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

Abstract

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 ...

Structure of the C-terminal region of an ERG channel and functional implications

“…5E). Our results indicate that N861H and N861I channels had a defect in maturation, similar to what was shown for LQTS mutations in the CNBHD, including N861I, which was proposed to be retained in the endoplasmic reticulum (41)(42)(43). The lack of hERG currents is consistent with an LQTS phenotype.…”

“…32). These LQTS mutations have been found to speed up deactivation of hERG channels or lead to defects in protein trafficking and folding, precluding formation of functional channels (35)(36)(37)(38)(39)(40)(41)(42)(43).…”

A Highly Conserved Motif at the COOH Terminus Dictates Endoplasmic Reticulum Exit and Cell Surface Expression of NKCC2

“…Moreover, there have been several reports demonstrating that COOH-terminal residues are important for correct protein targeting (12)(13)(14). Occasionally, COOH-terminal mutations are known to cause genetic disorders (15)(16)(17). Although studies of other ion transporters support the importance of the COOH-terminal signals in protein stability, maturation, surface delivery, and ER export (18 -22), little is known about the role of COOH-terminal signals in the biogenesis of NKCC2.…”

The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains

“…The truncation mutants rEag1-K848X and hErg-R1032X produced functional K ϩ channels with biophysical properties similar to those of respective WT (16) (data not shown). By contrast, neither rEag1-N673X nor hErg-N861X generated significant K ϩ currents (16,17). Despite the absence of CAD/TCC, all of the four truncation mutants preserved their native subfamily-specific assembly properties (Fig.…”

“…Likewise, subunit interaction for Erg channels may take place in the absence of the TCC domain (17)(18)(19)(20)(21). It is therefore possible that other regions within Eag and Erg may also contribute to subunit assembly and subfamily recognition.…”