Gail A. Robertson

Properties of HERG Channels Stably Expressed in HEK 293 Cells Studied at Physiological Temperature

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Novel Mechanism Associated With an Inherited Cardiac Arrhythmia Defective Protein Trafficking by the Mutant HERG (G601S) Potassium Channel

BACKGROUND The congenital long-QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential and a QT interval that leads to arrhythmia. Mutations in the human ether-a-go-go-related gene (HERG), which encodes the rapidly activating component of the delayed rectifier current (IKr), cause chromosome 7-linked LQTS (LQT2). Studies of mutant HERG channels in heterologous systems indicate that the mechanisms mediating LQT2 are varied and include mutant subunits that form channels with altered kinetic properties or nonfunctional mutant subunits. We recently reported a novel missense mutation of HERG (G601S) in an LQTS family that we have characterized in the present work. METHODS AND RESULTS To elucidate the electrophysiological properties of the G601S mutant channels, we expressed these channels in mammalian cells and Xenopus oocytes. The G601S mutant produced less current than wild-type channels but exhibited no change in kinetic properties or dominant-negative suppression when coexpressed with wild-type subunits. To examine the cellular trafficking of mutant HERG channel subunits, enhanced green fluorescent protein tagging and Western blot analyses were performed. These showed deficient protein trafficking of the G601S mutant to the plasma membrane. CONCLUSIONS Our results from both the Xenopus oocyte and HEK293 cell expression systems and green fluorescent protein tagging and Western blot analyses support the conclusion that the G601S mutant is a hypomorphic mutation, resulting in a reduced current amplitude. Thus, it represents a novel mechanism underlying LQT2.

Transfer of rapid inactivation and sensitivity to the class III antiarrhythmic drug E-4031 from HERG to M-eag channels

1 The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M‐eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M‐eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E‐4031. 2 We generated chimeric channels of HERG and M‐eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG. 3 Transfer of a small segment of the HERG polypeptide to M‐eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E‐4031 sensitivity to M‐eag. This region differs from the corresponding region in M‐eag by only fifteen residues. 4 Previous hypotheses that rapid inactivation of HERG channels occurs by a C‐type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C‐type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences. 5 In addition to sites homologous to those previously described for C‐type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C‐type inactivation. Although this site is equivalent to one implicated in P‐type inactivation in Kv2.1 channels, our data are most consistent with a single, C‐type inactivation mechanism.

Potassium Currents Expressed from Drosophila and Mouse eag cDNAs in Xenopus Oocytes

The ether-a-go-go (eag) gene family encodes a set of related ion channel polypeptides expressed in the excitable cells of organisms ranging from invertebrates to mammals. Earlier studies demonstrated that eag mutations in Drosophila cause an increase in membrane excitability in the nervous system. Mutations in the human eag-related gene (HERG) have been implicated in cardiac arrhythmia, and recent studies show that HERG subunits contribute to the channels mediating IKr and the terminal repolarization of the cardiac action potential. A physiological role for M-EAG, the mouse counterpart to Drosophila eag, has not been determined. Here, we describe basic properties of Eag and M-EAG channels expressed in frog oocytes, using two-electrode voltage clamp and patch clamp techniques. Both Eag and M-EAG channels are voltage-dependent, outwardly rectifying and highly selective for K+ over Na+ over Na+ ions. In contrast to previous reports, we found no evidence for Ca2+ flux through Eag channels. The most notable difference between these closely related channels is that Eag currents exhibit partial inactivation, whereas M-EAG currents are sustained for the duration of an activating voltage command. In addition, Eag currents run down more rapidly than do M-EAG currents in excised macropatches. Rundown is reversible by inserting the patch into the interior of the oocyte, indicating that a cytosolic factor regulates channel activity or stability. These studies should facilitate identification of currents mediated by Eag and M-EAG channels in vivo.

Physiological Properties of hERG 1a/1b Heteromeric Currents and a hERG 1b-Specific Mutation Associated With Long-QT Syndrome

Cardiac IKr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that IKr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of IKr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC50. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac IKr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.

The eag family of K+ channels in Drosophila and mammals.

ABSTRACT: Mutations of eag, first identified in Drosophila on the basis of their leg‐shaking phenotype, cause repetitive firing and enhanced transmitter release in motor neurons. The encoded EAG polypeptide is related both to voltage‐gated K+ channels and to cyclic nucleotide‐gated cation channels. Homology screens identified a family of eag‐related channel polypeptides, highly conserved from nematodes to humans, comprising three subfamilies: EAG, ELK, and ERG. When expressed in frog oocytes, EAG channels behave as voltage‐dependent, outwardly rectifying K+‐selective channels. Mutations of the human eag‐related gene (HERG) result in a form of cardiac arrhythmia that can lead to ventricular fibrillation and sudden death. Electrophysiological and pharmacological studies have provided evidence that HERG channels specify one component of the delayed rectifier, IKr, that contributes to the repolarization phase of cardiac action potentials. An important role or HERG channels in neuronal excitability is also suggested by the expression of these channels in brain tissue. Moreover, mutations of ERG‐type channels in the Drosophila sei mutant cause temperature‐induced convulsive seizures associated with aberrant bursting activity in the flight motor pathway. The in vivo function of ELK channels has not yet been established, but when these channels are expressed in frog oocytes, they display properties intermediate between those of EAG‐ and ERG‐type channels. Coexpression of the K+‐channel b subunit encoded by Hk with EAG in oocytes dramatically increases current amplitude and also affects the gating and modulation of these currents. Biochemical evidence indicates a direct physical interaction between EAG and HK proteins. Overall, these studies highlight the diverse properties of the eag family of K+ channels, which are likely to subserve diverse functions in vivo.

The Forms of a Task and Their Blends

Movement is categorized according to its goal. Scratching is a movement in which force is exerted against a specific site on the body surface. Locomotion is a movement in which the center of mass of an organism is transferred from one location in space to another. Movement performed to accomplish a specific goal is termed a task. A task may be performed in each of several ways. A human can scratch a site on the side of the thorax with either the hand or the elbow. A human can step in a forward, lateral, or backward direction. Each particular way that a task is accomplished is termed a form of that task (Mortin et al., 1985, Robertson et al., 1985).

Heteromeric Assembly of Human Ether-à-go-go-related Gene (hERG) 1a/1b Channels Occurs Cotranslationally via N-terminal Interactions

Alternate transcripts of the human ether-à-go-go-related gene (hERG1) encode two subunits, hERG 1a and 1b, which form potassium channels regulating cardiac repolarization, neuronal firing frequency, and neoplastic cell growth. The 1a and 1b subunits are identical except for their unique, cytoplasmic N termini, and they readily co-assemble in heterologous and native systems. We tested the hypothesis that interactions of nascent N termini promote heteromeric assembly of 1a and 1b subunits. We found that 1a and 1b N-terminal fragments bind in a direct and dose-dependent manner. hERG1 hetero-oligomerization occurs in the endoplasmic reticulum where co-expression of N-terminal fragments with hERG1 subunits disrupted oligomerization and core glycosylation. The disruption of core glycosylation, a cotranslational event, allows us to pinpoint these N-terminal interactions to the earliest steps in biogenesis. Thus, N-terminal interactions mediate hERG 1a/1b assembly, a process whose perturbation may represent a new mechanism for disease.

hERG Gating Microdomains Defined by S6 Mutagenesis and Molecular Modeling

Human ether-à-go-go–related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.