Identification of the cyclic-nucleotide-binding domain as a conserved determinant of ion-channel cell-surface localization

Abstract

Mutations of a putative cyclic-nucleotide-binding domain (CNBD) can disrupt the function of the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN2) and the human ether-a-go-go-related gene potassium channel (HERG). Loss of function caused by C-terminal truncation, which includes all or part of the CNBD in HCN and HERG, has been related to abnormal channel trafficking. Similar defects have been reported for several of the missense mutations of HERG associated with long QT syndrome type 2 (LQT2). Thus, we postulate that normal processing of these channels depends upon the presence of the CNBD. Here, we show that removal of the entire CNBD prevents Golgi transit, surface localization and function of HERG channel tetramers. This is also true when any of the structural motifs of the CNBD is deleted, suggesting that deletion of any highly conserved region along the entire length of the CNBD can disrupt channel trafficking. Furthermore, we demonstrate that defective trafficking is a consequence of all LQT2 mutations in the CNBD, including two mutations not previously assessed and two others for which there are conflicting results in the literature. The trafficking sensitivity of the CNBD might be of general significance for other ion channels because complete deletion of the CNBD or mutations at highly conserved residues within the CNBD of the related ERG3 channel and HCN2 also prevent Golgi transit. These results broadly implicate the CNBD in ion-channel trafficking that accounts for the commonly observed loss of function associated with CNBD mutants and provides a rationale for distinct genetic disorders.

Properties of WT and mutant hERG K+ channels expressed in neonatal mouse cardiomyocytes

“…Compared with mERG1a protein, hERG channel protein has 96% sequence homology at the amino acid level (25). In our Western Blot analyses, transfection of WT hERG or the LQT2 mutants into neonatal mouse cardiomyocytes resulted in the expression of new protein bands at 135 and 155 kDa, similar to previous reports (2,15,19,45) of hERG expression in noncardiac mammalian heterologous systems. For control conditions, WT hERG-and ⌬Y475 hERG-transfected cardiomyocytes generated both 135-and 155-kDa bands, whereas G601S hERG-and N470D hERG-transfected cardiomyocytes generated 135-kDa bands with weak or absent 155-kDa bands.…”

“…Cardiomyocytes transfected with WT hERG showed new protein bands at 135 and 155 kDa. Western blot analyses of WT hERG overexpression in several systems, including HEK-293 cells, have shown two protein bands at 135 kDa (immature) and 155 kDa (mature) that represent posttranslational core and complex glycosylation of hERG protein, respectively (2,15,19,45). We next transfected the missense mutations, G601S and N470D hERG (12,16,36), that form trafficking-deficient channel proteins when studied in noncardiac mammalian systems (3,16,45).…”

A splice site mutation in hERG leads to cryptic splicing in human long QT syndrome

“…It is noted that all LQT2 mutations in the cyclic nucleotide binding domain lead to defective trafficking of mutant channels [22,32]. The large insertion caused by the cryptic splicing involves a highly ordered structure in β5 region of the cyclic nucleotide binding domain, which has been shown to play an important role in hERG channel trafficking [32]. The present results also show that coexpression of mutant and WT channels leads to a dominant negative suppression of WT channel function by intracellular retention of heteromeric channels.…”

“…These trafficking deficient hERG mutants are retained in the endoplasmic reticulum due to prolonged association with chaperone proteins calnexin, Hsp70 or Hsp90, and are rapidly degraded by the ubiquitin proteasome pathway [29][30][31]. It is noted that all LQT2 mutations in the cyclic nucleotide binding domain lead to defective trafficking of mutant channels [22,32]. The large insertion caused by the cryptic splicing involves a highly ordered structure in β5 region of the cyclic nucleotide binding domain, which has been shown to play an important role in hERG channel trafficking [32].…”

Bag1 Co-chaperone Promotes TRC8 E3 Ligase-dependent Degradation of Misfolded Human Ether a Go-Go-related Gene (hERG) Potassium Channels

“…The most common mechanism is impaired hERG trafficking from the endoplasmic reticulum (ER) to the plasma membrane. Mutant channels, which fail to fold properly or form tetramers, are recognized by the ER quality control machinery, retained, and sent for ER-associated proteasomal degradation (ERAD) (2)(3)(4).…”