Abstract
KCNH2 encodes the Kv11.1 α-subunit that underlies the rapidly activating delayed-rectifier K+ current in the heart. Loss-of-function KCNH2 mutations cause long QT syndrome type 2 (LQT2), and most LQT2-linked missense mutations inhibit the trafficking of Kv11.1 channel protein to the cell surface membrane. Several trafficking-deficient LQT2 mutations (e.g., G601S) generate Kv11.1 proteins that are sequestered in a microtubule-dependent quality control (QC) compartment in the transitional endoplasmic reticulum (ER). We tested the hypothesis that the QC mechanisms that regulate LQT2-linked Kv11.1 protein trafficking are mutation-specific. Confocal imaging analyses of HEK293 cells stably expressing the trafficking-deficient LQT2 mutation F805C showed that, unlike G601S-Kv11.1 protein, F805C-Kv11.1 protein was concentrated in several transitional ER subcompartments. The microtubule depolymerizing drug nocodazole differentially affected G601S- and F805C-Kv11.1 protein immunostaining. Nocodazole caused G601S-Kv11.1 protein to distribute into peripheral reticular structures, and it increased the diffuse immunostaining of F805C-Kv11.1 protein around the transitional ER subcompartments. Proteasome inhibition also affected the immunostaining of G601S- and F805C-Kv11.1 protein differently. Incubating cells in MG132 minimally impacted G601S-Kv11.1 immunostaining, but it dramatically increased the diffuse immunostaining of F805C-Kv11.1 protein in the transitional ER. Similar results were seen after incubating cells in the proteasome inhibitor lactacystin. Differences in the cellular distribution of G601S-Kv11.1 and F805C-Kv11.1 protein persisted in transfected human inducible pluripotent stem cell derived cardiomyocytes. These are the first data to visually demonstrate mutation-specific differences in the trafficking-deficient LQT2 phenotype, and this study has identified a novel way to categorize trafficking-deficient LQT2 mutations based on differences in intracellular retention.
Highlights
Long QT syndrome (LQTS) is a disorder characterized by delayed ventricular repolarization, prolongation of the QT interval on an electrocardiogram (ECG), and an increased risk for the ventricular arrhythmia Torsades de Pointes (Crotti et al, 2008)
In this study we demonstrate that the trafficking-deficient long QT syndrome type 2 (LQT2) missense mutation F805C generated Kv11.1 protein that accumulates in discrete endoplasmic reticulum (ER) subcompartments
In Human Embryonic Kidney 293 (HEK293) cells F805C-Kv11.1 protein did not co-localize with several ER/ER Golgi Intermediate Complex (ERGIC) marker proteins, but it did co-localize with the transitional ER protein BAP31
Summary
Long QT syndrome (LQTS) is a disorder characterized by delayed ventricular repolarization, prolongation of the QT interval on an electrocardiogram (ECG), and an increased risk for the ventricular arrhythmia Torsades de Pointes (Crotti et al, 2008). A key finding is several drugs that bind to Kv11.1 channels and block IKr (e.g., E-4031) can act as “pharmacological chaperones” to improve the trafficking and functional expression for many LQT2 channels (pharmacological correction) (Zhou et al, 1999; Ficker et al, 2002; Anderson et al, 2006, 2014). This is an important finding because it indicates that some trafficking-deficient LQT2 channels are promising targets for therapeutic intervention to improve delivery of channels to the surface membrane. We tested the hypothesis that the ER QC control mechanisms that prevent the trafficking of G601Sand F805C-Kv11.1 proteins are different
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