KCNE1 Subunits Require Co-assembly with K+ Channels for Efficient Trafficking and Cell Surface Expression

Chandrasekhar, Kshama D.; Bas, Tuba; Kobertz, William R.

doi:10.1074/jbc.m604398200

Cited by 61 publications

(87 citation statements)

References 35 publications

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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.

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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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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.

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115

113

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“…To strengthen the notion that the two proteins assemble in the pharynx we assessed the effect that epigenetic inactivation of mps-4 by RNAi had on the expression of EXP-2 tagged by GFP. Generally, KCNE proteins are essential components of their respective complexes that become unstable and/or cannot traffic to the plasma membrane without them (22,31). As expected, mps-4 RNAi selectively attenuated GFP signals in isthmus and terminal bulb (Fig.…”

Section: Mps-4 and Exp-2 Co-assemble In Pharyngeal Muscle-the Findingsupporting

confidence: 68%

An Arrhythmia Susceptibility Gene in Caenorhabditis elegans

Park

Sesti

2007

Journal of Biological Chemistry

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kcne are evolutionarily conserved genes that encode accessory subunits of voltage-gated K ؉ (Kv) channels. Missense mutations in kcne1, kcne2, and kcne3 are linked to congenital and acquired channelopathies in Homo sapiens. Here we show an unique example of conservation of kcne activities at genetic, physiological, functional, and pathophysiological level in Caenorhabditis elegans. Thus, mps-4 is the homologue of kcne1 that operates in human heart and inner ear. Like its KCNE relatives, MPS-4 assembles with a Kv channel, EXP-2, to form a complex that controls pharyngeal muscle contractility. MPS-4 modulates EXP-2 function in a similar fashion as KCNE proteins endow human channels. When defective, MPS-4, can induce abnormal repolarization by mechanisms that resemble the way KCNE proteins are thought to provoke arrhythmia in human heart. Mutation of a conserved aspartate residue associated with human disease (MPS-4-D74N) alters the functional attributes of the C. elegans current. Taken together these data underscore a significant conservation of KCNE activities in different pumps. This implies that C. elegans can develop into a system to study the molecular and genetic basis of KCNE-mediated muscle contractility and disease states.KCNEs are evolutionary conserved ancillary subunits of voltage-gated potassium (Kv) channels (1, 2). They regulate the function, trafficking, modulation by signaling molecules, and pharmacology of their channel partners in multiple mammalian tissues including heart, stomach, skeletal muscle, auditory epithelium, and the central nervous system (2-9). In human left ventricle, KCNE subunits endow the Kv channels that conduct the repolarizing potassium current. Thus, KCNE1 assembles with KCNQ1 to form I Ks, whereas KCNE2 and possibly KCNE1 assemble with HERG to form I Kr (4, 10 -12). Mutations in kcne1 and kcne2 can decrease the repolarizing K ϩ current through impairing I Ks and/or I Kr function, causing congenital and acquired Long QT syndrome (4,(13)(14)(15)(16)(17)(18)(19)(20). This disease predisposes to polymorphic tachyarrhythmias, and life threatening ventricular fibrillation (19). An important feature of kcne genes is that they are conserved across phyla (21-23). There are four kcne-related genes in Caenorhabditis elegans termed mps genes that play a prominent role in the nervous system of the animal. The founding member of the family, MPS-1, assembles with the pore-forming subunit KVS-1 in a subset of sensory neurons to form a complex that contributes to both their maintenance and sensitivity (22). Two other members, MPS-2 and MPS-3, appear to form a ternary complex with KVS-1 that regulates taste sensitivity to sodium (23). In contrast, the role and the partner of the fourth member, MPS-4, which is the homolog of human KCNE1 (Fig. 1A) were not known. In an effort to characterize the physiological role of MPS-4, we found that this gene is expressed in the alimentary system of the worm, the pharynx. Notably, the pharynx is a pump that is considered a prototype of the human heart (24...

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“…2 A). The apparent molecular weight of Q1 was Ϸ70 kDa and that of E1 was 40 kDa, characteristic of maturely glycosylated and surface expressed E1 (31). The cross-linked heterodimer of Q1 and E1 was detected as a band of apparent molecular weight of 110 kDa (Fig.…”

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confidence: 93%

Location of KCNE1 relative to KCNQ1 in the I _KS potassium channel by disulfide cross-linking of substituted cysteines

Chung¹,

Chan²,

Bankston³

et al. 2009

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

114

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The cardiac-delayed rectifier K ؉ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cysflanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.arrhythmias ͉ cardiac repolarization ͉ electrophysiology ͉ atrial fibrillation ͉ S1T he slow, outwardly rectifying K ϩ current (I KS ) is one of two delayed rectifier K ϩ currents critical for repolarization of the heart, particularly during sympathetic nervous system stimulation (1, 2). The I KS channel is composed of four pore-forming KCNQ1 (Q1) subunits and two auxiliary KCNE1 (E1) subunits (3-5). Several human mutations in Q1 and E1 cause variants of long QT syndrome (6), short QT syndrome (7), or atrial fibrillation (8, 9).Although a tetramer of Q1 subunits alone forms a voltagegated channel, only Q1 and E1 together form a channel with the slow activation and deactivation kinetics and the minimal inactivation characteristics of I KS (10, 11). Furthermore, E1 is necessary for sympathetic modulation of I KS (12). How E1 exerts its effect on Q1 is not yet fully understood.There have been a number of conclusions about Q1-E1 interactions in the I KS channel, not all of which are compatible. There is evidence for (13) and against (14, 15) the contribution of E1 to the pore wall and its accessibility from the pore. There is also evidence that E1 interacts with the pore domain, although not necessarily exposed in the pore (16,17), that the E1 TM helix (E1-TM) interacts directly with Q1 S4 helix (18), that E1 modulates Q1 through its C terminus (19-21), and that E1 interacts with the cytoplasmic Q1 S4-S5 linker (22).More recently, a site of possible Q1-E1 interaction was suggested by the association of mutations in Q1 S...

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KCNE1 Subunits Require Co-assembly with K+ Channels for Efficient Trafficking and Cell Surface Expression

Cited by 61 publications

References 35 publications

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

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

An Arrhythmia Susceptibility Gene in Caenorhabditis elegans

Location of KCNE1 relative to KCNQ1 in the I _KS potassium channel by disulfide cross-linking of substituted cysteines

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KCNE1 Subunits Require Co-assembly with K+ Channels for Efficient Trafficking and Cell Surface Expression

Cited by 61 publications

References 35 publications

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

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

An Arrhythmia Susceptibility Gene in Caenorhabditis elegans

Location of KCNE1 relative to KCNQ1 in the I KS potassium channel by disulfide cross-linking of substituted cysteines

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

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

Location of KCNE1 relative to KCNQ1 in the I _KS potassium channel by disulfide cross-linking of substituted cysteines