What is the clinical evidence from this study?

Study design: Other. Population: Long-QT syndrome type-2 (LQT2). Intervention: PAS-domain mutations in hERG vs. Wild-type hERG. Primary outcome: Plasma membrane expression and cellular processing.

April 15, 2019Open Access

Mutation-specific peripheral and ER quality control of hERG channel cell-surface expression

BFBrian FooMcGill University CBCamille BarbierMcGill University KGKevin GuoHospital of the University of Pennsylvania

Key Result

Mildly-misfolded PAS-domain mutant hERG channels exhibit attenuated plasma membrane expression due to mutation-specific degradation by both endoplasmic reticulum and peripheral quality control systems.

Structured PICO

Population

HeLa cells expressing WT and mutant hERG1a (PAS domain mutants: F29L, I42N, R56Q, M124R, C64Y, T65P, A78P, I96T; severely misfolded: G601S, F805C) via lentivirus-transduction

Intervention

Pharmacochaperone E4031 (10 µM), low temperature (30°C), proteasomal inhibitors (MG132, Bortezomib, Ixazomib), lysosomal inhibitors (Bafilomycin A1), CHIP siRNA, or Ub-AllR overexpression

Comparator

Untreated WT-hERG or untreated mutant hERG cells

Outcome

Plasma membrane (PM) expression and cellular processing (maturation efficiency, degradation) of hERG channelssurrogate

The loss-of-expression phenotype of LQT2-associated hERG PAS-domain mutations is driven by both ER and post-ER quality control mechanisms, highlighting potential therapeutic targets for conformational correction.

Abstract

-channel is associated with Long-QT syndrome type-2 (LQT2) and increased risk of cardiac arrhythmia. Reduced PM-expression is primarily attributed to retention and degradation of misfolded channels by endoplasmic reticulum (ER) protein quality control (QC) systems. However, as the molecular pathogenesis of LQT2 was defined using severely-misfolded hERG variants with limited PM-expression, the potential contribution of post-ER (peripheral) QC pathways to the disease phenotype remains poorly established. Here, we investigate the cellular processing of mildly-misfolded Per-Arnt-Sim (PAS)-domain mutant hERGs, which display incomplete ER-retention and PM-expression defects at physiological temperature. We show that the attenuated PM-expression of hERG is dictated by mutation-specific contributions from both the ER and peripheral QC systems. At the ER, PAS-mutants experience inefficient conformational maturation coupled with rapid ubiquitin-dependent proteasomal degradation. In post-ER compartments, they are rapidly endocytosed from the PM via a ubiquitin-independent mechanism and rapidly targeted for lysosomal degradation. Conformational destabilization underlies aberrant cellular processing at both ER- and post-ER compartments, since conformational correction by a hERG-specific pharmacochaperone or low-temperatures can restore WT-like trafficking. Our results demonstrate that the post-ER QC alone or jointly with the ER QC determines the loss-of-PM-expression phenotype of a subset of LQT2 mutations.

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