Interaction with GM130 during HERG Ion Channel Trafficking

Abstract

Many mutations in the Human Ether-à -go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.Among the mechanisms underlying long QT syndrome (LQTS), 1 failed trafficking of potassium channels encoded by the Human Ether-à -go-go-Related Gene (HERG) is increasingly recognized as a leading cause of disease (1)(2)(3)(4)(5). Under normal conditions, HERG channels give rise to cardiac I Kr (6, 7), a current contributing to the repolarization of the ventricular action potential (8). Inherited mutations in HERG associated with type 2 Long QT Syndrome (LQT2) disrupt I Kr (9), leading to delayed repolarization and a prolonged QT interval. Untreated, LQTS can result in potentially fatal torsades de pointes arrhythmias (10,11). The mechanisms by which LQT2 mutations disrupt HERG channel trafficking are unknown.More than 90 different LQT2-causing HERG mutations have been reported, including deletions, insertions, and missense mutations scattered throughout the gene (3). Many give rise to defects in channel trafficking, manifest as a reduction in membrane current density, accumulation of the HERG protein in intracellular compartments and a failure of terminal glycosylation associated with the mature protein expressed at the membrane (1, 2). Even some mutant channels with altered gating properties exhibit significantly reduced current density, indicating that failure to traffic may contribute to the mechanism of disease in these cases as well...

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

“…It is possible that residues 860 -899 eliminate the interaction between HERG and GM130. However, dissociation of HERG ⌬860 -899 and GM130 cannot explain the phenotypes reported here because this interaction is not required for the perfunctory delivery of HERG channels to the plasma membrane (32).…”

“…In addition, previous studies have indicated that multiple regions throughout the subunits are required for assembly of ion channels and it is unlikely that deletion or a point mutation of selected residues of HERG would completely abolish its interaction with an auxiliary subunit (30,31). Interestingly, residues 860 -899 fall between two non-contiguous regions of HERG involved in the interaction between HERG and GM130, proposed to facilitate HERG transport (32). It is possible that residues 860 -899 eliminate the interaction between HERG and GM130.…”

Decreased Subunit Stability as a Novel Mechanism for Potassium Current Impairment by a KCNQ2 C Terminus Mutation Causing Benign Familial Neonatal Convulsions

“…6B). This result may suggest fundamental differences between the cellular processing of Q2 subunits and the K ϩ channels subunits encoded by the human Ether-a-gogo-Related gene 1 (hERG1), which have been shown to interact with GM-130 in biochemical, morphological, and functional studies (32). Closer inspection to the data revealed a weak but convincing cell-surface labeling only in EGFP-Q2-transfected cells (as indicated by the arrows).…”

The Enigmatic Cytoplasmic Regions of KCNH Channels

“…A yeast two-hybrid screen revealed the cis-Golgi protein GM130 was a hERG binding partner using the carboxyl terminus as bait 77 . The interaction was disrupted by deletions of the C-terminal-most 117 amino acids.…”

“…Interestingly, the disrupting LQT2 mutations reside in the CNBh domain. The mutant hERG proteins are trafficking-defective, as if hERG channel exit from the ER or access to the Golgi requires the integrity of two regions, one of poor secondary structure, and the other a highly ordered domain 77 .…”