Role of glycosylation in cell surface expression and stability of HERG potassium channels

Abstract

Gong, Qiuming, Corey L. Anderson, Craig T. January, and Zhengfeng Zhou. Role of glycosylation in cell surface expression and stability of HERG potassium channels. Am J Physiol Heart Circ Physiol 283: H77-H84, 2002. First published March 7, 2002 10.1152/ajpheart.00008. 2002 encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel in the heart. We previously showed that HERG channel protein is modified by N-linked glycosylation. HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). In this study, we used the approaches of site-directed mutagenesis and biochemical modification to inhibit N-linked glycosylation and studied the role of glycosylation in the cell surface expression and turnover of HERG channels. Our results show that N598 is the only site for N-linked glycosylation and that glycosylation is not required for the cell surface expression of functional HERG channels. In contrast, N629 is not used for glycosylation, but mutation of this site (N629Q) causes a protein trafficking defect, which results in its intracellular retention. Pulse-chase experiments show that the turnover rate of nonglycosylated HERG channel is faster than that of the glycosylated form, suggesting that N-linked glycosylation plays an important role in HERG channel stability.heart; arrhythmia; ion channels; mutations; patch clamp HUMAN ether-à -go-go-related gene (HERG) encodes a K ϩ channel that belongs to the EAG family of voltagegated ion channels (35). HERG channel current has properties similar to the rapidly activating delayed rectifier K ϩ current in the heart, which plays a central role in action potential repolarization (27,30,34,38). Mutations in HERG cause the chromosome 7-linked form of inherited long QT syndrome (LQT2) (4), and to date Ͼ100 HERG mutations have been identified in LQT2 patients (13,14,31). HERG also plays an important role in the acquired form of long QT syndrome, because most drugs that cause drug-induced long QT syndrome block HERG channels. Thus HERG is an important target in both the inherited and acquired forms of long QT syndrome.Although electrophysiological and pharmacological properties of HERG channels have been studied widely, less is known about the biochemical processing of HERG channel protein. We previously reported (37, 38) that wild-type HERG channel protein expressed in HEK293 cells exhibits two bands on Western blot analysis and that the generation of both bands involves asparagine (N)-linked glycosylation. We showed (37) that the larger-molecular-mass band is the fully glycosylated, mature form of HERG channel located in the plasma membrane and the smaller-molecular-mass band is a core-glycosylated, precursor form of HERG channel located in the endoplasmic reticulum (ER). The HERG protein sequence contains two extracellular consensus sites for N-linked glycosylation (N598, N629). Recently, Petrecca et al. (20) reported that both single (N598Q, N629Q) and double (N598Q-N629Q) mutations resulted in t...

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).…”

Rescue of Aberrant Gating by a Genetically Encoded PAS (Per-Arnt-Sim) Domain in Several Long QT Syndrome Mutant Human Ether-á-go-go-related Gene Potassium Channels

“…2, A and B) and one at 135 kDa (representing the immature, core-glycosylated form; bottom arrow in Fig. 2, A and B) (12,26,27). In the neutravidin-purified sample, Western blot analysis revealed a robust band for the mature form ( Fig.…”

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

“…1C). Second, SPP cleavage leading to TRC8 degradation requires a flexible luminal region unobstructed by glycosylation (48), whereas hERG only has short luminal loops between multiple transmembrane helices, and an N-linked glycosylation site (54). Third, all of the SPP-cleaved TRC8 substrates have single transmembrane helices so the cleaved products are released into the cytosol (33,34,48), and hERG is a tetramer of subunits with six transmembrane helices each.…”