Small GTPase Determinants for the Golgi Processing and Plasmalemmal
Expression of Human Ether-a-go-go Related (hERG) K+
Channels |
| |
Authors: | Brian P Delisle Heather A S Underkofler Brooke M Moungey Jessica K Slind Jennifer A Kilby Jabe M Best Jason D Foell Ravi C Balijepalli Timothy J Kamp and Craig T January |
| |
Institution: | ‡Department of Physiology, University of Kentucky, Lexington, Kentucky 40536 and the §Cellular and Molecular Arrhythmia Research Program, Departments of Medicine and Physiology, University of Wisconsin, Madison, Wisconsin 53706 |
| |
Abstract: | The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes
(LQT1–10). Approximately 40% of genotype-positive LQT patients
have LQT2, which is characterized by mutations in the human ether-a-go-go
related gene (hERG). hERG encodes the voltage-gated
K+ channel α-subunits that form the pore of the rapidly
activating delayed rectifier K+ current in the heart. The purpose
of this study was to elucidate the mechanisms that regulate the intracellular
transport or trafficking of hERG, because trafficking is impaired for about
90% of LQT2 missense mutations. Protein trafficking is regulated by small
GTPases. To identify the small GTPases that are critical for hERG trafficking,
we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293
cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER)
export of proteins in COPII and COPI vesicles, respectively. Expression of DN
Sar1 inhibited the Golgi processing of hERG, decreased hERG current
(IhERG) by 85% (n ≥ 8 cells per group, *, p
< 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of
DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the
coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited
the Golgi processing of hERG, decreased IhERG by 79% (n
≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal
staining of hERG. These data suggest that hERG undergoes ER export in COPII
vesicles and endosomal recycling prior to being processed in the Golgi. We
conclude that hERG trafficking involves a pathway between the ER and endosomal
compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene
(hERG)3
encodes the voltage-gated K+ channel α-subunits that
oligomerize to form the pore of the rapidly activating delayed rectifier
K+ current (IKr) in cardiac myocytes
(1–3).
Hundreds of hERG mutations are linked to the congenital
pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest
that these mutations result in a loss of normal hERG K+ channel
(hERG) function (4,
5). In LQT2, missense mutations
are the dominant abnormality and many LQT2 missense mutations reduce hERG
K+ current (IhERG) by decreasing the intracellular
transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell
surface membrane (plasmalemma)
(6). Therefore, disruption of
hERG K+ channel trafficking appears to be a principal mechanism for
disease.Movement of proteins between membrane-bound intracellular compartments is
mediated by small transport vesicles, which bud from a donor compartment to
fuse with an appropriate acceptor compartment. The trafficking of many
transmembrane and secretory proteins between the ER and Golgi compartments is
dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1,
which regulate the formation of coat-associated protein complex I (COPI) and
II (COPII) vesicles, respectively
(7–19).
These small GTPases facilitate the polymerization of transport vesicle protein
coats on the donor membrane. Vesicular cargo selection, docking, and fusion to
the target membrane are regulated by adaptor proteins, SNARE proteins, and Rab
GTPases. To rationally develop novel therapeutic targets that may increase the
expression of trafficking-deficient LQT2 mutant channels, the molecular
mechanisms that regulate the trafficking of hERG need to be explored. The
purpose of this study is to identify transport proteins that regulate the
trafficking of wild type (WT) hERG. We used a strategy of testing specific WT
GTPases or ones containing dominant negative (DN) mutations to interfere with
their function. |
| |
Keywords: | |
|
|