Matthew C. Trudeau

Rod Cyclic Nucleotide-Gated Channels Have a Stoichiometry of Three CNGA1 Subunits and One CNGB1 Subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.

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.

hERG potassium channel gating is mediated by N- and C-terminal region interactions

Human ether-á-go-go–related gene (hERG) potassium channels have voltage-dependent closing (deactivation) kinetics that are unusually slow. A Per-Arnt-Sim (PAS) domain in the cytoplasmic N-terminal region of hERG regulates slow deactivation by making a direct interaction with another part of the hERG channel. The mechanism for slow deactivation is unclear, however, because the other regions of the channel that participate in regulation of deactivation are not known. To identify other functional determinants of slow deactivation, we generated hERG channels with deletions of the cytoplasmic C-terminal regions. We report that hERG channels with deletions of the cyclic nucleotide–binding domain (CNBD) had accelerated deactivation kinetics that were similar to those seen in hERG channels lacking the PAS domain. Channels with dual deletions of the PAS domain and the CNBD did not show further acceleration in deactivation, indicating that the PAS domain and the CNBD regulate deactivation by a convergent mechanism. A recombinant PAS domain that we previously showed could directly regulate PAS domain–deleted channels did not regulate channels with dual deletions of the PAS domain and CNBD, suggesting that the PAS domain did not interact with CNBD-deleted channels. Biochemical protein interaction assays showed that glutathione S-transferase (GST)–PAS (but not GST) bound to a CNBD-containing fusion protein. Coexpression of PAS domain–deleted subunits (with intact C-terminal regions) and CNBD-deleted subunits (with intact N-terminal regions) resulted in channels with partially restored slow deactivation kinetics, suggesting regulatory intersubunit interactions between PAS domains and CNBDs. Together, these data suggest that the mechanism for regulation of slow deactivation in hERG channels is an interaction between the N-terminal PAS domain and the C-terminal CNBD.

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.

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

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 Förster 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.

An Intersubunit Interaction Regulates Trafficking of Rod Cyclic Nucleotide-Gated Channels and Is Disrupted in an Inherited Form of Blindness

A mutation in a cyclic nucleotide-gated channel (CNGA1) is associated with retinitis pigmentosa (RP), a common, inherited eye disease. Expression of mutant (CNGA1-RP) homomeric channels in Xenopus oocytes revealed no measurable differences compared to wild-type CNGA1 homomers. As native retinal rod CNG channels comprise CNGA1 and CNGB1 subunits, we coexpressed CNGA1-RP and CNGB1. Surprisingly, this subunit combination did not produce detectable channels at the membrane surface. We show that the mechanism underlying this defect involves an intersubunit interaction between CNGA1 and CNGB1 that was not formed between CNGA1-RP and CNGB1 subunits. In the absence of this interaction, a short N-terminal region in CNGB1 prevented membrane expression. Thus, disruption of a regulatory interaction by mutation in CNGA1 exposed a region of CNGB1 that disrupted surface expression of heteromeric CNGA1-RP/CNGB1 channels, accounting for this instance of RP.

Mechanism of calcium/calmodulin inhibition of rod cyclic nucleotide-gated channels

Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium/calmodulin (Ca2+/CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca2+/CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca2+/CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca2+/CaM. These results suggest that Ca2+/CaM-dependent inhibition of rod channels is caused by the direct binding of Ca2+/CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca2+/CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels.

Structure of the C-terminal region of an ERG channel and functional implications

The human ether-à-go-go–related gene (hERG) encodes a K+ channel crucial for repolarization of the cardiac action potential. EAG-related gene (ERG) channels contain a C-terminal cyclic nucleotide-binding homology domain coupled to the pore of the channel by a C-linker. Here, we report the structure of the C-linker/cyclic nucleotide-binding homology domain of a mosquito ERG channel at 2.5-Å resolution. The structure reveals that the region expected to form the cyclic nucleotide-binding pocket is negatively charged and is occupied by a short β-strand, referred to as the intrinsic ligand, explaining the lack of direct regulation of ERG channels by cyclic nucleotides. In hERG channels, the intrinsic ligand harbors hereditary mutations associated with long-QT syndrome (LQTS), a potentially lethal cardiac arrhythmia. Mutations in the intrinsic ligand affected hERG channel gating and LQTS mutations abolished hERG currents and altered trafficking of hERG channels, which explains the LQT phenotype. The structure also reveals a dramatically different conformation of the C-linker compared with the structures of the related ether-à-go-go–like K+ and hyperpolarization-activated cyclic nucleotide-modulated channels, suggesting that the C-linker region may be highly dynamic in the KCNH, hyperpolarization-activated cyclic nucleotide-modulated, and cyclic nucleotide-gated channels.

Functional Analysis of a Mouse Brain Elk-Type K+Channel

Members of the Ether à go-go (Eag) K+channel subfamilies Eag, Erg, and Elk are widely expressed in the nervous system, but their neural functions in vivoremain largely unknown. The biophysical properties of channels from the Eag and Erg subfamilies have been described, and based on their characteristic features and expression patterns, Erg channels have been associated with native currents in the heart. Little is known about the properties of channels from the Elk subfamily. We have identified a mouse gene, Melk2, that encodes a predicted polypeptide with 48% amino acid identity to Drosophila Elk but only 40 and 36% identity with mouse Erg (Merg) and Eag (Meag), respectively. Melk2 RNA appears to be expressed at high levels only in brain tissue. Functional expression ofMelk2 in Xenopus oocytes reveals large, transient peaks of current at the onset of depolarization. Like Meag currents, Melk2 currents activate relatively quickly, but they lack the nonsuperimposable Cole–Moore shift characteristic of the Eag subfamily. Melk2 currents are insensitive to E-4031, a class III antiarrhythmic compound that blocks the Human Ether-à-go-go-Related Gene (HERG) channel and its counterpart in native tissues, IKr. Melk2 channels exhibit inward rectification because of a fast C-type inactivation mechanism, but the slower rate of inactivation and the faster rate of activation results in less inward rectification than that observed in HERG channels. This characterization of Melk currents should aid in identification of native counterparts to the Elk subfamily of channels in the nervous system.