HERG potassium channel regulation by the N-terminal eag domain

Human ether-á-go-go related gene (hERG, K(v)11.1) potassium channels play a significant role in cardiac excitability. Like other K(v) channels, hERG is activated by membrane voltage; however, distinct from other K(v) channels, hERG channels have unusually slow kinetics of closing (deactivation). The mechanism for slow deactivation involves an N-terminal "eag domain" which comprises a PAS (Per-Arnt-Sim) domain and a short Cap domain. Here we review recent advances in understanding how the eag domain regulates deactivation, including several new Nuclear Magnetic Resonance (NMR) solution structures of the eag domain, and evidence showing that the eag domain makes a direct interaction with the C-terminal C-linker and Cyclic Nucleotide-Binding Homology Domain.     

hERG1a N-terminal eag domain-containing polypeptides regulate homomeric hERG1b and heteromeric hERG1a/hERG1b channels: a possible mechanism for long QT syndrome

Human ether-á-go-go-related gene (hERG) potassium channels are critical for cardiac action potential repolarization. Cardiac hERG channels comprise two primary isoforms: hERG1a, which has a regulatory N-terminal Per-Arnt-Sim (PAS) domain, and hERG1b, which does not. Isolated, PAS-containing hERG1a N-terminal regions (NTRs) directly regulate NTR-deleted hERG1a channels; however, it is unclear whether hERG1b isoforms contain sufficient machinery to support regulation by hERG1a NTRs. To test this, we constructed a series of PAS domain-containing hERG1a NTRs (encoding amino acids 1-181, 1-228, 1-319, and 1-365). The NTRs were also predicted to form from truncation mutations that were linked to type 2 long QT syndrome (LQTS), a cardiac arrhythmia disorder associated with mutations in the hERG gene. All of the hERG1a NTRs markedly regulated heteromeric hERG1a/hERG1b channels and homomeric hERG1b channels by decreasing the magnitude of the current-voltage relationship and slowing the kinetics of channel closing (deactivation). In contrast, NTRs did not measurably regulate hERG1a channels. A short NTR (encoding amino acids 1-135) composed primarily of the PAS domain was sufficient to regulate hERG1b. These results suggest that isolated hERG1a NTRs directly interact with hERG1b subunits. Our results demonstrate that deactivation is faster in hERG1a/hERG1b channels compared to hERG1a channels because of fewer PAS domains, not because of an inhibitory effect of the unique hERG1b NTR. A decrease in outward current density of hERG1a/hERG1b channels by hERG1a NTRs may be a mechanism for LQTS.     

Direct interaction of eag domains and cyclic nucleotide–binding homology domains regulate deactivation gating in hERG channels

Human ether-á-go-go (eag)-related gene (hERG) potassium channels play a critical role in cardiac repolarization and are characterized by unusually slow closing (deactivation) kinetics. The N-terminal “eag” domain and a C-terminal C-linker/cyclic nucleotide–binding homology domain (CNBHD) are required for regulation of slow deactivation. The region between the S4 and S5 transmembrane domains (S4–S5 linker) is also implicated in this process, but the mechanism for regulation of slow deactivation is unclear. Here, using an eag domain–deleted channel (hERG Δeag) fused to Citrine fluorescent protein, we found that most channels bearing individual alanine mutations in the S4–S5 linker were directly regulated by recombinant eag domains fused to a cyan fluorescent protein (N-eag-CFP) and had robust Förster resonance energy transfer (FRET). Additionally, a channel bearing a group of eight alanine residues in the S4–S5 linker was not measurably regulated by N-eag-CFP domains, but robust FRET was measured. These findings demonstrate that the eag domain associated with all of the S4–S5 linker mutant channels. In contrast, channels that also lacked the CNBHD (hERG Δeag ΔCNBHD-Citrine) were not measurably regulated by N-eag-CFP nor was FRET detected, suggesting that the C-linker/CNBHD was required for eag domains to directly associate with the channel. In a FRET hybridization assay, N-eag-CFP had robust FRET with a C-linker/CNBHD-Citrine, suggesting a direct and specific interaction between the eag domain and the C-linker/CNBHD. Lastly, coexpression of a hERG subunit lacking the CNBHD and the distal C-terminal region (hERG ΔpCT-Citrine) with hERG Δeag-CFP subunits had FRET and partial restoration of slow deactivation. Collectively, these findings reveal that the C-linker/CNBHD, but not the S4–S5 linker, was necessary for the eag domain to associate with the channel, that the eag domain and the C-linker/CNBHD were sufficient for a direct interaction, and that an intersubunit interaction between the eag domain and the C-linker/CNBHD regulated slow deactivation in hERG channels at the plasma membrane.     

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

Congenital long QT syndrome 2 (LQT2) is caused by loss-of-function mutations in the human ether-á-go-go-related gene (hERG) voltage-gated potassium (K(+)) channel. hERG channels have slow deactivation kinetics that are regulated by an N-terminal Per-Arnt-Sim (PAS) domain. Only a small percentage of hERG channels containing PAS domain LQT2 mutations (hERG PAS-LQT2) have been characterized in mammalian cells, so the functional effect of these mutations is unclear. We investigated 11 hERG PAS-LQT2 channels in HEK293 cells and report a diversity of functional defects. Most hERG PAS-LQT2 channels formed functional channels at the plasma membrane, as measured by whole cell patch clamp recordings and cell surface biotinylation. Mutations located on one face of the PAS domain (K28E, F29L, N33T, R56Q, and M124R) caused defective channel gating, including faster deactivation kinetics and less steady-state inactivation. Conversely, the other mutations caused no measurable differences in channel gating (G53R, H70R, and A78P) or no measurable currents (Y43C, C66G, and L86R). We used a genetically encoded hERG PAS domain (NPAS) to examine whether channel dysfunction could be corrected. We found that NPAS fully restored wild-type-like deactivation kinetics and steady-state inactivation to the hERG PAS-LQT2 channels. Additionally, NPAS rescued aberrant currents in hERG R56Q channels during a dynamic ramp voltage clamp. Thus, our results reveal a putative "gating face" in the PAS domain where mutations within this region form functional channels with altered gating properties, and we show that NPAS is a general means for rescuing aberrant gating in hERG LQT2 mutant channels and may be a potential biological therapeutic.     

Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels

The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg⁵⁶ of the PAS domain and Asp⁸⁰³ of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels.     

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.     

Concerted All-or-none Subunit Interactions Mediate Slow Deactivation of Human ether-à-go-go-related Gene K+ Channels

During the repolarization phase of a cardiac action potential, hERG1 K⁺ channels rapidly recover from an inactivated state then slowly deactivate to a closed state. The resulting resurgence of outward current terminates the plateau phase and is thus a key regulator of action potential duration of cardiomyocytes. The intracellular N-terminal domain of the hERG1 subunit is required for slow deactivation of the channel as its removal accelerates deactivation 10-fold. Here we investigate the stoichiometry of hERG1 channel deactivation by characterizing the kinetic properties of concatenated tetramers containing a variable number of wild-type and mutant subunits. Three mutations known to accelerate deactivation were investigated, including R56Q and R4A/R5A in the N terminus and F656I in the S6 transmembrane segment. In all cases, a single mutant subunit induced the same rapid deactivation of a concatenated channel as that observed for homotetrameric mutant channels. We conclude that slow deactivation gating of hERG1 channels involves a concerted, fully cooperative interaction between all four wild-type channel subunits.     

Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance

Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.     

The N-linker region of hERG1a upregulates hERG1b potassium channels

A major physiological role of hERG1 (human Ether-á-go-go-Related Gene 1) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the potassium current I_Kr in cardiomyocytes. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, long QT syndrome, and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labeled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the N-terminal 1b domain did not have a measurable increase in current or biotinylated protein when coexpressed with hERG1a N-terminal regions, indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a N-terminal region and the hERG1b N-terminal region. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216 to 220 within the hERG1a “N-linker” region that were necessary for the upregulation of hERG1b. We propose that direct structural interactions between the hERG1a N-linker region and the hERG1b 1b domain increase hERG1b at the plasma membrane. Mechanisms regulating hERG1a and hERG1b are likely critical for cardiac function, may be disrupted by long QT syndrome mutants, and serve as potential targets for therapeutics.     

The S4-S5 linker acts as a signal integrator for HERG K+ channel activation and deactivation gating

Human ether-à-go-go-related gene (hERG) K(+) channels have unusual gating kinetics. Characterised by slow activation/deactivation but rapid inactivation/recovery from inactivation, the unique gating kinetics underlie the central role hERG channels play in cardiac repolarisation. The slow activation and deactivation kinetics are regulated in part by the S4-S5 linker, which couples movement of the voltage sensor domain to opening of the activation gate at the distal end of the inner helix of the pore domain. It has also been suggested that cytosolic domains may interact with the S4-S5 linker to regulate activation and deactivation kinetics. Here, we show that the solution structure of a peptide corresponding to the S4-S5 linker of hERG contains an amphipathic helix. The effects of mutations at the majority of residues in the S4-S5 linker of hERG were consistent with the previously identified role in coupling voltage sensor movement to the activation gate. However, mutations to Ser543, Tyr545, Gly546 and Ala548 had more complex phenotypes indicating that these residues are involved in additional interactions. We propose a model in which the S4-S5 linker, in addition to coupling VSD movement to the activation gate, also contributes to interactions that stabilise the closed state and a separate set of interactions that stabilise the open state. The S4-S5 linker therefore acts as a signal integrator and plays a crucial role in the slow deactivation kinetics of the channel.     

Voltage-sensing domain mode shift is coupled to the activation gate by the N-terminal tail of hERG channels

Human ether-a-go-go-related gene (hERG) potassium channels exhibit unique gating kinetics characterized by unusually slow activation and deactivation. The N terminus of the channel, which contains an amphipathic helix and an unstructured tail, has been shown to be involved in regulation of this slow deactivation. However, the mechanism of how this occurs and the connection between voltage-sensing domain (VSD) return and closing of the gate are unclear. To examine this relationship, we have used voltage-clamp fluorometry to simultaneously measure VSD motion and gate closure in N-terminally truncated constructs. We report that mode shifting of the hERG VSD results in a corresponding shift in the voltage-dependent equilibrium of channel closing and that at negative potentials, coupling of the mode-shifted VSD to the gate defines the rate of channel closure. Deletion of the first 25 aa from the N terminus of hERG does not alter mode shifting of the VSD but uncouples the shift from closure of the cytoplasmic gate. Based on these observations, we propose the N-terminal tail as an adaptor that couples voltage sensor return to gate closure to define slow deactivation gating in hERG channels. Furthermore, because the mode shift occurs on a time scale relevant to the cardiac action potential, we suggest a physiological role for this phenomenon in maximizing current flow through hERG channels during repolarization.     

Eag Domains Regulate LQT Mutant hERG Channels in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (I_Kr) current in the heart. The N-terminal ‘eag’ domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.     

Sequence of Gating Charge Movement and Pore Gating in hERG Activation and Deactivation Pathways

K_V11.1 voltage-gated K⁺ channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ∼threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.     

26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

Mutations of the cyclic nucleotide binding domain (CNBD) may disrupt human ether-a-go-go-related gene (hERG) K(+) channel function and lead to hereditary long QT syndrome (LQTS). We identified a novel missense mutation located in close proximity to the CNBD, hERG R744P, in a patient presenting with recurrent syncope and aborted cardiac death triggered by sudden auditory stimuli. Functional properties of wild type (WT) and mutant hERG R744P subunits were studied in Xenopus laevis oocytes using two-electrode voltage clamp electrophysiology and Western blot analysis. HERG R744P channels exhibited reduced activating currents compared to hERG WT (1.48±0.26 versus 3.40±0.29μA; n=40). These findings were confirmed by tail current analysis (hERG R744P, 0.53±0.07μA; hERG WT, 0.97±0.06μA; n=40). Cell surface trafficking of hERG R744P protein subunits was not impaired. To simulate the autosomal-dominant inheritance associated with LQTS, WT and R744P subunits were co-expressed in equimolar ratio. Mean activating and tail currents were reduced by 32% and 25% compared to hERG WT (n=40), indicating that R744P protein did not exert dominant-negative effects on WT channels. The half-maximal activation voltage was not significantly affected by the R744P mutation. This study highlights the significance of in vitro testing to provide mechanistic evidence for pathogenicity of mutations identified in LQTS. The functional defect associated with hERG R744P serves as molecular basis for LQTS in the index patient.     

Molecular determinants of interactions between the N-terminal domain and the transmembrane core that modulate hERG K+ channel gating

A conserved eag domain in the cytoplasmic amino terminus of the human ether-a-go-go-related gene (hERG) potassium channel is critical for its slow deactivation gating. Introduction of gene fragments encoding the eag domain are able to restore normal deactivation properties of channels from which most of the amino terminus has been deleted, and also those lacking exclusively the eag domain or carrying a single point mutation in the initial residues of the N-terminus. Deactivation slowing in the presence of the recombinant domain is not observed with channels carrying a specific Y542C point mutation in the S4-S5 linker. On the other hand, mutations in some initial positions of the recombinant fragment also impair its ability to restore normal deactivation. Fluorescence resonance energy transfer (FRET) analysis of fluorophore-tagged proteins under total internal reflection fluorescence (TIRF) conditions revealed a substantial level of FRET between the introduced N-terminal eag fragments and the eag domain-deleted channels expressed at the membrane, but not between the recombinant eag domain and full-length channels with an intact amino terminus. The FRET signals were also minimized when the recombinant eag fragments carried single point mutations in the initial portion of their amino end, and when Y542C mutated channels were used. These data suggest that the restoration of normal deactivation gating by the N-terminal recombinant eag fragment is an intrinsic effect of this domain directed by the interaction of its N-terminal segment with the gating machinery, likely at the level of the S4-S5 linker.     

Structural Properties of PAS Domains from the KCNH Potassium Channels

KCNH channels form an important family of voltage gated potassium channels. These channels include a N-terminal Per-Arnt-Sim (PAS) domain with unknown function. In other proteins PAS domains are implicated in cellular responses to environmental queues through small molecule binding or involvement in signaling cascades. To better understand their role we characterized the structural properties of several channel PAS domains. We determined high resolution structures of PAS domains from the mouse EAG (mEAG), drosophila ELK (dELK) and human ERG (hERG) channels and also of the hERG domain without the first nine amino acids. We analyzed these structures for features connected to ligand binding and signaling in other PAS domains. In particular, we have found cavities in the hERG and mEAG structures that share similarities with the ligand binding sites from other PAS domains. These cavities are lined by polar and apolar chemical groups and display potential flexibility in their volume. We have also found that the hydrophobic patch on the domain β-sheet is a conserved feature and appears to drive the formation of protein-protein contacts. In addition, the structures of the dELK domain and of the truncated hERG domain revealed the presence of N-terminal helices. These helices are equivalent to the helix described in the hERG NMR structures and are known to be important for channel function. Overall, these channel domains retain many of the PAS domain characteristics known to be important for cell signaling.     

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.     

Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking.     

The Roles of N- and C-terminal determinants in the activation of the Kv2.1 potassium channel

The human and rat forms of the Kv2.1 channel have identical amino acids over the membrane-spanning regions and differ only in the N- and C-terminal intracellular regions. Rat Kv2.1 activates much faster than human Kv2.1. Here we have studied the role of the N- and C-terminal residues that determine this difference in activation kinetics between the two channels. For this, we constructed mutants and chimeras between the two channels, expressed them in oocytes, and recorded currents by two-electrode voltage clamping. In the N-terminal region, mutation Q67E in the rat channel displayed a slowing of activation relative to rat wild type, whereas mutation D75E in the human channel showed faster activation than human wild type. In the C-terminal region, we found that some residues within the region of amino acids 740-853 ("CTA" domain) were also involved in determining activation kinetics. The electrophysiological data also suggested interactions between the N and C termini. Such an interaction was confirmed directly by using a glutathione S-transferase (GST) fusion protein with the N terminus of Kv2.1, which we showed to bind to the C terminus of Kv2.1. Taken together, these data suggest that exposed residues in the T1 domain of the N terminus, as well as the CTA domain in the C terminus, are important in determining channel activation kinetics and that these N- and C-terminal regions interact.     

Mechanistic insight into human ether-à-go-go-related gene (hERG) K+ channel deactivation gating from the solution structure of the EAG domain

Human ether-à-go-go-related gene (hERG) K(+) channels have a critical role in cardiac repolarization. hERG channels close (deactivate) very slowly, and this is vital for regulating the time course and amplitude of repolarizing current during the cardiac action potential. Accelerated deactivation is one mechanism by which inherited mutations cause long QT syndrome and potentially lethal arrhythmias. hERG deactivation is highly dependent upon an intact EAG domain (the first 135 amino acids of the N terminus). Importantly, deletion of residues 2-26 accelerates deactivation to a similar extent as removing the entire EAG domain. These and other experiments suggest the first 26 residues (NT1-26) contain structural elements required to slow deactivation by stabilizing the open conformation of the pore. Residues 26-135 form a Per-Arnt-Sim domain, but a structure for NT1-26 has not been forthcoming, and little is known about its site of interaction on the channel. In this study, we present an NMR structure for the entire EAG domain, which reveals that NT1-26 is structurally independent from the Per-Arnt-Sim domain and contains a stable amphipathic helix with one face being positively charged. Mutagenesis and electrophysiological studies indicate that neutralizing basic residues and breaking the amphipathic helix dramatically accelerate deactivation. Furthermore, scanning mutagenesis and molecular modeling studies of the cyclic nucleotide binding domain suggest that negatively charged patches on its cytoplasmic surface form an interface with the NT1-26 domain. We propose a model in which NT1-26 obstructs gating motions of the cyclic nucleotide binding domain to allosterically stabilize the open conformation of the pore.