N‐linked glycosylation sites determine HERG channel surface membrane expression

Section: Discussionmentioning

confidence: 99%

Loss of N-Linked Glycosylation Reduces Urea Transporter UT-A1 Response to Vasopressin

Chen

Fröhlich

Yang

et al. 2006

“…Glycosylation of ion channels has been shown to affect the intracellular transport, cell membrane targeting/stability, and activity of ion channels (16,18,19,(21)(22)(23). For example, the gating properties of a neuronal Na ϩ channel change as the glycosyl moiety of the channel protein changes during development (21).…”

Section: Discussionmentioning

confidence: 99%

“…Glycosylation site mutant channels, although properly folded and assembled, are degraded through cytoplasmic proteasomes (22). Similarly, N-linked glycosylation of the HERG channel is required for its cell surface expression (23).…”

mentioning

confidence: 99%

Reciprocal Modulation between the α and β4 Subunits of hSlo Calcium-dependent Potassium Channels

Jin

Weiger

Levitan

2002

Large conductance Ca2؉ -dependent potassium (K Ca or maxi K) channels are composed of a pore-forming ␣ subunit and an auxiliary ␤ subunit. We have shown that the brain-specific ␤4 subunit modulates the voltage dependence, activation kinetics, and toxin sensitivity of the hSlo channel (Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W. J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., SilosSantiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570). We investigated here the N-linked glycosylation of the ␤4 subunit and its effect on the modulation of the hSlo ␣ subunit. When expressed alone in HEK293 cells, the ␤4 subunit runs as a single molecular weight band on an SDS gel. However, when coexpressed with the hSlo ␣ subunit, the ␤4 subunit appears as two different molecular weight bands. Enzymatic deglycosylation or mutation of the N-linked glycosylation residues in ␤4 converts it to a single lower molecular weight band, even in the presence of the hSlo ␣ subunit, suggesting that the ␤4 subunit can be present as an immature, core glycosylated form and a mature, highly glycosylated form. Blockage of protein transport from the endoplasmic reticulum to the Golgi compartment with brefeldin A abolishes the mature, highly glycosylated ␤4 band. Glycosylation of the ␤4 subunit is not required for its binding to the hSlo channel ␣ subunit. It also is not necessary for cell membrane targeting of the ␤4 subunit, as demonstrated by surface biotinylation experiments. However, the double glycosylation site mutant ␤4 (␤4 N53A/N90A) protects the channel less against toxin blockade, as compared with the hSlo channel coexpressed with wild type ␤4 subunit. Taken together, these data show that the pore-forming ␣ subunit of the hSlo channel promotes N-linked glycosylation of its auxiliary ␤4 subunit, and this in turn influences the modulation of the channel by the ␤4 subunit.Ion channels are composed of pore-forming ␣ subunits and auxiliary subunits (2). The auxiliary subunits modulate diverse functions of ion channels, ranging from their biosynthesis to channel activity (1,(3)(4)(5)(6)(7)(8). The large conductance Ca 2ϩ -dependent potassium (K Ca or maxi K) channels are ubiquitously expressed in neurons and many other tissues. They contribute to action potential repolarization and influence neurotransmitter release (9 -12) (for review, see Ref. 13). The pore-forming ␣ subunit of the K Ca channel associates with a group of auxiliary ␤ subunits. The ␤ subunits influence such diverse aspects of K Ca channel function as kinetic behavior, voltage dependence, and sensitivity to toxins and other modulators (1,3,5,7,14,15).Many ion channels are heavily glycosylated (16 -20). Glycosylation modulates the activity, intracellular trafficking and targeting, and cell surface expression of ion channels. For instance, glycosylation of a neuronal Na ϩ channel is developmentally regulated and modulates steady state inactivation of the channel (21). The cell surface stability an...

“…18). Briefly, cells were placed on the stage of an inverted microscope (Zeiss IM35) at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Identification of a COOH-terminal Segment Involved in Maturation and Stability of Human Ether-a-go-go-related Gene Potassium Channels

Akhavan

Atanasiu

Shrier

2003

Self Cite

Mutations in the potassium channel encoded by the human ether-a-go-go-related gene (HERG) have been linked to the congenital long QT syndrome (LQTS), a cardiac disease associated with an increased preponderance of ventricular arrhythmias and sudden death. The COOH terminus of HERG harbors a large number of LQTS mutations and its removal prevents functional expression for reasons that remain unknown. In this study, we show that the COOH terminus of HERG is required for normal trafficking of the ion channel. We have identified a region critical for trafficking between residues 860 and 899 that includes a novel missense mutation at amino acid 861 (HERG N861I ). Truncations or deletion of residues 860 -899, characterized in six different expression systems including a cardiac cell line, resulted in decreased expression levels and an absence of the mature glycosylated form of the HERG protein. Deletion of this region did not interfere with the formation of tetramers but caused retention of the assembled ion channels within the endoplasmic reticulum. Consequently, removal of residues 860 -899 resulted in the absence of the ion channels from the cell surface and a more rapid turnover rate than the wild type channels, which was evident very early in biogenesis. This study reveals a novel role of the COOH terminus in the normal biogenesis of HERG channels and suggests defective trafficking as a common mechanism for abnormal channel function resulting from mutations of critical COOH-terminal residues, including the LQTS mutant HERG N861I . The long QT syndrome (LQTS)1 is a congenital heart disorder characterized by delayed cardiac action potential repolarization and a prolongation of the QT interval. This leads to an increased susceptibility of the heart to potentially sustained ventricular tachyarrhythmias that cause syncope and sudden death. Molecular genetic studies have identified five genes linked to LQTS including the human ether-a-go-go-related gene (HERG) (1). HERG encodes the ␣-subunit of the rapidly activating delayed rectifier current I Kr and consists of six transmembrane domains as well as NH 2 -and COOH-terminal cytoplasmic tails (2, 3). Over 90 mutations distributed throughout HERG have been linked to the LQTS, most of which reside in the intracellular tail regions of the channel protein (4, 5). Earlier electrophysiological and structural analysis have emphasized abnormal HERG function as a common manifestation of mutations in the NH 2 terminus (6 -8). In contrast, studies of COOH-terminal mutations suggest that the pathology is because of the absence of HERG channels from the cell surface (9 -12). This phenotype may be caused by improper folding of newly synthesized HERG polypeptides, in a fashion similar to that described for the ⌬F508 allele of CFTR (CFTR⌬F508). CFTR⌬F508 is recognized by the endoplasmic reticulum (ER) quality control machinery and is rapidly degraded before being processed in the Golgi apparatus (13). As a consequence, these molecules are prevented from journeying through the secretory ...