Uncovering the Mechanism of Potassium Channel Folding and Assembly with Implications for the Molecular Basis of Cardiac Arrhythmia

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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.