Glycosylation, Assembly and Trafficking of Cardiac Potassium Channel Complexes: A Dissertation

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes

A-kinase anchoring proteins: temporal and spatial regulation of intracellular signal transduction in the cardiovascular system

The prototypical intracellular second messenger cAMP is locally controlled by multimolecular protein complexes organized by A-kinase anchoring proteins (AKAPs). AKAPs serve as scaffolds for different sets of cAMP-metabolizing enzymes that control cAMP gradients and regulate cellular responses. In addition to adenylyl cyclases and phosphodiesterases, AKAPs bind signaling enzymes and ion channels that are important regulators of cardiac contractility and pathophysiological myocyte remodeling and hypertrophy. Compartmentation increases the local concentration of cAMP signaling components, providing faster, higher fidelity regulation of cellular processes. This review series highlights the contribution of AKAPs in the heart as scaffold proteins integrating protein kinases, phosphatases and other effector molecules involved in cAMP-dependent signaling

KCNE1 subunits require co-assembly with K+ channels for efficient trafficking and cell surface expression

KCNE peptides are a class of type I transmembrane beta subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K(+) channels. Accordingly, mutations that disrupt the assembly and trafficking of KCNE-K(+) channel complexes give rise to disease. The cellular mechanisms responsible for ensuring that KCNE peptides assemble with voltage-gated K(+) channels have yet to be elucidated. Using enzymatic deglycosylation, immunofluorescence, and quantitative cell surface labeling experiments, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with specific K(+) channel subunits; co-assembly mediates KCNE1 progression through the secretory pathway and results in cell surface expression. We also address an apparent discrepancy between our results and a previous study in human embryonic kidney cells, which showed wild type KCNE1 peptides can reach the plasma membrane without exogenously expressed K(+) channel subunits. By comparing KCNE1 trafficking in three cell lines, our data suggest that the errant KCNE1 trafficking observed in human embryonic kidney cells may be due, in part, to the presence of endogenous voltage-gated K(+) channels in these cells

A-kinase Anchoring Proteins: Temporal and Spatial Regulation of Intracellular Signal Transduction in the Cardiovascular System

The prototypical intracellular second messenger cAMP is locally controlled by multimolecular protein complexes organized by A-kinase anchoring proteins (AKAPs). AKAPs serve as scaffolds for different sets of cAMP-metabolizing enzymes that control cAMP gradients and regulate cellular responses. In addition to adenylyl cyclases and phosphodiesterases, AKAPs bind signaling enzymes and ion channels that are important regulators of cardiac contractility and pathophysiological myocyte remodeling and hypertrophy. Compartmentation increases the local concentration of cAMP signaling components, providing faster, higher fidelity regulation of cellular processes. This review series highlights the contribution of AKAPs in the heart as scaffold proteins integrating protein kinases, phosphatases and other effector molecules involved in cAMP-dependent signaling