Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobility–mass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.One of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N- and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3–9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation—i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patch-clamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13–15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach.     

Properties of Slo1 K+ channels with and without the gating ring

High-conductance Ca²⁺- and voltage-activated K⁺ (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca²⁺ and Mg²⁺ sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca²⁺- and Mg²⁺-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca²⁺ and Mg²⁺ activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.Slo1 channels are expressed in most human tissues and play key roles in many important physiological processes, including smooth muscle contraction, neurotransmitter release, neuronal excitability, hair cell tuning, and action potential termination (1–6). Slo1 channels also are named BK (Big K⁺) or MaxiK channels because of their high single-channel conductance (∼300 pS in 150-mM symmetrical K⁺). Slo1 channels are activated synergistically by both depolarization and intracellular calcium (7–9), linking these two activators in a negative feed-back system to restore negative membrane potential which, in turn, closes voltage-activated Ca²⁺ channels. The dual regulation by voltage and calcium led Hille (10) to predict that BK channels function like the classical Hodgkin–Huxley delayed rectifier channel, except that the range of voltage activation was set by the intracellular Ca²⁺ concentration. The cloning (11) and analysis of the Slo1 channel structure seemed to validate this prediction, in that Slo1 appeared to be modular in its construction, having a core domain containing a voltage sensor controlling a K⁺-selective pore and a long C-terminal tail forming a gating ring structure comprised of four pairs of regulators of the conductance of K⁺ (RCK) domains for sensing and transducing the effect of Ca²⁺ binding to the core.One of the four identical α subunits that assemble to form the Slo1 WT channel (Slo1-WT) is shown in Fig. 1 Top. For the mbr5 cDNA (12) used in this study, the “core” consists of 342 residues including seven transmembrane segments (S0–S6) and the S6–RCK1 linker sequence, which is attached to a long tail of 827 residues. The tail sequence of Slo1-WT is distinct from the cytoplasmic domains of other members of the K⁺ channel extended family. Structure–function studies of the tail have shown the existence of two high-affinity Ca²⁺ binding sites (13, 14) and one low-affinity Mg²⁺ site (14, 15). Modulation of the channel also occurs by additional biological factors, including protons, heme, carbon monoxide, phosphorylation, and oxidation (16–20), all of which may function via their interaction with the tail. Thus, the large tail accommodates a variety of regulatory domains which sense different intracellular factors, leading to pushing or tugging against the core to facilitate or inhibit channel gating. These complicated allosteric interactions between core and tail almost certainly involve several transduction pathways (21–23), all of which alter the properties of the core. Thus, a logical starting point to begin investigating the allosteric interactions would be to understand the baseline properties of the isolated core. However, this approach has been hampered by the inability to express functional cores in the absence of the tail. Previous analysis of truncated expression constructs of Slo1 channels found that their processing stalls in the endoplasmic reticulum (ER), they are not assembled into tetramers, they fail to be exported to the plasma membrane, or they are nonfunctional (24). We now show that core constructs without gating rings can be expressed by leaving a short region required for subunit tetramerization and by appending a small tail domain which facilitates processing and efficient export to the plasma membrane. Thus, we now are able to investigate gating in the absence of a gating ring.Open in a separate windowFig. 1.Slo1 channel constructs used in this study. The Slo1 channel constructs used in this study are based on the mouse mbr5 cDNA (12) and the mouse Shaker family Kv1.4 channel (25). The “Slo1 core and tail” refers to the first 342 and the last 827 amino acid residues. The “Kv1.4 tail” refers to the last 74 amino acid residues of Kv1.4. The different channel constructs are designated as follows: Slo1-WT is Slo1 full-length WT; Slo1C-KvT is a Slo1 core with a 74-residue Kv1.4 tail; Slo1C-Kv-minT is a Slo1 core with a Kv1.4 11-residue mini tail; Slo1C-KvT_NAFQ is a Slo1 core with a 74-residue Kv1.4 tail with NAFQ substituted for KKFR in the tail; Slo1C-KvT R207E is Slo1C-KvT with R207E in S4 in the core.     

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6–8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3₁₀-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4–S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.Voltage-gated sodium (Na_V) channels are responsible for initiation and propagation of action potentials in nerve, muscle, and endocrine cells (1, 2). They are members of the structurally homologous superfamily of voltage-gated ion channel proteins that also includes voltage-gated potassium (K_V), voltage-gated calcium (Ca_V), and cyclic nucleotide-gated (CNG) channels (3). Mammalian Na_V and Ca_V channels consist of four homologous domains (I through IV), each containing six transmembrane segments (S1 through S6) and a membrane-reentrant pore loop between the S5 and S6 segments (1, 3). Segments S1–S4 of the channel form the voltage-sensing domain (VSD), and segments S5 and S6 and the membrane-reentrant pore loop form the pore. The bacterial Na_V channel NaChBac and its relatives consist of tetramers of four identical subunits, which closely resemble one domain of vertebrate Na_V and Ca_V channels, but provide much simpler structures for studying the mechanism of voltage sensing (4, 5). The hallmark feature of the voltage-gated ion channels is the steep voltage dependence of activation, which derives from the voltage-driven outward movement of gating charges in response to the membrane depolarization (6, 7). The S4 transmembrane segment in the VSD has four to seven arginine residues spaced at 3-aa intervals, which serve as gating charges in the voltage-sensing mechanism (8–15). The intracellular S4–S5 linker that connects the VSD to the pore plays a key role in coupling voltage-dependent conformational changes in the VSD to opening and closing of the pore (16). The gating charges are pulled in by the internally negative transmembrane electric field and released to move out on depolarization. Their outward movement must be catalyzed by the voltage sensor to reduce the large thermodynamic barrier to movement of charged amino acid residues across the membrane. The molecular mechanism by which the gating charges are stabilized in the hydrophobic transmembrane environment and the catalytic mechanism through which they are transported across the membrane in response to changes in membrane potential are the subjects of intense research efforts.Progress has been made in determining high-resolution structures of voltage sensors of K_V and Na_V channels in activated states (17–20). However, high-resolution structures of resting and intermediate states of voltage sensors are unknown. The majority of evidence supports a sliding helix model of the voltage-dependent gating in which the gating charge-carrying arginines in S4 are proposed to sequentially form ion pairs with negatively charged residues in S1–S3 segments during activation of the channel (9–11, 21). However, the structural basis for stabilization of the gating charges in the membrane and catalysis of their movement through the hydrophobic membrane environment remain uncertain. Here, we have integrated bioinformatics analysis of Na_V and K_V channel families using the HHPred homology detection server (22–24), high-resolution structural modeling using the Rosetta Membrane (25–27) and Rosetta Symmetry methods (28), the X-ray structures of the K_v1.2-K_v2.1 chimeric channel and NavAb with activated VSDs (19, 20) and the MlotiK1 CNG channel in the resting state (29), and experimental data showing sequential state-dependent interactions between gating charges in S4 and negatively charged residues in S1–S3 (this work and refs. 30–33). Predictions of the resulting voltage-sensing model are confirmed in this work by disulfide-locking studies and mutant cycle analysis of the interactions of hydrophobic residues in the S4 segment. This model reveals structural details of the voltage-dependent conformational changes in the VSD that stabilize and catalyze gating charge movement and are coupled to opening and closing of the intracellular activation gate of the ion-conducting pore.     

Dynamic PIP2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

The S4 segment and the S4–S5 linker of voltage-gated potassium (Kv) channels are crucial for voltage sensing. Previous studies on the Shaker and Kv1.2 channels have shown that phosphatidylinositol-4,5-bisphosphate (PIP₂) exerts opposing effects on Kv channels, up-regulating the current amplitude, while decreasing the voltage sensitivity. Interactions between PIP₂ and the S4 segment or the S4–S5 linker in the closed state have been highlighted to explain the effects of PIP₂ on voltage sensitivity. Here, we show that PIP₂ preferentially interacts with the S4–S5 linker in the open-state KCNQ2 (Kv7.2) channel, whereas it contacts the S2–S3 loop in the closed state. These interactions are different from the PIP₂–Shaker and PIP₂–Kv1.2 interactions. Consistently, PIP₂ exerts different effects on KCNQ2 relative to the Shaker and Kv1.2 channels; PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. Disruption of the interaction of PIP₂ with the S4–S5 linker by a single mutation decreases the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop does not alter voltage sensitivity. These results provide insight into the mechanism of PIP₂ action on KCNQ channels. In the closed state, PIP₂ is anchored at the S2–S3 loop; upon channel activation, PIP₂ interacts with the S4–S5 linker and is involved in channel gating.A series of ion channels, such as inward rectifier K⁺ (Kir) channels, transient receptor potential channels, and voltage-gated channels, are sensitive to the presence of phosphatidylinositol-4,5-bisphosphate (PIP₂) in membranes (1–4). Structural studies on Kir channels (1, 2, 5) demonstrated that PIP₂ directly interacts with the channels. Subsequent studies supported that PIP₂ also interacts directly with voltage-gated potassium (Kv) channels (6–19). Several positive residues that may be critical for PIP₂ activity have been identified (7, 11, 18, 20–24). Previous studies on Kv1.2 and Shaker channels showed that PIP₂ exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18). The S4 segment and the S4–S5 linker of Kv channels are crucial for voltage sensing. The interactions of PIP₂ with the S4 segments and the S4–S5 linkers of the closed-state Shaker and Kv1.2 channels underlie the loss-of-function effect of PIP₂ on voltage sensitivity (7, 18).The KCNQ (Kv7) family of slowly activated outwardly rectifying potassium channels is one of the Kv channel families that are sensitive to the presence of PIP₂ in the membrane. KCNQ channels have been widely studied because of their important biological and pharmacological functions. Retigabine, a first-in-class K⁺ channel opener used for the treatment of epilepsy, adopts a unique mechanism to enhance the activity of KCNQ channels (25). PIP₂ is important for the functions of KCNQ channels. Reduction of PIP₂ affinity caused by congenic mutations of KCNQ channels is associated with long QT syndrome, suggesting critical physiological implications of PIP₂ on KCNQ channels (23, 26). We reported that PIP₂ also alters the pharmacological selectivity of KCNQ potassium channels (6). Zaydman et al. (27) showed that the coupling of voltage sensing and pore opening in the KCNQ1 channel requires PIP₂ and suggested there is a PIP₂ interaction site at the interface between the voltage-sensing domain (VSD) and the central pore domain (PD). However, the effects and interactions of PIP₂ on KCNQ channels are not well understood.Here, by combining molecular dynamics (MD) simulations, mutagenesis, and electrophysiological determinations, we observed that the effects and interactions of PIP₂ on KCNQ2 are different relative to the Shaker and Kv1.2 channels. PIP₂ up-regulates both the current amplitude and voltage sensitivity of the KCNQ2 channel. PIP₂ preferentially interacts with the S4–S5 linker of the open-state KCNQ2 channel and does not interact with the S4 segment or S4-S5 linker of the closed state. In the closed state, PIP₂ only interacts with the S2–S3 loop. Furthermore, our electrophysiological experiments suggest that disruption of the interaction of PIP₂ with the S4–S5 linker may decrease the voltage sensitivity and current amplitude, whereas disruption of the interaction with the S2–S3 loop only alters the current amplitude of the channel. These results provide insights into the mechanism of PIP₂ action on Kv channels.     

Dynamics transitions at the outer vestibule of the KcsA potassium channel during gating

In K⁺ channels, the selectivity filter, pore helix, and outer vestibule play a crucial role in gating mechanisms. The outer vestibule is an important structurally extended region of KcsA in which toxins, blockers, and metal ions bind and modulate the gating behavior of K⁺ channels. Despite its functional significance, the gating-related structural dynamics at the outer vestibule are not well understood. Under steady-state conditions, inactivating WT and noninactivating E71A KcsA stabilize the nonconductive and conductive filter conformations upon opening the activation gate. Site-directed fluorescence polarization of 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labeled outer vestibule residues shows that the outer vestibule of open/conductive conformation is highly dynamic compared with the motional restriction experienced by the outer vestibule during inactivation gating. A wavelength-selective fluorescence approach shows a change in hydration dynamics in inactivated and noninactivated conformations, and supports a possible role of restricted/bound water molecules in C-type inactivation gating. Using a unique restrained ensemble simulation method, along with distance measurements by EPR, we show that, on average, the outer vestibule undergoes a modest backbone conformational change during its transition to various functional states, although the structural dynamics of the outer vestibule are significantly altered during activation and inactivation gating. Taken together, our results support the role of a hydrogen bond network behind the selectivity filter, side-chain conformational dynamics, and water molecules in the gating mechanisms of K⁺ channels.The functional behavior of K⁺ channels is defined by a series of structural rearrangements associated with the processes of activation and inactivation gating (1–6). In response to a prolonged stimulus and in the absence of an N-terminal inactivating particle, most K⁺ channels become nonconductive through a process known as C-type inactivation (7). This C-type inactivation is crucial in controlling the firing patterns in excitable cells and is fundamental in determining the length and frequency of the cardiac action potential (8). C-type inactivation is inhibited by high extracellular K⁺ (9, 10), and the blocker tetraethylammonium (TEA) (11) can also be slowed down in the presence of permeant ions with a long residence time in the selectivity filter (Rb⁺, Cs⁺, and NH⁴⁺) (10).The prokaryotic pH-gated K⁺ channel KcsA shares most of the mechanistic properties of C-type inactivation in voltage-dependent K⁺ channels (5, 6, 12–16). Recent crystal structures of open/inactivated KcsA reveal that there is a remarkable correlation between the degree of opening at the activation gate and the conformation and ion occupancy of the selectivity filter (5). In KcsA, the selectivity filter is stabilized by a hydrogen bond network, with key interactions between residues Glu71, Asp80, and Trp67 and a bound water molecule (17). Disrupting this hydrogen bond network favors the conductive conformation of the selectivity filter (12, 13, 15).Early electrophysiological experiments have suggested that the outer vestibule (around T449 residue in Shaker and Y82 residue in KcsA) undergoes significant conformational rearrangement during C-type inactivation gating (16, 18, 19). However, comparison of the WT KcsA crystal structure, where the filter is in its conductive conformation, with either the structure obtained with low K⁺ (collapsed filter) (17) or the crystal structure of open-inactivated KcsA with maximum opening (inactivated filter) (5) does not show major conformational changes in the outer vestibule that would explain these results (Fig. 1A). We have suggested that this apparent discrepancy can be understood if we take into consideration the potential differences in the dynamic behavior of the outer vestibule changes as the K⁺ channel undergoes its gating cycle (16).Open in a separate windowFig. 1.Comparison of outer vestibule conformation in KcsA structures with conductive and collapsed/inactivated filters. (A) High-K⁺ KcsA structure [Protein Data Bank (PDB) ID code 1K4C; yellow] is compared with a low-K⁺ KcsA structure (PDB ID code 1K4D; blue) in the closed state (Left) and open/inactivated conformation (PDB ID code 3F5W; green) (Right). The outer vestibule residues are depicted as red spheres, and relevant residues are labeled. (B) Schematic representation of typical macroscopic currents elicited by pH-jump experiments in WT (inactivating) and E71A (noninactivating) KcsA channels at a depolarizing membrane potential is shown. Conditions that stabilize the closed, open/inactivated, and open/conductive conformations at the steady state are indicated with a black circle. (C) Effect of opening the lower gate on the mobility of spin-labeled outer vestibule residues in palmitoyloleoylphosphatidyl choline/palmitoyloleoylphosphatidyl glycerol (POPC/POPG) (3:1, moles/moles) reconstituted WT (Left) and noninactivating mutant E71A (Right) backgrounds for the closed (pH 7, red) and open (pH 4, black) states of KcsA, as determined by continuous wave (CW) EPR. The spectra shown are amplitude-normalized. Details are provided in SI Materials and Methods.We have probed the gating-induced structural dynamics at the outer vestibule of KcsA using site-directed fluorescence and site-directed spin labeling and pulsed EPR approaches in combination with a recently developed computational method, restrained ensemble (RE) simulations. RE simulation was used to constrain the outer vestibule using experimentally derived distance histograms in different functional states (closed, open/inactivated, and open/conductive) and to monitor the extent of backbone conformational changes during gating. To this end, we took advantage of our ability to stabilize both the open/conductive (E71A mutant) and the open/inactivated (WT) conformations of KcsA upon opening the activation gate under steady-state conditions (Fig. 1B).Our data show that the outer vestibule in the open/conductive conformation is highly dynamic. In addition, the red edge excitation shift (REES) points to a change in hydration dynamics between conductive and nonconductive outer vestibule conformations, suggesting a role of restricted water molecules in C-type inactivation gating. We suggest that, on average, the backbone conformation of the outer vestibule does not change significantly between different functional states but that local dynamics change significantly, underlining the importance of the hydrogen bond network behind the selectivity filter and the microscopic observables (e.g., dynamics of hydration) in K⁺ channel gating and C-type inactivation.     

Structural basis for gating mechanisms of a eukaryotic P-glycoprotein homolog

P-glycoprotein is an ATP-binding cassette multidrug transporter that actively transports chemically diverse substrates across the lipid bilayer. The precise molecular mechanism underlying transport is not fully understood. Here, we present crystal structures of a eukaryotic P-glycoprotein homolog, CmABCB1 from Cyanidioschyzon merolae, in two forms: unbound at 2.6-Å resolution and bound to a unique allosteric inhibitor at 2.4-Å resolution. The inhibitor clamps the transmembrane helices from the outside, fixing the CmABCB1 structure in an inward-open conformation similar to the unbound structure, confirming that an outward-opening motion is required for ATP hydrolysis cycle. These structures, along with site-directed mutagenesis and transporter activity measurements, reveal the detailed architecture of the transporter, including a gate that opens to extracellular side and two gates that open to intramembranous region and the cytosolic side. We propose that the motion of the nucleotide-binding domain drives those gating apparatuses via two short intracellular helices, IH1 and IH2, and two transmembrane helices, TM2 and TM5.Multidrug transporters of the ATP-binding cassette (ABC) superfamily, such as P-glycoprotein (P-gp; MDR1; ABCB1), MRP1 (ABCC1), and ABCG2 (BCRP), transport a large number of structurally unrelated compounds with molecular weights ranging up to several thousand Daltons (1, 2). These transporters not only play important roles in normal physiology by protecting tissues from various toxic xenobiotics and endogenous metabolites but also contribute to multidrug resistance (MDR) in tumors, a major obstacle to effective chemotherapeutic treatment (1, 3–7). Their functional forms consist of a minimum of four core domains: two transmembrane domains (TMDs) that create the translocation pathway for substrates and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP to power the transport process (8, 9). These four domains can exist either as two separate polypeptides (half-size) or fused together in a single large polypeptide with an internal duplication (full-size). The crystal structures of mouse and nematode P-gps, as well as their bacterial homologs (10–14), have been determined, and they have provided important insights into the relationships between protein structure and the functional and biochemical characteristics of P-gp. However, the detailed architecture of the TMD machinery and the gating mechanism during the transition between the inward- and outward-open states are poorly understood.Here, we report the structures of a eukaryotic P-gp homolog, unlocked (at 2.6-Å resolution) and locked allosterically with a tailor-made peptide at 2.4-Å resolution. Although CmABCB1 is not a full-length ABC transporter but a half-sized ABC transporter adopting a homodimeric architecture, CmABCB1 showed quite similar functional properties to those of human P-gp (hP-gp). Based on these structures, we propose mechanisms by which the intramembranous entrance gate can take up various substrates from the inner leaflet of the membrane bilayer. Although the entrance and exit gates are assembled from distinct transmembrane helices, and their gating motions are in opposite directions, the mechanisms powered by the dimerization motions of the NBDs enable one gate to close while the other gate simultaneously opens with the aid of two short intracellular helices and two transmembrane helices, which act as a lever arm. This mechanism is totally different from that of solute carrier (SLC) transporters (15–19) and ABC importers (20), in which the intra- and extracellular gating apparatuses are constructed from the same transmembrane helices. Furthermore, a part of the intramembranous entrance gate has another function gating the pathway from the inside of the transporter to the cytosol. The mode of action of the novel inhibitor, which disables the diverging outward motions of the TM helices by clamping them from the outside of the transporter, supports our proposed gating mechanism.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Understanding the mechanism of proteasome 20S core particle gating

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments.The 20S proteasome core particle (20S CP) is responsible for most of the nonlysosomal protein degradation in the cell, thereby ensuring protein homeostasis (1–4). Regulated protein hydrolysis is critical for cell viability through removal of potentially toxic misfolded or otherwise damaged proteins before they accumulate to levels leading to aggregation. This important molecular machine also plays a critical role in regulating the cell cycle by controlling the concentrations of key proteins in a time-dependent manner and in the immune response by producing antigenic peptides. Not surprisingly, it has emerged as a major drug target in the fight against certain types of cancer (5).To ensure that the proteasome does not inadvertently proteolyze proteins in the cell, the 20S CP assumes a barrel-like architecture composed of four stacked heptameric rings (6, 7), as shown in Fig. 1A. The two outer rings are composed of α-subunits (α₇), with catalytic sites localized to the inner β₇-rings and facing the lumen of the barrel. Access to the β-subunit catalytic sites is blocked by the first 12 amino acids of the α-subunit, which form a gate that occludes entry of substrate through the narrow pore in the α-ring, called the α-annulus, Fig. 1B (1, 6, 8). Substrate entry into the catalytic chamber of the proteasome is controlled further through the binding of regulatory particles (RPs) to each of the barrel ends. These include the 19S RP that recognizes ubiquitin-tagged proteins that are earmarked for degradation (3) and other protein systems that function in an ATP-independent manner, including proteasome activator 200 kDa (PA200) and PA28 (eukaryotic 20S CPs), as well as AAA protein complexes such as PAN and VAT (archaeal 20S CPs) (1, 7, 9–12).Open in a separate windowFig. 1.Architecture of the N-terminal gating residues of the T. acidophilum proteasome. (A) Cross-sectional side view of the 20S CP proteasome (α₇β₇β₇α₇) showing the barrel-like structure of the CP [Protein Data Bank (PDB) ID code 1YA7] (52). Two subunits have been removed from each of the rings so that the antechambers (α₇-β₇) and catalytic chamber (β₇-β₇) are visible. (B) α₇-ring highlighting gating residues in the in (closed) and out (open) states. The α-annulus is shown in a space-filling representation, with helix H0 labeled for reference (PDB ID code 2KU1) (17). (C) Stabilizing interactions between M1 and V129 in the in state, as established from NOE experiments recorded on a 20S CP sample. (D) The reverse-turn interaction formed between conserved residues, Y8, D9, P17, and Y26, from adjacent α-subunits (teal and blue) in the out state (52).The role of the ubiquitin-dependent protein degradation pathway, and hence the significance of the 19S–20S proteasome complex in eukaryotic organisms, is well established (3, 4, 10). Recent studies further clarified that the naked 20S CP also may play a significant role in in vivo protein lysis, particularly that involving intrinsically disordered proteins or folded proteins with regions of significant disorder (13, 14). It is estimated that up to 20% of all cellular proteins may serve as substrates for the 20S CP proteasome, with the gating residues assuming a critical role in controlling proteolysis (15). The importance of gating is emphasized further by studies of yeast cells with gateless proteasomes, showing very low survival rates under conditions of prolonged starvation, in contrast to their counterparts with wild-type 20S CPs (16).The significance of gating to proteasome function has led us to study how gating termini control access to the 20S CP, by using proteasomes from the archaea Thermoplasma acidophilum as a model system. We previously showed by solution NMR spectroscopy that although the gating residues of the T. acidophilum 20S CP are disordered, they can assume distinct conformations in which the gate is either localized outside the lumen and above the annulus (the “out” position) or inside the lumen (the “in” position) (Fig. 1B) (17). These in and out termini exchange stochastically on the seconds timescale, with a population of two in and five out, on average, for the wild-type protein. Gating termini that penetrate the α-annulus decrease the surface area available for substrate entry, leading to decreased rates of hydrolysis (17).Paramagnetic relaxation enhancement NMR studies establish that in the in state, there are large numbers of transient interactions between gating residues and the lumen of the antechamber (17). Furthermore, methyl-NOESY spectra show a clear contact between M1 of the gate and V129 of the antechamber (Fig. 1C). In addition, X-ray studies indicate that the out state is stabilized by a reverse-turn interaction between four highly conserved residues (Y8, D9, P17, and Y26) in pairs of adjacent α-subunits (Fig. 1D) (18). Thus, although structural details about the end points of the gate exchange reaction have been elucidated and the importance of the exchange between multiple gating conformations for regulating substrate entry into the archaeal 20S CP is clear, the mechanism of gate exchange remains to be discovered. For example, is this process driven through interactions with solvent, or are there significant internal (intraproteasome) frictional forces in play? A second question relating to the mechanism of gating concerns the average size of the structural units that take part in the transition leading to the interconversion between in and out states. Does the exchange process occur in a series of only a few steps involving large pieces of the protein, or are many smaller steps and correspondingly smaller protein segments involved? Answers to these basic questions have important implications for understanding how the rate of gate exchange might vary in vivo as a function of cellular protein concentration, for example.To address these mechanistic aspects of proteasome gating, we have used magnetization exchange NMR spectroscopy to measure the rate of gate exchange as a function of viscogen concentration, using a pair of viscogens of different sizes. The rate vs. viscogen profiles so obtained have been analyzed using the Kramers rate equation valid in the high-friction limit (19, 20), establishing that water plays a critical role in driving gate exchange and that the process proceeds through very small step sizes, smaller than an amino acid. Rates of proteasome gating also have been measured in an archeal lysate from the thermophile Thermus thermophilus. Despite the large number of potential proteasome interacting partners, exchange kinetics are little changed and populations of in and out states remain unaltered relative to buffer. Measured viscosity values as a function of probe size in cellular lysate have been used to construct a molecular ruler for estimating how rates of reactions quantified in solutions of buffer, in general, translate to in-cell. For processes driven by solvent and occurring via small protein segments, the ruler predicts only small rate decreases in lysate so long as it is inert (≤30% for a lysate protein concentration of 100 g/L), consistent with the changes in kinetics observed here for α₇ gating.     

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.Ion conduction through the pore domain of cation channels is regulated by two gates: an inner gate at the bundle crossing of the pore-lining transmembrane helices and an outer gate located at the selectivity filter (Fig. 1 B and C). These two gates are functionally coupled as demonstrated by C-type inactivation, in which channel opening triggers loss of conduction at the selectivity filter (1–4). A structural model for C-type inactivation has been developed for KcsA, with selectivity filter collapse occurring upon channel opening (4–10). In the reverse pathway, inactivation of the selectivity filter has been linked to changes at the inner gate (5–14). However, flux-dependent inactivation occurs in Na⁺ and Ca²⁺ channels as well and would likely require a structurally different mechanism to explain coupling between the selectivity filter and inner gate (7, 13–18).Open in a separate windowFig. 1.Crystal structures of the nonselective cation channel NaK and the potassium-selective NaK2K mutant show structural changes restricted to the area of the selectivity filter. Alignment of the WT NaK (gray; PDB 3E8H) and NaK2K (light blue; PDB 3OUF) selectivity filters shows a KcsA-like four-ion-binding-site selectivity filter is created by the NaK2K mutations (D66Y and N68D) (A), but no structural changes occur outside the vicinity of the selectivity filter (B). (C) Full-length NaK (green; PDB 2AHZ) represents a closed conformation. Alignment of this structure with NaK (gray) highlights the changes in the M2 hinge (arrow), hydrophobic cluster (residues F24, F28, and F94 shown as sticks), and constriction point (arrow; residue Q103 shown as sticks) upon channel opening. Two (A) or three monomers (B and C) from the tetramer are shown for clarity.This study provides experimental evidence of structural and dynamic coupling between the inner gate and selectivity filter in the NaK channel, a nonselective cation channel from Bacillus cereus (19). These results were entirely unexpected given the available high-resolution crystal structures (20, 21). The NaK channel has the same basic pore architecture as K⁺ channels (Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence (₆₃TVGDGN₆₈) and structure (19–23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21, 24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence (₆₃TVGYGD₆₈; Fig. 1 A and B), leading to K⁺ selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21, 23). This K⁺-selective mutant of NaK is called NaK2K. Outside of the immediate vicinity of the two mutations in the selectivity filter, high-resolution crystal structures of NaK and NaK2K are essentially identical (Fig. 1B) with an all-atom rmsd of only 0.24 Å.NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating because there is no evidence for any collapse or structural change in the selectivity filter. The NaK selectivity filter structure is identical in Na⁺ or K⁺ (22) and even in low-ion conditions (25), consistent with its nonselective behavior. Even the selective NaK2K filter appears structurally stable in all available crystal structures (25). Here we use NMR spectroscopy to study bicelle-solubilized NaK. Surprisingly, we find significant differences in the NMR spectra of NaK and NaK2K that extend throughout the protein and are not localized to the selectivity filter region. This, combined with NMR dynamics studies of NaK, suggests a dynamic pathway for transmembrane coupling between the inner gate and selectivity filter of NaK.     

SLO3 auxiliary subunit LRRC52 controls gating of sperm KSPER currents and is critical for normal fertility

Following entry into the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that results in competence to fertilize ova. Associated with capacitation is an increase in membrane conductance to both Ca²⁺ and K⁺, leading to an elevation in cytosolic Ca²⁺ critical for activation of hyperactivated swimming motility. In mice, the Ca²⁺ conductance (alkalization-activated Ca²⁺-permeable sperm channel, CATSPER) arises from an ensemble of CATSPER subunits, whereas the K⁺ conductance (sperm pH-regulated K⁺ current, KSPER) arises from a pore-forming ion channel subunit encoded by the slo3 gene (SLO3) subunit. In the mouse, both CATSPER and KSPER are activated by cytosolic alkalization and a concerted activation of CATSPER and KSPER is likely a common facet of capacitation-associated increases in Ca²⁺ and K⁺ conductance among various mammalian species. The properties of heterologously expressed mouse SLO3 channels differ from native mouse KSPER current. Recently, a potential KSPER auxiliary subunit, leucine-rich-repeat-containing protein 52 (LRRC52), was identified in mouse sperm and shown to shift gating of SLO3 to be more equivalent to native KSPER. Here, we show that genetic KO of LRRC52 results in mice with severely impaired fertility. Activation of KSPER current in sperm lacking LRRC52 requires more positive voltages and higher pH than for WT KSPER. These results establish a critical role of LRRC52 in KSPER channels and demonstrate that loss of a non-pore-forming auxiliary subunit results in severe fertility impairment. Furthermore, through analysis of several genotypes that influence KSPER current properties we show that in vitro fertilization competence correlates with the net KSPER conductance available for activation under physiological conditions.Upon entry into the female reproductive tract, mammalian sperm undergo a sequence of maturational steps, collectively termed capacitation, to become competent to fertilize an egg (1, 2). Two important components of this process, thought to be shared among mammalian species, are cytosolic alkalization (3–5) and then an associated increase in cytosolic Ca²⁺ (6, 7). These events are coupled with changes in ionic fluxes in sperm membrane. Over the past 10 y, the application of patch-clamp recording to individual sperm (8) has allowed identification of ionic currents that respond to alkalization and/or Ca²⁺ (9–12). In mouse sperm, alkalization leads to activation of two sperm-specific channels, the Ca²⁺-permeable CATSPER channel (8–10, 13) and the K⁺-permeable KSPER K⁺ channel (14, 15). KSPER and CATSPER currents are also present in human sperm (16–18), although intriguingly there seem to be differences in regulation of each channel type between mice and humans (11, 12, 17).Despite the species-specific differences in the details of their regulation, KSPER and CATSPER are of central importance in sperm function and fertility in both humans and mice. In mouse sperm, KSPER and CATSPER together account for all patch-clamp measurable cation current activated by voltage and alkalization (19) and are thought to act in concert to mediate the changes in membrane cation conductance and Ca²⁺ influx that occur during the onset of capacitation (14, 20). The critical role of both KSPER and CATSPER in the mouse has been established by demonstration that genetic KO of the pore-forming subunits of each channel [KSPER (15, 21) and CATSPER (22–26)] results in infertility and the CATSPER auxiliary δ subunit is also required for fertility (27).For mouse KSPER, the pore-forming subunit is termed SLO3, encoded by the kcnu1 (slo3) gene (28). SLO3, which is exclusively expressed in testis (15, 28), is a homolog of the Ca²⁺- and voltage-activated, large-conductance (BK)-type K⁺ channel (29) and heterologously expressed SLO3 results in voltage- and alkalization-activated K⁺ currents (28). However, heterologously expressed mouse SLO3 channels differ from mouse KSPER current in important ways. Specifically, whereas pH 7 substantially activates KSPER in mouse sperm at membrane potentials (V_m) between −10 and −50 mV, activation of SLO3 channels at pH 7 is scarcely observed even at an activation potential of +100 mV (30, 31). This discrepancy raised the possibility that an additional regulatory partner of SLO3 may be present in mouse sperm. Guided by the discovery of a new type of auxiliary subunit of the BK channel (32), we recently showed that a related subunit, LRRC52 (leucine-rich-repeat-containing protein 52) is selectively expressed in sperm (33). When LRRC52 is coexpressed with SLO3, the gating range of the resulting channels at a given pH is more like that of KSPER in mouse sperm. LRRC52 protein has also been shown to be present in human sperm and, when coexpressed with human SLO3, LRRC52 results in currents with properties similar to those of human KSPER (12). To evaluate the importance of the LRRC52 subunit, we have now generated lrrc52^−/− (LRRC52 KO) mice. LRRC52 KO mice have a severe fertility deficit that is associated with a shift to more positive potentials in the KSPER current activation range in the LRRC52 KO sperm. Furthermore, measurement of the net KSPER current in sperm from two other genotypes, slo3^+/− and a slo3-eGFP, suggests that sperm reproductive capacity strongly depends on the KSPER conductance available over physiological potentials. These results confirm that LRRC52 is a critical component of the KSPER channel complex and is essential for male fertility.     

Functional evolution of Erg potassium channel gating reveals an ancient origin for IKr

Mammalian Ether-a-go-go related gene (Erg) family voltage-gated K⁺ channels possess an unusual gating phenotype that specializes them for a role in delayed repolarization. Mammalian Erg currents rectify during depolarization due to rapid, voltage-dependent inactivation, but rebound during repolarization due to a combination of rapid recovery from inactivation and slow deactivation. This is exemplified by the mammalian Erg1 channel, which is responsible for I_Kr, a current that repolarizes cardiac action potential plateaus. The Drosophila Erg channel does not inactivate and closes rapidly upon repolarization. The dramatically different properties observed in mammalian and Drosophila Erg homologs bring into question the evolutionary origins of distinct Erg K⁺ channel functions. Erg channels are highly conserved in eumetazoans and first evolved in a common ancestor of the placozoans, cnidarians, and bilaterians. To address the ancestral function of Erg channels, we identified and characterized Erg channel paralogs in the sea anemone Nematostella vectensis. N. vectensis Erg1 (NvErg1) is highly conserved with respect to bilaterian homologs and shares the I_Kr-like gating phenotype with mammalian Erg channels. Thus, the I_Kr phenotype predates the divergence of cnidarians and bilaterians. NvErg4 and Caenorhabditis elegans Erg (unc-103) share the divergent Drosophila Erg gating phenotype. Phylogenetic and sequence analysis surprisingly indicates that this alternate gating phenotype arose independently in protosomes and cnidarians. Conversion from an ancestral I_Kr-like gating phenotype to a Drosophila Erg-like phenotype correlates with loss of the cytoplasmic Ether-a-go-go domain. This domain is required for slow deactivation in mammalian Erg1 channels, and thus its loss may partially explain the change in gating phenotype.Voltage-gated ion channel families are highly conserved across the Eumetazoa (cnidarians and bilaterians) (1, 2). Vertebrates recently expanded the number of ion channel genes within each of the conserved families because of vertebrate-specific gene duplications. Additionally, phylogenetically restricted duplications of ion channel genes appear common throughout the Eumetazoa (1, 3–5). Thus, there is little 1:1 gene orthology between the eumetazoan phyla (1). However, numerous studies show extremely high functional conservation, including family-specific gating properties. For example, Shaker-related voltage-gated K⁺ channels first cloned in Drosophila show a high fidelity of gating phenotype to their mammalian counterparts (6). Subsequent studies have shown this functional conservation extends to cnidarians (4, 7–10), which separated from bilaterians near the base of the eumetazoan tree over 500 Mya (11). One exception to this pattern of high conservation is the Ether-a-go-go related gene (Erg) family (or Kv11) of voltage-gated K⁺ channels. The three mammalian Erg orthologs show striking gating differences compared with Drosophila Erg (seizure, DmErg).The mammalian Erg gating phenotype is typified by human Erg1 (HsErg1), which underlies I_Kr, a K⁺ current that repolarizes the late plateau phase of ventricular action potentials (12, 13). HsErg1 loss-of-function mutations prolong the QT interval in ECG recordings, indicating impaired action potential repolarization (14). Several key gating features adapt Erg1 for ventricular action potential plateau repolarization. First, Erg1 channels inactivate rapidly in response to depolarization (Fig. 1 A–C). Second, recovery from inactivation through the open state is extremely rapid (Fig. 1B), whereas channel deactivation is slow (Fig. 1D); the combination produces a jump in Erg1 current in response to repolarization (15). The net effect is that peak Erg1 current flow is delayed and specifically accelerates cardiac action potential plateau repolarization (15), and the length of the plateau is dependent on Erg1 current density (16). The physiological role of mammalian Erg2 and Erg3 channels has not been extensively characterized, but they share an I_Kr-like gating phenotype (17).Open in a separate windowFig. 1.Comparison of HsErg1 and DmErg gating phenotypes. (A) Families of outward currents recorded from Xenopus oocytes expressing HsErg1 (Left) and DmErg + DAO (Right) in response to depolarizations (Inset). Scale bars indicate time and current amplitude. Currents elicited by a step to +60 mV are highlighted, and arrows indicate (1) rectification of HsErg1 during depolarization by inactivation, (2) rebound in HsErg1 current in response to repolarization due to rapid recovery and slow deactivation, and (3) rapid DmErg deactivation. (B) Comparison of HsErg1 (black) and DmErg (red) currents during a protocol in which channels were first activated by a 1 s step to +60 mV, returned to –100 mV for 10 ms, and then returned to +60 mV. Currents are normalized in peak amplitude for comparison. HsErg1 is inactivated at the end of the first depolarization, recovers to the open state at −100 mV, and inactivates rapidly from a high peak during the second pulse. DmErg1 remains active throughout the first +60 mV pulse, closes at –100 mV, and reactivates during the second +60 mV pulse. (C) Peak HsErg1 current during an initial depolarization (* in B) normalized to peak current after recovery from inactivation (# in B): inactivation reduces the HsErg1 current >20-fold during the first step. Data show mean ± SEM, n = 6 cells. (D) Time constant of deactivation (Tau_DEACT) measured from tail currents recorded at the indicated voltages for HsErg1 (black) and DmErg (red). Data show mean ± SEM, n = 6–7 cells. (E) Normalized GV curves for HsErg1 and DmErg fit with a single Boltzmann distribution (parameters in SI Methods. Scale bar indicates that time and current amplitudes have been normalized.In contrast, DmErg does not inactivate during depolarization (Fig. 1 A and B) and deactivates rapidly upon repolarization (Fig. 1D) (18). The voltage-activation curve (GV) of DmErg is shifted to hyperpolarized potentials, suggesting influence on subthreshold excitability (Fig. 1E). Modeled HsErg1 and DmErg responses to a crude plateau action potential waveform (Fig. 1F and Fig. S1) point to distinct physiological roles. HsErg1 current is attenuated during the plateau by inactivation and rebounds sharply as the plateau decays. These features allow HsErg1 to accelerate late repolarization without blocking the plateau itself (15). Peak DmErg current flows during the plateau, and the current decays rapidly during repolarization. DmErg would therefore directly combat plateau formation. Loss of HsErg1 inactivation in humans indeed leads to a shortened QT interval based on premature action potential repolarization (16). The specific contribution of DmErg to firing patterns in native cells is unknown, but its gating features are consistent with regulation of subthreshold excitability or rapid action potential repolarization. Temperature-sensitive mutations in the seizure locus that encodes DmErg cause bursts of uncoordinated motor output (19) suggestive of changes in subthreshold excitability. The Caenorhabditis elegans Erg ortholog (CeErg, encoded by unc-103) has not been functionally expressed, but genetic analysis demonstrates that it regulates the excitation threshold of vulva muscles in females and protractor muscles in males (20–23).The Erg, Ether-a-go-go (Eag), and Elk gene families comprise the EAG superfamily of voltage-gated K⁺ channels. These gene families are highly conserved in eumetazoan genomes, and Eag channels display a high functional conservation in the bilaterians. Given the distinct gating phenotypes of the Erg genes in Drosophila and mammals, we decided to explore the functional evolution of the Erg gene family to determine the origins of the distinct I_Kr-like and DmErg gating phenotypes in the Erg gene family. We functionally characterized CeErg and Erg paralogs from the starlet sea anemone Nematostella vectensis. We examined CeErg to determine whether the DmErg gating phenotype was present in multiple protostome invertebrate phyla. We reasoned that comparison of bilaterian and Nematostella Erg channels would provide insight into ancestral Erg gating phenotypes present before the cnidarian/bilaterian divergence. Functional and phylogenetic analysis presented here supports an I_Kr-like phenotype as the ancestral gating pattern. An alternate DmErg-like gating phenotype has emerged independently at least twice during metazoan evolution (once in cnidarians and at least once in protostomes) and correlates with loss of the cytoplasmic eag gating domain.     

Understanding the kinetic mechanism of RNA single base pair formation

RNA functions are intrinsically tied to folding kinetics. The most elementary step in RNA folding is the closing and opening of a base pair. Understanding this elementary rate process is the basis for RNA folding kinetics studies. Previous studies mostly focused on the unfolding of base pairs. Here, based on a hybrid approach, we investigate the folding process at level of single base pairing/stacking. The study, which integrates molecular dynamics simulation, kinetic Monte Carlo simulation, and master equation methods, uncovers two alternative dominant pathways: Starting from the unfolded state, the nucleotide backbone first folds to the native conformation, followed by subsequent adjustment of the base conformation. During the base conformational rearrangement, the backbone either retains the native conformation or switches to nonnative conformations in order to lower the kinetic barrier for base rearrangement. The method enables quantification of kinetic partitioning among the different pathways. Moreover, the simulation reveals several intriguing ion binding/dissociation signatures for the conformational changes. Our approach may be useful for developing a base pair opening/closing rate model.RNAs perform critical cellular functions at the level of gene expression and regulation (1–4). RNA functions are determined not only by RNA structure or structure motifs [e.g., tetraloop hairpins (5, 6)] but also by conformational distributions and dynamics and kinetics of conformational changes. For example, riboswitches can adopt different conformations in response to specific conditions of the cellular environment (7, 8). Understanding the kinetics, such as the rate and pathways for the conformational changes, is critical for deciphering the mechanism of RNA function (9–19). Extensive experimental and theoretical studies on RNA folding kinetics have provided significant insights into the kinetic mechanism of RNA functions (19–36). However, due to the complexity of the RNA folding energy landscape (37–46) and the limitations of experimental tools (47–55), many fundamental problems, including single base flipping and base pair formation and fraying, remain unresolved. These unsolved fundamental problems have hampered our ability to resolve other important issues, such as RNA hairpin and larger structure folding kinetics. Several key questions remain unanswered, such as whether the hairpin folding is rate-limited by the conformational search of the native base pairs, whose formation leads to fast downhill folding of the whole structure, or by the breaking of misfolded base pairs before refolding to the native structure (18, 19, 54–73).Motivated by the need to understand the basic steps of nucleic acids folding, Hagan et al. (74) performed forty-three 200-ps unfolding trajectories at 400 K and identified both on- and off-pathway intermediates and two dominant unfolding pathways for a terminal C-G base pair in a DNA duplex. In one of the pathways, base pairing and stacking interactions are broken concomitantly, whereas in the other pathway, base stacking is broken after base pairing is disrupted. Furthermore, the unfolding requires that the Cyt diffuse away from the pairing Gua to a distance such that the C-G hydrogen bond cannot reform easily. More recently, Colizzi and Bussi (75) performed molecular dynamics (MD) pulling simulations for an RNA duplex and construct free energy landscape from the pulling simulation. The simulation showed that the base pair opening reaction starts with the unbinding of the 5′-base, followed by the unbinding of the 3′-base (i.e., the 5′-base is less stable than the 3′-base). These previous unfolding simulations offered significant insights into the pathways and transition states. However, as shown below, several important issues remain.One intriguing problem is the rate model for base pairing. There are currently three main types of models. In the first type of model, the barrier ΔG+‡ for closing a base pair is dominated by the entropic cost ΔS for positioning the nucleotides to the base-paired configuration and the barrier ΔG−‡ for opening a base pair is the enthalpic cost ΔH for disrupting the hydrogen bonds and base stacking interactions (18, 59, 60). In the second type of model, ΔG+‡ is the net free energy change for base pairing ΔG = ΔH ? TΔS and ΔG−‡ is zero (76, 77). In the third type of model, ΔG±‡=±ΔG/2 is used (78). In addition to the above three main types, other models, such as more sophisticated hybrid rate models, have been proposed (29).In this paper, we report a hybrid method (see Fig. 1) to investigate the single base pairing process. In contrast to the previous simulations for temperature- or force-induced unfolding reactions, we directly model the folding process here (i.e., the base pair closing process). Specifically, we use MD simulations to identify the conformational clusters. Based on the network of the conformational clusters as a reduced conformational ensemble, we apply kinetic Monte Carlo (KMC) and master equation (ME) methods to elucidate the detailed roles of base pairing and stacking interactions, as well as the roles of water and ions (79–82). The study reveals previously unidentified kinetics pathways, misfolded states, and rate-limiting steps. A clear understanding of the microscopic details of the elementary kinetic move is a prerequisite for further rigorous study of large-scale RNA kinetic studies. The method described here may provide a feasible way to develop a rate model for the base pair/stack-based kinetic move set. Furthermore, the mechanism of RNA single base folding may provide useful insights into many biologically significant processes, such as nucleotide flipping (83) in helicases and base pair fraying (84) (as the possible first step for nucleic duplex melting in nucleic acid enzymatic processes).Open in a separate windowFig. 1.(A) Folding of a single nucleotide (G1, red) from the unfolded (Left) to the native folded (Right) state. (B) Exhaustive sampling for the (discrete) conformations of the G1 nucleotide (Right) through enumeration of the torsion angles (formed by the blue bonds). (C) Schematic plot shows the trajectories on the energy landscape (depicted with two reaction coordinates for clarity) explored by the MD simulations. The lines, open circles, and hexagons denote the trajectories; the initial states; and the (centroid structures of the) clusters, respectively. (D) Conformational network based on six clusters. (E) The rmsds to the different clusters provide information about the structural changes in a MD trajectory.     

Detection of genomic variations and DNA polymorphisms and impact on analysis of meiotic recombination and genetic mapping

DNA polymorphisms are important markers in genetic analyses and are increasingly detected by using genome resequencing. However, the presence of repetitive sequences and structural variants can lead to false positives in the identification of polymorphic alleles. Here, we describe an analysis strategy that minimizes false positives in allelic detection and present analyses of recently published resequencing data from Arabidopsis meiotic products and individual humans. Our analysis enables the accurate detection of sequencing errors, small insertions and deletions (indels), and structural variants, including large reciprocal indels and copy number variants, from comparisons between the resequenced and reference genomes. We offer an alternative interpretation of the sequencing data of meiotic products, including the number and type of recombination events, to illustrate the potential for mistakes in single-nucleotide polymorphism calling. Using these examples, we propose that the detection of DNA polymorphisms using resequencing data needs to account for nonallelic homologous sequences.DNA polymorphisms are ubiquitous genetic variations among individuals and include single nucleotide polymorphisms (SNPs), insertions and deletions (indels), and other larger rearrangements (1–3) (Fig. 1 A and B). They can have phenotypic consequences and also serve as molecular markers for genetic analyses, facilitating linkage and association studies of genetic diseases, and other traits in humans (4–6), animals, plants, (7–10) and other organisms. Using DNA polymorphisms for modern genetic applications requires low-error, high-throughput analytical strategies. Here, we illustrate the use of short-read next-generation sequencing (NGS) data to detect DNA polymorphisms in the context of whole-genome analysis of meiotic products.Open in a separate windowFig. 1.(A) SNPs and small indels between two ecotype genomes. (B) Possible types of SVs. Col genotypes are marked in blue and Ler in red. Arrows indicate DNA segments involved in SVs between the two ecotypes. (C) Meiotic recombination events including a CO and a GC (NCO). Centromeres are denoted by yellow dots.There are many methods for detecting SNPs (11–14) and structural variants (SVs) (15–25), including NGS, which can capture nearly all DNA polymorphisms (26–28). This approach has been widely used to analyze markers in crop species such as rice (29), genes associated with diseases (6, 26), and meiotic recombination in yeast and plants (30, 31). However, accurate identification of DNA polymorphisms can be challenging, in part because short-read sequencing data have limited information for inferring chromosomal context.Genomes usually contain repetitive sequences that can differ in copy number between individuals (26–28, 31); therefore, resequencing analyses must account for chromosomal context to avoid mistaking highly similar paralogous sequences for polymorphisms. Here, we use recently published datasets to describe several DNA sequence features that can be mistaken as allelic (32, 33) and describe a strategy for differentiating between repetitive sequences and polymorphic alleles. We illustrate the effectiveness of these analyses by examining the reported polymorphisms from the published datasets.Meiotic recombination is initiated by DNA double-strand breaks (DSBs) catalyzed by the topoisomerase-like SPORULATION 11 (SPO11). DSBs are repaired as either crossovers (COs) between chromosomes (Fig. 1C), or noncrossovers (NCOs). Both COs and NCOs can be accompanied by gene conversion (GC) events, which are the nonreciprocal transfer of sequence information due to the repair of heteroduplex DNA during meiotic recombination. Understanding the control of frequency and distribution of CO and NCO (including GC) events has important implications for human health (including cancer and aneuploidy), crop breeding, and the potential for use in genome engineering. COs can be detected relatively easily by using polymorphic markers in the flanking sequences, but NCO products can only be detected if they are accompanied by a GC event. Because GCs associated with NCO result in allelic changes at polymorphic sites without exchange of flanking sequences, they are more difficult to detect. Recent advances in DNA sequencing have made the analysis of meiotic NCOs more feasible (30–32, 34); however, SVs present a challenge in these analyses. We recommend a set of guidelines for detection of DNA polymorphisms by using genomic resequencing short-read datasets. These measures improve the accuracy of a wide range of analyses by using genomic resequencing, including estimation of COs, NCOs, and GCs.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.