Project Summary/Abstract
Potassium channels are membrane proteins critical for the electrochemical regulation and function of cardiac cells.
Many diseases are associated with mutations in human potassium channels, including Long-QT Syndrome, Short-QT
Syndrome, Brugada Syndrome, Lev-Lenegre Syndrome, and Idiopathic Ventricular Fibrillation. The molecular basis of
these diseases remains poorly understood, and many arrhythmia-associated mutations may directly disrupt protein folding.
Therefore, it is essential to study the mechanism and biophysical determinants of potassium channel folding to understand
how these mutations may result in arrhythmia.
Preliminary work is presented here on the in vitro folding of the KcsA transmembrane pore domain, a robust model
system for human potassium channels such as hERG and Kv1.2. This work suggests that KcsA rapidly inserts as monomers
into a protein-dense region within the lipid membrane, and tetramerization kinetics are protein concentration-independent,
implying a unimolecular rate-limiting step despite the tetrameric nature of the channel. These observations raise the
following questions: What is the role of the protein-dense region in potassium channel folding? What are the structural
events in potassium channel folding, specifically regarding the rate-limiting step? Lastly, and most relevant to cardiac health,
how might missense mutations of the pore helix, such as A614V, L615V, and T623I of hERG, disrupt folding and lead to
arrhythmia? The proposed work will investigate the protein-dense region using super-resolution light and scanning-probe
microscopy in reconstituted membranes and live HL-1 cardiomyocytes to evaluate the hypothesis that the protein-dense
region functions to quickly concentrate channel monomers in the membrane and thus increase the speed and efficiency of
folding. To determine the structural events in channel folding, we will use a novel hydrogen-exchange mass spectrometry
(HXMS) technique alongside other biophysical methods to evaluate the hypothesis that folding must occur by one of two
possible mechanisms: (1) a “native assembly model” in which four natively-folded channel monomers assemble in a single,
concerted step, or (2) a “keystone model” in which the transmembrane helices of each monomer initially tetramerize into a
transmembrane bundle, and then the pore helix and selectivity filters insert into and stabilize the channel like the keystone
of an arch. Pulse-labeling and native state HXMS will probe the folding dynamics and stability, respectively, of channel
variants associated with Long-QT Syndrome to evaluate the hypothesis that pore helix missense mutations can cause disease
by preventing proper pore helix folding. These approaches will be complemented by computational coarse-grained and all-
atom techniques, including a novel “committor” analysis method to study the reactive flux between metastable folded and
unfolded potassium channel states.
The proposed work is high impact: It uses innovative and interdisciplinary techniques such as HXMS to uncover
the mechanism of potassium channel folding and its implications for cardiac arrhythmia. These insights will inform future
studies of membrane protein folding biophysics as well as the pathogenesis of heart rhythm disorders.
