A recombinant N-terminal domain fully restores deactivation gating in N-truncated and long QT syndrome mutant hERG potassium channels

Abstract

Human ether á go-go related gene (hERG) potassium channels play a central role in cardiac repolarization where channel closing (deactivation) regulates current density during action potentials. Consequently, mutations in hERG that perturb deactivation are linked to long QT syndrome (LQTS), a catastrophic cardiac arrhythmia. Interactions between an N-terminal domain and the pore-forming ''core'' of the channel were proposed to regulate deactivation, however, despite its central importance the mechanistic basis for deactivation is unclear. Here, to more directly examine the mechanism for regulation of deactivation, we genetically fused N-terminal domains to fluorescent proteins and tested channel function with electrophysiology and protein interactions with Fö rster resonance energy transfer (FRET) spectroscopy. Truncation of hERG N-terminal regions markedly sped deactivation, and here we report that reapplication of gene fragments encoding N-terminal residues 1-135 (the ''eag domain'') was sufficient to restore regulation of deactivation. We show that fluorophore-tagged eag domains and N-truncated channels were in close proximity at the plasma membrane as determined with FRET. The eag domains with Y43A or R56Q (a LQTS locus) mutations showed less regulation of deactivation and less FRET, whereas eag domains restored regulation of deactivation gating to full-length Y43A or R56Q channels and showed FRET. This study demonstrates that direct, noncovalent interactions between the eag domain and the channel core were sufficient to regulate deactivation gating, that an LQTS mutation perturbed physical interactions between the eag domain and the channel, and that small molecules such as the eag domain represent a novel method for restoring function to channels with disease-causing mutations.eag domain ͉ FRET ͉ LQTS H uman ether á go-go related gene (hERG) potassium channels were originally cloned from a human hippocampus library based on homology to mammalian ether á go-go (eag) potassium channels (1). hERG forms the major subunits of the ''rapid component of the cardiac delayed rectifier potassium current'' (I Kr ) in the heart (2, 3). The physiological role of I Kr is to repolarize the late phase of cardiac action potentials (4). The clinical significance of hERG channels and I Kr in the heart is emphasized by mutations in the hERG gene, which are linked to long QT syndrome (LQTS), a cardiac arrhythmia (5). hERG channels are also the targets for inhibitory compounds that cause acquired arrhythmias (6).hERG channels exhibit unusual kinetics that help to specialize them for their role in the heart. With membrane depolarization, hERG channels activate relatively slowly and inactivate rapidly, which limits current amplitude. With subsequent membrane repolarization, hERG channels rapidly recover from inactivation and then very slowly deactivate, which gives rise to a large tail current. The resurgent tail current helps to repolarize the late phase of cardiac action potentials (2-4, 7).The molecular basis for the slow deacti...

Demonstration of Physical Proximity between the N Terminus and the S4-S5 Linker of the Human ether-à-go-go-related Gene (hERG) Potassium Channel

“…Therefore, it sheds significant new insight into the molecular basis of the protein-protein interactions between the N-terminal distal regions and the S4-S5 linker involved in the control of gating properties and hormonal modulation of hERG (1,2,24,(27)(28)(29)(30)(31)(32)(33)36).…”

“…It has been repeatedly proposed that an interaction between N-terminal domains and the S4-S5 linker is the cause of the strong influence exerted by the N terminus in the gating properties (1, 16, 23-25, 27-33, 37-39). Nevertheless, evidence for this interpretation is mostly indirect, including (i) the essential role of the S4-S5 linker coupling voltage sensor activation to the activation gate (38,40), (ii) the strong parallelism between the alterations in gating caused by mutations in the N terminus and those in the S4-S5 linker (1, 16, 24, 27-33, 37, 41), (iii) the prevention of deactivation slowing by the N terminus after mutation of Gly-546 in the S4-S5 linker to cysteine and its subsequent modification by the addition of the sulfhydryl reagent N-ethylmaleimide (29), (iv) the impairment of the cytoplasmic domain-dependent modulation of gating in response to hormonal treatments by single-point mutations in the initial portion of the S4-S5 linker (2), (v) the restoration of slow deactivation gating by the addition of synthetic or recombinant N-terminal domains to channels lacking the N terminus (28,31,32), and (vi) the closer positioning of the N-terminal-most segments of the hERG N terminus toward the transmembrane channel core structures compared with other cytoplasmic regions of the channel (3). Note, however, that despite these findings, a direct demonstration of a physical interaction between the distal portion of the N terminus and the S4-S5 linker in the entire channel protein is lacking.…”

“…Thus, the important role of the distal eag/PAS and exclusive proximal domains of the N terminus in the activation properties and their modulation by hormones has been demonstrated (1,2,(23)(24)(25)(26). It is known that the initial region of hERG N terminus determines its slow deactivation, whereas the proximal domain is the functionally critical region for slowing activation gating (24,(27)(28)(29)(30)(31)(32)(33). Also, it has recently been proposed that some residues in the N-terminal portions of the C terminus (either in the named "C-linker" between the last transmembrane segment (S6) and the cyclic nucleotide-binding domain (cNBD) homologous region or in the cNBD itself) could be involved in the control of deactivation gating, possibly through their interaction with the N-terminal-most initial regions of the N terminus (34 -36).…”

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

“…Previously, we showed that slow deactivation could be restored in LQT2 mutant hERG R56Q channels by application of a genetically encoded PAS domain (NPAS) in Xenopus oocytes (15). Here, we sought to determine whether NPAS was a general mechanism for rescue of LQT2 mutant channels.…”

“…hERG channels containing LQT2 mutations in the PAS domain (hERG PAS-LQT2) exhibit robust currents when studied in Xenopus oocytes (6,(13)(14)(15); however, most channels with LQT2 mutations located outside the PAS domain do not have measurable currents and show defects in maturation and trafficking when studied in mammalian cells (12, 16 -21). As only 5 hERG PAS-LQT2 channels have been functionally characterized in mammalian cells (20 -24), the mechanism for how PAS domain mutations disrupt hERG function when expressed in more physiological conditions remains unclear.…”