An LQT mutant minK alters KvLQT1 trafficking

Krumerman, Andrew; Gao, Xiaohong; Bian, Jin-Song; Melman, Yonathan F.; Kagan, Anna; McDonald, Thomas V.

doi:10.1152/ajpcell.00275.2003

Cited by 69 publications

(84 citation statements)

References 45 publications

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“…Indeed, previous studies clearly showed that KCNE1 increased KCNQ1 current and modified the electrophysiological properties of the KCNQ1 channel (half-activation potential and kinetics of inactivation) (19, 28 -30). In addition, although more contradictory, previous studies also reported increased trafficking of KCNQ1 when co-expressed with KCNE1 (31). Finally, the chaperone role that we propose for KCNE1 is consistent with previous publications showing that ␤-subunits stabilize and enhance the trafficking of other channels (32,33).…”

Section: Discussionsupporting

confidence: 90%

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

Péroz

Dahimène

Baró

et al. 2009

Journal of Biological Chemistry

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Mutations in the potassium channel KCNQ1 that determine retention of the mutated proteins in the endoplasmic reticulum (ER) are associated with the autosomal dominant negative Romano-Ward LQT1 cardiac syndrome. In the present study, we have analyzed the consequences and the potential molecular mechanisms involved in the ER retention of three RomanoWard mutations located in KCNQ1 N terminus (Y111C, L114P, and P117L). We showed that the mutant KCNQ1 proteins exhibited reduced expression levels with respect to wild-type (WT)-KCNQ1. Radiolabeling pulse-chase experiments revealed that the lower expression levels did not result from reduced rate of synthesis. Instead, using a combination of Western blot and pulse-chase experiments, we showed that the mutant channel Y111C-KCNQ1, used as a model, was ubiquitinated and degraded in the proteasome more rapidly (t1 ⁄ 2 ‫؍‬ 82 min) than WT-KCNQ1 channel (t1 ⁄ 2 ‫؍‬ 113 min). On the other hand, KCNQ1 degradation did not appear to involve the GTP-dependent pathway. We also showed that KCNE1 stabilized both wildtype and Y111C proteins. To identify potential actors involved in KCNQ1 degradation, we studied the implication of the ERresident protein Derlin-1 in KCNQ1 degradation. We showed that although KCNQ1 and Derlin-1 share the same molecular complex and co-immunoprecipitate when co-expressed in HEK293FT cells, Derlin-1 did not affect KCNQ1 steady state expression and degradation. These data were confirmed in T84 cells that express endogenous KCNQ1 and Derlin-1. Small interfering RNA knock-down of Derlin-1 did not modify KCNQ1 expression level, and no interaction between endogenous KCNQ1 and Derlin-1 could be detected.

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Section: Discussionsupporting

confidence: 90%

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

Péroz

Dahimène

Baró

et al. 2009

Journal of Biological Chemistry

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“…For KCNQ1 channels, coassembly could occur later in biosynthesis because the subunit specificity domain for tetramerization resides in the C terminus (27). In either case, to exploit the dimer-of-dimers pathway, coassembly would have to occur in the endoplasmic reticulum, which is consistent with previous assembly studies with wild-type (28) and mutant KCNE1 peptides (29).…”

Section: Discussionsupporting

confidence: 84%

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

Morin

Kobertz

2008

Proc. Natl. Acad. Sci. U.S.A.

115

113

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Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting ␣-subunits with specific regulatory ␤-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of ␣-and ␤-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K ؉ channel inhibitor and its iterative application to tally the number of ␤-subunits in a KCNQ1/KCNE1 K ؉ channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K ؉ channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE ␤-subunit modulation of voltage-gated K ؉ channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K ؉ channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE ␤-subunits.membrane proteins that open and close in response to changes in membrane potential. To regulate cellular potassium flow in both excitable and nonexcitable tissues, Q1 channels coassemble with regulatory KCNE peptides, forming membraneembedded K ϩ channel complexes with various voltage-sensing and gating properties (1). The importance of forming a properly assembled Q1-KCNE complex is underscored by the mutations that give rise to long QT syndrome and congenital hearing loss (2, 3). Although the fourfold arrangement of the Q1 ␣-subunits along the ion conduction pathway is established and unquestioned, the number of KCNE ␤-subunits in the K ϩ channel complex has been a long-standing and heated debate (4-7). Goldstein and coworkers (6) have strongly argued that Q1 channels average two KCNE peptides per complex; however, they could not definitively rule out K ϩ channel complexes containing a solitary KCNE peptide. Another complicating factor is the notion that Q1 channels form complexes with KCNE ␤-subunits with multiple stoichiometries (5, 8). Thus, approaches that measure the average number of KCNE peptides in the Q1 channel complex at the cell surface are unable to readily discern between fixed and various stoichiometries.To determine the stoichiometry or stoichiometries of Q1-KCNE K ϩ channel complexes, we devised an iterative cell surface modification scheme where the labeling reagent specifically binds to the outer vestibule of the K ϩ conduction pore while covalently modifying a cysteine-bearing KCNE peptide in the channel complex (Fig. 1). Once th...

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“…A chaperone effect may contribute, as has been suggested for other KCNE interactions (19). However, we did not consistently observe increased recovery of HCN2 protein in the membrane fraction of ventricular cultures co-expressing HCN2 and HA-MiRP1, compared with those expressing HCN2 alone.…”

Section: Discussioncontrasting

confidence: 42%

MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes

Qu¹,

Kryukova²,

Potapova

et al. 2004

Journal of Biological Chemistry

120

106

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MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotidegated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between ؊60 and ؊95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a ␤ subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.MinK-related protein (MiRP1 or KCNE2) is purported to be a ␤ subunit for several voltage-gated potassium channels, including the rapid delayed rectifier (1), slow delayed rectifier (2), and transient outward current (3). In addition, we have reported that it can act as a ␤ subunit for the hyperpolarizationactivated, cyclic nucleotide-gated (HCN) 1 family of pacemaker channels (4). We found that co-expression of MiRP1 with HCN1 or HCN2 in Xenopus oocytes resulted in larger and more rapidly activating currents than when either HCN isoform was expressed alone but that MiRP1 did not shift the midpoint of activation of either isoform. Further, co-immunoprecipitation experiments involving HA-tagged MiRP1 and HCN1 demonstrated that the two proteins interact when co-expressed in oocytes.A more recent study demonstrated MiRP1 interaction with the HCN4 isoform. Here, co-expression with MiRP1, either in oocytes or Chinese hamster ovary cells, also resulted in increased current amplitude, but this was associated with slowing of kinetics and a negative shift of the midpoint of activation (5). However, another laboratory did not detect any effect of MiRP1 when co-expressed in HEK293 cells with either HCN4 or an HCN1-HCN4 tandem construct (6). Finally, a study of MiRP1 and HCN2 co-expression in Chinese hamster ovary cells reported a reduced time-dependent current component and increased instantaneous ...

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An LQT mutant minK alters KvLQT1 trafficking

Cited by 69 publications

References 45 publications

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes

MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes

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An LQT mutant minK alters KvLQT1 trafficking

Cited by 69 publications

References 45 publications

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

Counting membrane-embedded KCNE β-subunits in functioning K + channel complexes

MiRP1 Modulates HCN2 Channel Expression and Gating in Cardiac Myocytes

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Counting membrane-embedded KCNE β-subunits in functioning K ⁺ channel complexes