Gate Domain Research Articles

Applying Voltage Clamp Fluorometry to Track SCN5A Voltage Sensor Movement

Functional eukaryotic Na+ channels are composed of a single monomer with four homologous domains (DI-DIV), each with six transmembrane segments (S1-S6). In each domain, the fourth segment, S4, contains positive charges that transduce changes in voltage into channel gating. Previously, voltage clamp fluorometry (VCF) was applied to track the S4 voltage sensors in the rat muscle sodium channel, rNaV1.4 (Cha et al, Neuron, 1999 22(1):73-87). Our aim was to apply a similar methodology to the human cardiac sodium channel, hNaV1.5, which initiates the cardiac action potential, is the target of numerous anti-arrhythmic drugs and carries >100 mutations that are linked to inherited cardiac diseases. VCF reports on protein motion via an extracellular cysteine that is conjugated with a small fluorescent molecule, in our case tetramethylrhodamine maleimide. Within the extracellular S4 of each NaV1.5 domain, we have identified positions for cysteine substitutions that, when fluorescently tagged, display a voltage-sensitive change in fluorescence. Consistent with motions previously identified in rNaV1.4, and reflecting the role of each domain in gating, initial results show ultra-rapid outward movement of the S4 segments in DI, DII and DIII correlating with activation, but much slower translocation of the DIV S4 that is closer to the time scale of inactivation.

Proton-Coupled Transition between Outward-Facing and Inward-Facing States in the Uracil Transporter

The bacterial nucleobase-ascorbate transporters are proton-driven membrane symporters. The solved structure of the bacterial uracil transporter, which demonstrates an inward-facing open state, is spatially organized into a core domain and a gate domain. Two glutamate residues (Glu241 and Glu290) anchor the substrate uracil in its binding site at the interface between the two domains. However, the outward-facing conformation and the conformational transition between the inward-facing and the outward-facing states are completely unknown. Furthermore, and mechanistically more important, how protons drive this transition remains also elusive. In the present study, we apply molecular dynamics simulations and free energy perturbation (FEP) to address some of these questions. The results of equilibrium simulations show that the empty transporter adopts an outward-facing open conformation through a rigid-body movement of TM5 and TM12. Uracil binding and protonation of Glu241 lead to an occluded state of the transporter. Deprotonation of Glu241 and protonation of His245 (as a result of proton transfer between these side chains) induce the displacement of TM12 from the core domain, exposing the bound uracil to the cytoplasm. Our FEP calculations show that the energy in the uracil bound state with protonated His245 is ∼25.9 kcal/mol lower than that with protonated Glu241, suggesting a favorable proton transfer from Glu241 to His245. Lastly, the release of the proton from protonated His245 in the uracil-bound state causes a large separation of TM12 from the core domain, thus allowing the substrate to move out of its binding site and into the cytoplasm. Based on our simulations, we propose that TM5 and TM12 play the role of extracellular and intracellular gates, respectively, and that proton transfer between Glu241 and His245 regulates the conformational transition between the inward-facing and outward-facing states.

Type a Bax Channels: The Highly Voltage-Gated Form of this Killer Protein

Bax, a pro-apoptotic protein, translocates from the cytosol to the mitochondrial outer membrane (MOM) where it oligomerizes and forms channels. When reconstituted into planar phospholipid membranes, Bax forms two types of channels: Type A and Type B. The former is voltage-gated and is the focus of this presentation. Electrophysiological studies of a single Type A channel show a complex gating pattern: a trimer of conductance decrements each with distinct voltage gating. The results are interpreted in terms of a linear trimer of strongly interacting subunit channels with the middle subunit oriented in the opposite direction from the others. The closing of the first subunit occurred around +70 mV with slow kinetics and steep voltage dependence. The second subunit didn't gate until the first one closed, after which, it closed with n = 13 and V0 ∼ −22 mV. Only with the second subunit closed, did the third subunit can start to gate. It closed with n = 32 and V0 ∼ +25 mV. Note that all the gating events are extremely voltage-dependent probably due to the oligomeric nature of the subunit channel. The restricted gating indicates that the gating domains are restricted by the state of neighboring subunits. Whereas the first and third subunits closed at positive voltage, the second subunit closed at negative indicating an opposite orientation. Adjacent trimeric channels provide opportunities for further interaction. A higher n value (20) was observed for gating of the second subunit in multi-channel membranes. This complex functional behavior provides insights into the properties and interactions of Bax proteins in membranes. These properties likely contribute to the decision-making process leading either to the formation large Bax channels and apoptosis or small Bax channels and cell survival. (supported by NSF grant MCB-1023008)

Role of the Wrist Domain in the Response of the Epithelial Sodium Channel to External Stimuli

The epithelial Na(+) channel (ENaC) is regulated by a variety of external factors that alter channel activity by inducing conformational changes within its large extracellular region that are transmitted to the gate. The wrist domain consists of small linkers connecting the extracellular region to the transmembrane domains, where the channel pore and gate reside. We employed site-directed mutagenesis combined with two-electrode voltage clamp to investigate the role of the wrist domain in channel gating in response to extracellular factors. Channel inhibition by external Na(+) was reduced by selected mutations within the wrist domain of the α subunit, likely reflecting an increase in channel open probability. The most robust changes were observed when Cys was introduced at αPro-138 and αSer-568, sites immediately adjacent to the palm domain. In addition, one of these Cys mutants exhibited an enhanced response to shear stress. In the context of channels that have a low open probability due to retention of an inhibitory tract, the response to external Na(+) was reduced by Cys substitutions at both αPro-138 and αSer-568. We observed a significant correlation between changes in channel inhibition by external Na(+) and the relative response to shear stress for the α subunit mutants that were examined. Mutants that exhibited reduced inhibition by external Na(+) also showed an enhanced response to shear stress. Together, our data suggest that the wrist domain has a role in modulating the channel's response to external stimuli.

Identification of the Substrate Recognition and Transport Pathway in a Eukaryotic Member of the Nucleobase-Ascorbate Transporter (NAT) Family

Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H+ symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.

Metabolic and thermal stimuli control K2P2.1 (TREK-1) through modular sensory and gating domains

K(2P)2.1 (TREK-1) is a polymodal two-pore domain leak potassium channel that responds to external pH, GPCR-mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K(2P)2.1 (TREK-1) intracellular C-terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C-type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore-forming core, we further demonstrate that Ct is the primary heat-sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K(2P)2.1 (TREK-1) in which there is a clear crosstalk between the C-type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature-sensing domains in temperature-sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature-sensitive channels.

Structural, Biochemical, and Functional Characterization of the Cyclic Nucleotide Binding Homology Domain from the Mouse EAG1 Potassium Channel

KCNH channels are voltage-gated potassium channels with important physiological functions. In these channels, a C-terminal cytoplasmic region, known as the cyclic nucleotide binding homology (CNB-homology) domain displays strong sequence similarity to cyclic nucleotide binding (CNB) domains. However, the isolated domain does not bind cyclic nucleotides. Here, we report the X-ray structure of the CNB-homology domain from the mouse EAG1 channel. Through comparison with the recently determined structure of the CNB-homology domain from the zebrafish ELK (eag-like K(+)) channel and the CNB domains from the MlotiK1 and HCN (hyperpolarization-activated cyclic nucleotide-gated) potassium channels, we establish the structural features of CNB-homology domains that explain the low affinity for cyclic nucleotides. Our structure establishes that the "self-liganded" conformation, where two residues of the C-terminus of the domain are bound in an equivalent position to cyclic nucleotides in CNB domains, is a conserved feature of CNB-homology domains. Importantly, we provide biochemical evidence that suggests that there is also an unliganded conformation where the C-terminus of the domain peels away from its bound position. A functional characterization of this unliganded conformation reveals a role of the CNB-homology domain in channel gating.

Gating properties of the P2X2a and P2X2b receptor channels: Experiments and mathematical modeling

Adenosine triphosphate (ATP)-gated P2X2 receptors exhibit two opposite activation-dependent changes, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of calcium and the C-terminal domain in gating, we combined biophysical and mathematical approaches using two splice forms of receptors: the full-size form (P2X2aR) and the shorter form missing 69 residues in the C-terminal domain (P2X2bR). Both receptors developed conductivity for N-methyl-d-glucamine within 2–6 s of ATP application. However, pore dilation was accompanied with a decrease rather than an increase in the total conductance, which temporally coincided with rapid and partial desensitization. During sustained agonist application, receptors continued to desensitize in calcium-independent and calcium-dependent modes. Calcium-independent desensitization was more pronounced in P2X2bR, and calcium-dependent desensitization was more pronounced in P2X2aR. In whole cell recording, we also observed use-dependent facilitation of desensitization of both receptors. Such behavior was accounted for by a 16-state Markov kinetic model describing ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two to three ATP molecules bind and undergo calcium-independent desensitization, causing a decrease in the total conductance, or pore dilation, causing a shift in the reversal potential. In calcium-containing media, receptor desensitization is facilitated and the use-dependent desensitization can be modeled by a calcium-dependent toggle switch. The experiments and the model together provide a rationale for the lack of sustained current growth in dilating P2X2Rs and show that receptors in the dilated state can also desensitize in the presence of calcium.

Highly Conserved Salt Bridge Stabilizes Rigid Signal Patch at Extracellular Loop Critical for Surface Expression of Acid-sensing Ion Channels

Acid-sensing ion channels (ASICs) are non-selective cation channels activated by extracellular acidosis associated with many physiological and pathological conditions. A detailed understanding of the mechanisms that govern cell surface expression of ASICs, therefore, is critical for better understanding of the cell signaling under acidosis conditions. In this study, we examined the role of a highly conserved salt bridge residing at the extracellular loop of rat ASIC3 (Asp(107)-Arg(153)) and human ASIC1a (Asp(107)-Arg(160)) channels. Comprehensive mutagenesis and electrophysiological recordings revealed that the salt bridge is essential for functional expression of ASICs in a pH sensing-independent manner. Surface biotinylation and immunolabeling of an extracellular epitope indicated that mutations, including even minor alterations, at the salt bridge impaired cell surface expression of ASICs. Molecular dynamics simulations, normal mode analysis, and further mutagenesis studies suggested a high stability and structural constrain of the salt bridge, which serves to separate an adjacent structurally rigid signal patch, important for surface expression, from a flexible gating domain. Thus, we provide the first evidence of structural requirement that involves a stabilizing salt bridge and an exposed rigid signal patch at the destined extracellular loop for normal surface expression of ASICs. These findings will allow evaluation of new strategies aimed at preventing excessive excitability and neuronal injury associated with tissue acidosis and ASIC activation.

Differential state-dependent modification of inactivation-deficient Nav1.6 sodium channels by the pyrethroid insecticides S-bioallethrin, tefluthrin and deltamethrin

Pyrethroid insecticides disrupt nerve function by modifying the gating kinetics of transitions between the conducting and nonconducting states of voltage-gated sodium channels. Pyrethroids modify rat Na(v)1.6+β1+β2 channels expressed in Xenopus oocytes in both the resting state and in one or more states that require channel activation by repeated depolarization. The state dependence of modification depends on the pyrethroid examined: deltamethrin modification requires repeated channel activation, tefluthrin modification is significantly enhanced by repeated channel activation, and S-bioallethrin modification is unaffected by repeated activation. Use-dependent modification by deltamethrin and tefluthrin implies that these compounds bind preferentially to open channels. We constructed the rat Na(v)1.6Q3 cDNA, which contained the IFM/QQQ mutation in the inactivation gate domain that prevents fast inactivation and results in a persistently open channel. We expressed Na(v)1.6Q3+β1+β2 sodium channels in Xenopus oocytes and assessed the modification of open channels by pyrethroids by determining the effect of depolarizing pulse length on the normalized conductance of the pyrethroid-induced sodium tail current. Deltamethrin caused little modification of Na(v)1.6Q3 following short (10ms) depolarizations, but prolonged depolarizations (up to 150ms) caused a progressive increase in channel modification measured as an increase in the conductance of the pyrethroid-induced sodium tail current. Modification by tefluthrin was clearly detectable following short depolarizations and was increased by long depolarizations. By contrast modification by S-bioallethrin following short depolarizations was not altered by prolonged depolarization. These studies provide direct evidence for the preferential binding of deltamethrin and tefluthrin (but not S-bioallethrin) to Na(v)1.6Q3 channels in the open state and imply that the pyrethroid receptor of resting and open channels occupies different conformations that exhibit distinct structure-activity relationships.

The Distal C-Terminal Region of the KcsA Potassium Channel Is a pH-Dependent Tetramerization Domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.

Understanding the Kinetics of ATP-Activated P2X2A and P2X2B Receptor Channels using a Markov State Model

ATP-gated P2X2 receptors exhibit two opposite activation-dependent changes during sustained agonist application, pore dilation and pore closing (desensitization), through a process that is incompletely understood. To address this issue and to clarify the roles of Ca2+ and the C-terminal domain in gating, we combined biophysical and mathematical approaches using the full size receptor (labeled P2X2aR) and the splice form missing 69 residues in the C-terminal domain (labeled the P2X2bR). Both forms of the receptor developed conductivity for large organic cations within 2-6 s of ATP application and desensitized in a Ca2+ influx-dependent manner, whereas P2X2bR also desensitized in a Ca2+ influx-independent manner. In whole-cell recording with broken membranes, we also observed use-dependent facilitation of desensitization, reflecting the altered Ca2+ handling by cells. Such behavior was accounted for by a Markov state kinetic model with 12 states describing the ATP binding/unbinding and activation/desensitization. The model assumes that naive receptors open when two ATP molecules bind and slowly dilates to a higher conductance state when a third ATP binds, generating a shift to less negative reversal potential observed experimentally in organic cation-containing medium. The use-dependent desensitization is modeled by a Ca2+-dependent toggle switch, whereas the P2X2bR model also exhibits fast Ca2+-independent desensitization. The model is extended to include memory to previous stimulations that not only explained the decrease in the slope of the IV-curves during −80 to +80 voltage ramps delivered twice per second, but also captured the effect of ATP stimulation when cells were held at positive holding potential.

Role of the KcsA Channel Cytoplasmic Domain in pH-Dependent Gating

The KcsA channel is a representative potassium channel that is activated by changes in pH. Previous studies suggested that the region that senses pH is entirely within its transmembrane segments. However, we recently revealed that the cytoplasmic domain also has an important role, because its conformation was observed to change dramatically in response to pH changes. Here, to investigate the effects of the cytoplasmic domain on pH-dependent gating, we made a chimera mutant channel consisting of the cytoplasmic domain of the KcsA channel and the transmembrane region of the MthK channel. The chimera showed a pH dependency similar to that of KcsA, indicating that the cytoplasmic domain can act as a pH sensor. To identify how this region detects pH, we substituted certain cytoplasmic domain amino acids that are normally negatively charged at pH 7 for neutral ones in the KcsA channels. These mutants opened independently of pH, suggesting that electrostatic charges have a major role in the cytoplasmic domain's ability to sense and respond to pH.

Structure and mechanism of the uracil transporter UraA

The nucleobase/ascorbate transporter (NAT) proteins, also known as nucleobase/cation symporter 2 (NCS2) proteins, are responsible for the uptake of nucleobases in all kingdoms of life and for the transport of vitamin C in mammals. Despite functional characterization of the NAT family members in bacteria, fungi and mammals, detailed structural information remains unavailable. Here we report the crystal structure of a representative NAT protein, the Escherichia coli uracil/H(+) symporter UraA, in complex with uracil at a resolution of 2.8 Å. UraA has a novel structural fold, with 14 transmembrane segments (TMs) divided into two inverted repeats. A pair of antiparallel β-strands is located between TM3 and TM10 and has an important role in structural organization and substrate recognition. The structure is spatially arranged into a core domain and a gate domain. Uracil, located at the interface between the two domains, is coordinated mainly by residues from the core domain. Structural analysis suggests that alternating access of the substrate may be achieved through conformational changes of the gate domain.

Deletion of the Amino-Terminus Uncovers an Inactivated State in Eag1 Channels

Ether-a-go-go (Eag) family channels, which include hErg1, are voltage-gated K+ channels that are important in cardiac and neural function and have a well-documented role in disease. Eag1 channels in particular are found throughout the central nervous system and are crucial regulators of cell cycle and tumor progression. Wild-type Eag1 channels exhibit voltage-gated activation and deactivation, but no apparent inactivation. Here we find that deletion of the entire intracellular amino-terminal domain uncovers an inactivated channel state at depolarizing potentials. We characterized this new inactivated state in inside-out patches excised from Xenopus oocytes expressing the mutant Eag1 channels. Intriguingly, we find that application of the membrane-impermeable cysteine modifying reagent MTSES to the intracellular side of the amino-terminal deletion mutant abolishes this inactivation. We have localized this cysteine-modifying effect to one or more of three cysteine residues in the intracellular carboxy-terminal domain. One of these residues resides in a domain that shares sequence similarity with the cyclic nucleotide binding domain (CNBD) of other channels and enzymes, and the other two reside in the C-linker which connects the CNBD-like region to the S6 transmembrane domain. As the CNBD-like region in does not bind to, and these channels are not regulated by cyclic nucleotides, the role of domain in channel gating is unknown. Our results suggest that the entire amino-terminus, or a fraction thereof, may act as a ligand that binds to the CNBD-like region to prevent inactivation in the wild-type channel.

A Salt Bridge Crucial for Glycine Receptor Function

The glycine receptor (GlyR) chloride channel is a member of the pentameric ligand-gated ion channel (pLGIC) receptor family. These channels mediate fast inhibitory transmission in the nervous system. Recent structural data and a wide body of functional data have underlined the importance of the interface between the ligand-binding domain and the transmembrane domain in channel gating. A number of charged residues at this interface have been shown to form salt bridges critical for channel function in other pLGICs. However, no such interaction has been demonstrated in the GlyR. Mutations at D148 (Cys-loop) and R218 (pre-M1) in the homomeric alpha 1 GlyR can have drastic effects on GlyR function and the single mutants D148C and R218C failed to produce glycine-induced currents in Xenopus oocytes. However, the D148C, R218C double mutant resulted in receptors that produced glycine-gated currents with only a small decrease in glycine sensitivity compared to WT. Importantly, sub-saturating glycine-evoked currents were dramatically increased by the reducing agent dithiothreitol. This suggests that D148C and R218C can form a disulphide bridge, possibly indicating an electrostatic interaction between D148 and R218 that stabilizes the channel closed state. The possibilty of a state-dependent salt bridge between D148 and R218 raised the question how the charges are stabilised in the open state. Interestingly, structural studies have suggested that R218 is flanked by two highly conserved phenylalanine side chains. To test, if these aromatic amino acids interact with R218 electrostically via a cation-pi interaction, we incorporated a series of fluorinated Phe derivatives using the in vivo non-sense suppression method. However, the fluorination-induced changes in the glycine sensitivity were not consistent with a cation-pi interaction. Taken together, these results point towards a crucial salt bridge between D148 and R218 but no electrostatic contributions by the flanking phenylalanines.

Temperature Dependence of Mutant Vanilloid Receptor (tRPV) Channels

Temperature-gated transient receptor potential (TRP) channels exhibit exceedingly large energetic changes in activation. However, the source of energy and its structural basis are not known. Several studies have recently suggested that the pore domain of the channels plays an essential role in temperature gating. In particular, multiple mutations in the outer pore of the TRPV3 channel, which is activated by heat above 30oC, were found to selectively abrogate the heat response of the channel while leaving the response to chemical agonists largely intact. Vanilloid receptors (TRPV) have a membrane topology resembling that of voltage-gated ion channels, and these mutant residues are located at the S6-linker region. The profound and yet selective effects of the region on thermal sensitivity of TRPV3 suggest that the outer pore of the channel may be responsible for heat sensing. In support of the hypothesis, the replacement of a pore turret region in TRPV1 by a glycine-based fragment was also shown to specifically eliminate the heat sensitivity of the channel without compromising its capsaicin response. Alternatively, the “heat sensors” of the channels, if any, may reside elsewhere, while the pore domain is involved in the downsteam pathway of activation such as allosteric coupling between the heat sensors and the gate of the channel. In this study, we will attempt to discern such two mechanisms. We will show the remaining of temperature dependence in the mutant channels and explore mechanisms and the role of the pore domain in temperature gating.

The Role of the Cytoplasimic Domain in pH-Dependent Gating by the KCSA Channel

Ion channels are membrane-spanning proteins that regulate ion flow across the membrane by responding to environmental changes. These changes are sensed by a sensor region, which then conveys the information to the pore region, which induces channel opening or closing. However, the mechanism for this conveyance remains unknown.The KcsA channel is a representative potassium channel activated by protons (changes in pH). It is a tetramer with four homologous subunits, each of which has two transmembrane segments which form a transmembrane pore, an α-helix at the N terminus, and a cytoplasmic domain made of 35 amino acids at the C terminus. Studies have argued the region that senses protons and regulates gating is entirely within the transmembrane segments. However, we have found that the cytoplasmic domain may also be involved in these two functions.Here, we show that the charged amino acids in the cytoplasmic domain, which makes up about half of this domain's amino acids, play an important role in pH-dependent gating. To investigate their effects, we made these amino acids neutral and measured gate activity. The mutant channel could be activated at what is normally inactive pH. This suggests that charges on the cytoplasmic domain generate electrostatic repulsion or attraction within the tetramer and influence KcsA channel activation in response to pH changes.

Activation of the epithelial sodium channel by the metalloprotease meprin β subunit

The Epithelial Na+ Channel (ENaC) is an apical heteromeric channel that mediates Na+ entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH2-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin β (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin β or α/β in Xenopus oocytes increased amiloride-sensitive Na+ currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin β, but not the α subunit. Meprin β promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin β regulates the activity of ENaC in a metalloprotease-dependent fashion.

PIASy-dependent SUMOylation regulates DNA topoisomerase IIalpha activity.

DNA topoisomerase IIα (TopoIIα) is an essential chromosome-associated enzyme with activity implicated in the resolution of tangled DNA at centromeres before anaphase onset. However, the regulatory mechanism of TopoIIα activity is not understood. Here, we show that PIASy-mediated small ubiquitin-like modifier 2/3 (SUMO2/3) modification of TopoIIα strongly inhibits TopoIIα decatenation activity. Using mass spectrometry and biochemical analysis, we demonstrate that TopoIIα is SUMOylated at lysine 660 (Lys660), a residue located in the DNA gate domain, where both DNA cleavage and religation take place. Remarkably, loss of SUMOylation on Lys660 eliminates SUMOylation-dependent inhibition of TopoIIα, which indicates that Lys660 SUMOylation is critical for PIASy-mediated inhibition of TopoIIα activity. Together, our findings provide evidence for the regulation of TopoIIα activity on mitotic chromosomes by SUMOylation. Therefore, we propose a novel mechanism for regulation of centromeric DNA catenation during mitosis by PIASy-mediated SUMOylation of TopoIIα.