Regulation Of Channel Gating Research Articles

Cryo-EM structures of prokaryotic ligand-gated ion channel GLIC provide insights into gating in a lipid environment

GLIC, a proton-activated prokaryotic ligand-gated ion channel, served as a model system for understanding the eukaryotic counterparts due to their structural and functional similarities. Despite extensive studies conducted on GLIC, the molecular mechanism of channel gating in the lipid environment requires further investigation. Here, we present the cryo-EM structures of nanodisc-reconstituted GLIC at neutral and acidic pH in the resolution range of 2.6 – 3.4 Å. In our apo state at pH 7.5, the extracellular domain (ECD) displays conformational variations compared to the existing apo structures. At pH 4.0, three distinct conformational states (C1, C2 and O states) are identified. The protonated structures exhibit a compacted and counter-clockwise rotated ECD compared with our apo state. A gradual widening of the pore in the TMD is observed upon reducing the pH, with the widest pore in O state, accompanied by several layers of water pentagons. The pore radius and molecular dynamics (MD) simulations suggest that the O state represents an open conductive state. We also observe state-dependent interactions between several lipids and proteins that may be involved in the regulation of channel gating. Our results provide comprehensive insights into the importance of lipids impact on gating.

Structure and dynamics of the proton-selective histidine and the gating tryptophan in an inward rectifying hybrid influenza B and A virus M2 proton channel.

The M2 proteins of influenza A and B viruses form acid-activated proton channels that are essential for the virus lifecycle. Proton selectivity is achieved by a transmembrane (TM) histidine whereas gating is achieved by a tryptophan residue. Although this functional apparatus is conserved between AM2 and BM2 channels, AM2 conducts protons exclusively inward whereas BM2 conducts protons in either direction depending on the pH gradient. Previous studies showed that in AM2, mutations of D44 abolished inward rectification of AM2, suggesting that the tryptophan gate is destabilized. To elucidate how charged residues C-terminal to the tryptophan regulates channel gating, here we investigate the structure and dynamics of H19 and W23 in a BM2 mutant, GDR-BM2, in which three BM2 residues are mutated to the corresponding AM2 residues, S16G, G26D and H27R. Whole-cell electrophysiological data show that GDR-BM2 conducts protons with inward rectification, identical to wild-type (WT) AM2 but different from WT-BM2. Solid-state NMR 15N and 13C spectra of H19 indicate that the mutant BM2 channel contains higher populations of cationic histidine and neutral τ tautomers compared to WT-BM2 at acidic pH. Moreover, 19F NMR spectra of 5-19F-labeled W23 resolve three peaks at acidic pH, suggesting three tryptophan sidechain conformations. Comparison of these spectra with the tryptophan spectra of other M2 peptides suggests that these indole sidechain conformations arise from interactions with the C-terminal charged residues and with the N-terminal cationic histidine. Taken together, these solid-state NMR data show that inward rectification in M2 proton channels is accomplished by tryptophan interactions with charged residues on both its C-terminal and N-terminal sides. Gating of these M2 proton channels is thus accomplished by a multi-residue complex with finely tuned electrostatic and aromatic interactions.

Temperature sensitive contact modes allosterically gate TRPV3.

TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channel's functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gate's state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.

Twisting and Protonation of Retinal Chromophore Regulate Channel Gating of Channelrhodopsin C1C2.

Channelrhodopsins (ChRs) are light-gated ion channels and central optogenetic tools that can control neuronal activity with high temporal resolution at the single-cell level. Although their application in optogenetics has rapidly progressed, it is unsolved how their channels open and close. ChRs transport ions through a series of interlocking elementary processes that occur over a broad time scale of subpicoseconds to seconds. During these processes, the retinal chromophore functions as a channel regulatory domain and transfers the optical input as local structural changes to the channel operating domain, the helices, leading to channel gating. Thus, the core question on channel gating dynamics is how the retinal chromophore structure changes throughout the photocycle and what rate-limits the kinetics. Here, we investigated the structural changes in the retinal chromophore of canonical ChR, C1C2, in all photointermediates using time-resolved resonance Raman spectroscopy. Moreover, to reveal the rate-limiting factors of the photocycle and channel gating, we measured the kinetic isotope effect of all photoreaction processes using laser flash photolysis and laser patch clamp, respectively. Spectroscopic and electrophysiological results provided the following understanding of the channel gating: the retinal chromophore highly twists upon the retinal Schiff base (RSB) deprotonation, causing the surrounding helices to move and open the channel. The ion-conducting pathway includes the RSB, where inflowing water mediates the proton to the deprotonated RSB. The twisting of the retinal chromophore relaxes upon the RSB reprotonation, which closes the channel. The RSB reprotonation rate-limits the channel closing.

Charged residues in the GABA-a receptor intracellular domain impact channel activation and desensitization.

Opening and closing of GABA receptor (GABAR) channels regulate inhibitory signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. Although GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, little is understood about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR homology model based on the 5HT-type-3 receptor, which revealed a helix (MX) at the C-terminal end of M3 that is lysine rich in the α1 (6 lysines) and γ2 (8 lysines) subunits. Charged residues in the α and γ subunits were mutated to alanine. Effects on GABA EC50 were measured using two-electrode voltage clamping of oocytes expressing α1β2γ2L WT and mutant GABARs. αK327A significantly increased GABA EC50 6-fold in comparison to WT and significantly reduced GABAR macroscopic desensitization. Extent of current desensitization after 10 seconds in saturating GABA (10 mM) was 6.86 ± 2.28% (n=7) versus WT (23.7 ± 7.29%, n=10). Neutralizing charge at αK328, K330 and K335 had no functional effects. γK328A, γ2K330A and γ2K332A significantly increased GABA EC50 by 2 to 3 fold. γ2K330A reduced GABAR desensitization (extent after 10 seconds 7.36 ± 2.37%, n=2). Our data indicate structural perturbations in the α and γ MX regions of the GABAR ICD alter GABAR channel activation and desensitization. Interestingly, in our GABAR structural model, α1R312 (in MX) interacts with α1R249 (M1-M2 linker). Single charge reversals at these positions increased GABA EC50 and the rate of GABA desensitization. A charge swap (R312E - E249R) rescued receptor function to WT suggesting a functional electrostatic interaction and that these residues form a salt bridge, providing support for our homology model.

Optimized tight binding between the S1 segment and KCNE3 is required for the constitutively open nature of the KCNQ1-KCNE3 channel complex.

Tetrameric voltage-gated K⁺ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K⁺ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues (volume scanning), we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

Nicotinic acetylcholine receptor redux: Discovery of accessories opens therapeutic vistas

The neurotransmitter acetylcholine (ACh) acts in part through a family of nicotinic ACh receptors (nAChRs), which mediate diverse physiological processes including muscle contraction, neurotransmission, and sensory transduction. Pharmacologically, nAChRs are responsible for tobacco addiction and are targeted by medicines for hypertension and dementia. Nicotinic AChRs were the first ion channels to be isolated. Recent studies have identified molecules that control nAChR biogenesis, trafficking, and function. These nAChR accessories include protein and chemical chaperones as well as auxiliary subunits. Whereas some factors act on many nAChRs, others are receptor specific. Discovery of these regulatory mechanisms is transforming nAChR research in cells and tissues ranging from central neurons to spinal ganglia to cochlear hair cells. Nicotinic AChR-specific accessories also enable drug discovery on high-confidence targets for psychiatric, neurological, and auditory disorders.

Accessory proteins and nicotinic receptors

Neuroscience Acetylcholine was the first neurotransmitter identified, and nicotinic acetylcholine receptors (nAChRs) were the first neurotransmitter receptors isolated. Recent studies have identified a multitude of molecules and mechanisms that regulate nAChRs in different tissues. In a Review, Matta et al. discuss these discoveries and their implications for the cell biology and medicinal pharmacology of nACHRs. Many accessory factors promote the assembly and function of diverse nAChRs. Some factors are small molecules, some are proteins, some control receptor biogenesis, and some regulate channel gating. These protein chaperones and auxiliary subunits elucidate the pharmacological and physiological processes regulated by acetylcholine. Science , abg6539, this issue p. [eabg6539][1] [1]: /lookup/doi/10.1126/science.abg6539

Influence of GABA-A Receptor Intracellular Domain on Channel Activation and Desensitization

Rapid opening and closing of GABA-A receptor (GABAR) channels regulate signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, but little information exists about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR ICD homology model based on a cryo-EM 5HT3AR structure, which revealed a helix (MX) at the C-terminal end of M3 and a helix (MA) at the N-terminal end of M4 projecting intracellularly. Four residues with potential interactions between these helices were mutated to alanine (α1K327A in MX, α1K390A in MA, and γ2K409A and γ2Y413A in MA). Mutations effects on GABA-EC50 and current desensitization were measured using two-electrode voltage clamping of oocytes expressing wild-type (WT) and mutant GABARs. α1K327A increased GABA EC50 ∼6-fold compared to α1β2γ2 WT (35.98 ± 1.03 μM, n=9) and significantly reduced GABAR desensitization. Extent of current desensitization after 60 seconds in saturating GABA (10mM) was 30.16 ± 2.24 % (n=9) versus WT (62.96 ± 4.15 %, n=13). α1K390A had no effect on GABA EC50 or desensitization, and mutation of the aligned residue in γ2 subunit, γ2K409A, decreased GABA EC50 ∼2 fold without affecting desensitization. γ2Y413A significantly increased desensitization and increased GABA EC50 by 2-fold. Desensitization extent after 10 seconds was 76.57 ± 2.09 % (n=18) compared to WT (23.71 ± 2.30 %, n=10). These data indicate that structural perturbations of the α1MX and γ2MA regions in the GABAR ICD can influence macroscopic GABAR current desensitization. Interestingly, GABAR MX regions are lysine rich. We speculate that mechanisms underlying GABAR gating transitions may involve charged interactions between MX and other regions of the GABAR ICD (e.g. MA). Experiments are underway to test this idea.

Some calcium is required for binding

Structural Biology The melanocortin-4 receptor (MC4R) coordinates food intake and energy expenditure and is a target for treating obesity. MC4R is an unusual G protein–coupled receptor, in part because it binds either an endogenous agonist or an endogenous antagonist, leading to reduced appetite or increased food intake, respectively. Yu et al. determined the structure of MC4R bound to an antagonist (see the Perspective by Chaturvedi and Shukla). This structure revealed a calcium ion coordinated by the receptor and the antagonist. Biochemical studies showed that the calcium ion also increased the affinity for endogenous agonist, which translated into increased potency. The authors also confirm a previous finding that MC4R directly couples to the ion channel Kir7.1 and regulates channel gating. Science , this issue p. [428][1]; see also p. [369][2] [1]: /lookup/doi/10.1126/science.aaz8995 [2]: /lookup/doi/10.1126/science.abb4393

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

Veratridine binding to a transmembrane helix of sodium channel Nav1.4 determined by solid-state NMR.

The multi-step ligand action to a target protein is an important aspect when understanding mechanisms of ligand binding and discovering new drugs. However, structurally capturing such complex mechanisms is challenging. This is particularly true for interactions between large membrane proteins and small molecules. One such large membrane of interest is Nav1.4, a eukaryotic voltage-gated sodium channel. Domain 4 segment 6 (D4S6) of Nav1.4 is a transmembrane α-helical segment playing a key role in channel gating regulation, and is targeted by a neurotoxin, veratridine (VTD). VTD has been suggested to exhibit a two-step action to activate Nav1.4. Here, we determine the NMR structure of a selectively 13C-labeled peptide corresponding to D4S6 and its VTD binding site in lipid bilayers determined by using magic-angle spinning solid-state NMR. By 13C NMR, we obtain NMR structural constraints as 13C chemical shifts and the 1H-2H dipolar couplings between the peptide and deuterated lipids. The peptide backbone structure and its location with respect to the membrane are determined under the obtained NMR structural constraints aided by replica exchange molecular dynamics simulations with an implicit membrane/solvent system. Further, by measuring the 1H-2H dipolar couplings to monitor the peptide-lipid interaction, we identify a VTD binding site on D4S6. When superimposed to a crystal structure of a bacterial sodium channel NavRh, the determined binding site is the only surface exposed to the protein exterior and localizes beside the second-step binding site reported in the past. Based on these results, we propose that VTD initially binds to these newly-determined residues on D4S6 from the membrane hydrophobic domain, which induces the first-step channel opening followed by the second-step blocking of channel inactivation of Nav1.4. Our findings provide new detailed insights of the VTD action mechanism, which could be useful in designing new drugs targeting D4S6.

Phosphorylation Alters the Residual Structure and Interactions of the Regulatory L1 Linker Connecting NBD1 to the Membrane-Bound Domain in SUR2B.

ATP-sensitive potassium (KATP) channels in vascular smooth muscle are comprised of four pore-forming Kir6.1 subunits and four copies of the sulfonylurea receptor 2B (SUR2B), which acts as a regulator of channel gating. Recent electron cryo-microscopy (cryo-EM) structures of the pancreatic KATP channel show a central Kir6.2 pore that is surrounded by the SUR1 subunits. Mutations in the L1 linker connecting the first membrane-spanning domain and the first nucleotide binding domain (NBD1) in SUR2B cause cardiac disease; however, this part of the protein is not resolved in the cryo-EM structures. Phosphorylation of the L1 linker, by protein kinase A, disrupts its interactions with NBD1, which increases the MgATP affinity of NBD1 and KATP channel gating. To elucidate the mode by which the L1 linker regulates KATP channels, we have probed the effects of phosphorylation on its structure and interactions using nuclear magnetic resonance (NMR) spectroscopy and other techniques. We demonstrate that the L1 linker is an intrinsically disordered region of SUR2B but possesses residual secondary and compact structure, both of which are disrupted with phosphorylation. NMR binding studies demonstrate that phosphorylation alters the mode by which the L1 linker interacts with NBD1. The data show that L1 linker residues with the greatest α-helical propensity also form the most stable interaction with NBD1, highlighting a hot spot within the L1 linker. This hot spot is the site of disease-causing mutations and is associated with other processes that regulate KATP channel gating. These data provide insights into the mode by which the phospho-regulatory L1 linker regulates KATP channels.

Translocatable voltage-gated Ca2+ channel β subunits in α1–β complexes reveal competitive replacement yet no spontaneous dissociation

β subunits of high voltage-gated Ca2+ (CaV) channels promote cell-surface expression of pore-forming α1 subunits and regulate channel gating through binding to the α-interaction domain (AID) in the first intracellular loop. We addressed the stability of CaV α1B-β interactions by rapamycin-translocatable CaV β subunits that allow drug-induced sequestration and uncoupling of the β subunit from CaV2.2 channel complexes in intact cells. Without CaV α1B/α2δ1, all modified β subunits, except membrane-tethered β2a and β2e, are in the cytosol and rapidly translocate upon rapamycin addition to anchors on target organelles: plasma membrane, mitochondria, or endoplasmic reticulum. In cells coexpressing CaV α1B/α2δ1 subunits, the translocatable β subunits colocalize at the plasma membrane with α1B and stay there after rapamycin application, indicating that interactions between α1B and bound β subunits are very stable. However, the interaction becomes dynamic when other competing β isoforms are coexpressed. Addition of rapamycin, then, switches channel gating and regulation by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipid. Thus, expression of free β isoforms around the channel reveals a dynamic aspect to the α1B-β interaction. On the other hand, translocatable β subunits with AID-binding site mutations are easily dissociated from CaV α1B on the addition of rapamycin, decreasing current amplitude and PI(4,5)P2 sensitivity. Furthermore, the mutations slow CaV2.2 current inactivation and shift the voltage dependence of activation to more positive potentials. Mutated translocatable β subunits work similarly in CaV2.3 channels. In sum, the strong interaction of CaV α1B-β subunits can be overcome by other free β isoforms, permitting dynamic changes in channel properties in intact cells.

Partial rescue of F508del-cystic fibrosis transmembrane conductance regulator channel gating with modest improvement of protein processing, but not stability, by a dual-acting small molecule.

Rescue of F508del-cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the most common CF mutation, requires small molecules that overcome protein processing, stability and channel gating defects. Here, we investigate F508del-CFTR rescue by CFFT-004, a small molecule designed to independently correct protein processing and channel gating defects. Using CFTR-expressing recombinant cells and CF patient-derived bronchial epithelial cells, we studied CFTR expression by Western blotting and channel gating and stability with the patch-clamp and Ussing chamber techniques. Chronic treatment with CFFT-004 improved modestly F508del-CFTR processing, but not its plasma membrane stability. By contrast, CFFT-004 rescued F508del-CFTR channel gating better than C18, an analogue of the clinically used CFTR corrector lumacaftor. Subsequent acute addition of CFFT-004, but not C18, potentiated F508del-CFTR channel gating. However, CFFT-004 was without effect on A561E-CFTR, a CF mutation with a comparable mechanism of CFTR dysfunction as F508del-CFTR. To investigate the mechanism of action of CFFT-004, we used F508del-CFTR revertant mutations. Potentiation by CFFT-004 was unaffected by revertant mutations, but correction was abolished by the revertant mutation G550E. These data suggest that correction, but not potentiation, by CFFT-004 might involve nucleotide-binding domain 1 of CFTR. CFFT-004 is a dual-acting small molecule with independent corrector and potentiator activities that partially rescues F508del-CFTR in recombinant cells and native airway epithelia. The limited efficacy and potency of CFFT-004 suggests that combinations of small molecules targeting different defects in F508del-CFTR might be a more effective therapeutic strategy than a single agent.

Stargazin and cornichon-3 relieve polyamine block of AMPA receptors by enhancing blocker permeation.

Most ligand- and voltage-gated ion channels assemble as signaling complexes consisting of pore-forming and auxiliary subunits. In the mammalian brain, AMPA-type ionotropic glutamate receptors (AMPARs) coassemble with several families of auxiliary subunits that regulate channel gating as well as ion channel block and permeation. Previous work has shown that auxiliary proteins stargazin (or γ2) and cornichon-3 (CNIH-3) attenuate the cytoplasmic polyamine channel block of AMPARs, although the underlying mechanism has yet to be established. Here, we show that γ2 and CNIH-3 relieve channel block by enhancing the rate of blocker permeation. Surprisingly, the relative permeability of the polyamine spermine (Spm) through the pore of the AMPAR-γ2 or -CNIH-3 complexes is considerably more than AMPARs expressed alone. Spm permeability is comparable to that of Na+ for the GluA2-γ2 complex and four times greater than Na+ with GluA2 + CNIH-3. A modified model of permeant channel block fully accounts for both the voltage- and time-dependent nature of Spm block. Estimates of block rate constants reveal that auxiliary subunits do not attenuate block by shifting the location of the block site within the membrane electric field, and they do not affect the blocker's ability to reach it. Instead, γ2 and CNIH-3 relieve channel block by facilitating the blocker's exit rates from the open channel. From a physiological perspective, the relief of channel block exerted by γ2 and CNIH-3 ensures that there is unfettered signaling by AMPARs at glutamatergic synapses. Moreover, the pronounced ability of AMPARs to transport polyamines may have an unexpected role in regulating cellular polyamine levels.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Mechanisms of noncovalent β subunit regulation of NaV channel gating.

Voltage-gated Na+ (NaV) channels comprise a macromolecular complex whose components tailor channel function. Key components are the non-covalently bound β1 and β3 subunits that regulate channel gating, expression, and pharmacology. Here, we probe the molecular basis of this regulation by applying voltage clamp fluorometry to measure how the β subunits affect the conformational dynamics of the cardiac NaV channel (NaV1.5) voltage-sensing domains (VSDs). The pore-forming NaV1.5 α subunit contains four domains (DI-DIV), each with a VSD. Our results show that β1 regulates NaV1.5 by modulating the DIV-VSD, whereas β3 alters channel kinetics mainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the β3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to β3 at the same position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD. Together, these results provide compelling evidence that β3 binds proximally to the DIII-VSD. Molecular-level differences in β1 and β3 interaction with the α subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting NaV channel regulation of cell excitability.

Novel Mechanism of Gating in the TrkH-TrkA Complex

The superfamily of K+ transporters (SKT) is ubiquitous in bacteria, fungi and plants. SKT proteins are required for survival of bacteria in low K+ conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two components, a membrane embedded protein that resembles an ion channel and a cytosolic protein that regulates channel gating [1]. Crystal structures of two bacterial SKT systems were reported recently [2,3]. In both structures, the membrane embedded component forms a homodimer onto which a homotetrameric ring of the cytosolic protein docks. Single-channel activities of one of the complexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, were recorded and analyzed: ATP or ATP analogs such as AMPPNP activates the channel while ADP closes it [2]. The structure of the TrkH-TrkA complex is likely in a closed conformation because it was crystallized in the presence of NADH, a ligand that does not activates the channel. In order to understand how ATP or its analogs induces channel opening, we solved the structure of the TrkH-TrkA complex in the presence of AMPPNP to 3.29 A by x-ray crystallography. When compared to the previous structures, the new structure shows that each TrkA protomer binds to two AMPPNP molecules, and that the TrkA tetramer assumes an elongated conformation that likely induces a change in the TrkH. Conformational changes in the TrkH involve significant changes in the dimer interface and are different from any other channels of known structure. These new observations and hypotheses will be validated and tested by mutational and functional analyses.[1] Levin EJ, Zhou M. Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr Opin Struct Biol. 2014;27:95-101.[2] Cao Y, Pan Y, Huang H, et al. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature. 2013;496(7445):317-22.[3] Vieira-pires RS, Szollosi A, Morais-cabral JH. The structure of the KtrAB potassium transporter. Nature. 2013;496(7445):323-8.

Role of Tank-Binding Kinase in Neurodegeneration Produced by Spinocerebellar Ataxia Type 13

Inherited mutations in the Kv3.3 potassium channel, such as the G952R Kv3.3 mutation, Kv3.3 mutations result in Spinocerebellar Ataxia 13 (SCA13). In contrast to wild type Kv3.3 channels, G592R mutant channels have slowed inactivation and fail to trigger the formation of a Hax-1- and Arp2/3- dependent actin cytoskeleton under the plasma membrane, but significantly increase the rate of cell death in transfected cells. To determine which transduction pathways are activated by G592R Kv3.3 we carried out an in situ kinase profiling screen to compare levels of activity of protein kinases in the cerebella of G592R-knock-in mice with those of wild type animals. The activity of TBK1 was found to be significantly elevated in the cerebellum but not the cerebral cortex of G592R Kv3.3 mice. This result was confirmed by western blotting. We found that enhanced TBK1 activity accounts for the slowed inactivation of the mutant channel. Pharmacological inhibition of TBK1 increased the rate of inactivation of Kv3.3 currents, while TBK1 overexpression slowed their inactivation rate. In order to explore the role of TBK1 in cell death caused by G592R Kv3.3, we measured levels of cleaved caspase 7 in different conditions, and found that the level of cleaved caspase 7 is increased in cells expressing mutant Kv3.3 channels compared with that in cells expressing wild type channels, particularly under condition of depolarization with high K+. Pretreatment with the TBK1 inhibitor, however, reduced expression levels of cleaved caspase 7 under high K+ stimulation in both types of cells, but not in untransfected cells. Our results suggest that the interactions of the Kv3.3 potassium channel with TBK1 and Hax-1 regulate channel gating and may link the channel to signaling pathways that protect cerebellar neurons from apoptosis.