicate that hERG and KvLQT1 physically interact via their –C termini. The Darling lab has characterized the interaction of hERG and KvLQT1 via apFRET experiments and found there to be a positive FRET efficiency for the hERG-CFP + KvLQT1-YFP pair. This thesis aimed to investigate whether interchanging the fluorophores attached to hERG and KvLQT1 affects the FRET efficiency. In addition, this work addresses whether the concatenation of minK to KvLQT1, so the construct can produce full IKs current, alters the interaction between KvLQT1 and hERG, as measured via apFRET. In order to address these questions, apFRET experiments were performed on KvLQT1-CFP + hERG-YFP and hERG-CFP + minK-KvLQT1-mYFP FRET pairs. It was found that the mean FRET efficiencies of KvLQT1-CFP-YFP (positive control, n=24), hERG-CFP (negative control, n=30), hERG-CFP + KvLQT1-YFP (experimental control, n=24) were 27.6% ± 7.2%, -0.7% ± 6.6%, and 0.3% ± 5.6% respectively. The mean FRET efficiencies for the KvLQT1-CFP + hERG-YFP (n=15) and hERG-CFP + minK-KvLQT1-YFP pairs were -0.1% ± 4.9% and -2.2% ± 6.1% (n=20) respectively. The observed FRET efficiency close to 0% for the experimental control, hERG-CFP + KvLQT1-YFP, is an unexpected result based on previously published and ongoing work in the Darling lab. Thus, these data suggest that the apFRET technique needs further optimization before evaluating the FRET efficiencies of the KvLQT1-CFP + hERG-YFP and hERG-CFP + minK-KvLQT1-YFP FRET pairs.

Share

COinS