Folding of prestin's anion-binding site and the mechanism of outer hair cell electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by the amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and help define prestin's unique voltage-sensing mechanism and electromotility.

Conformation-specific Synthetic Antibodies Discriminate Multiple Functional States of the Ion Channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg2+, and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg2+. Two sABs from these selections, C12 and C18, showed different degrees of Mg2+-sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg2+-depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg2+-depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg2+] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Conformation-specific synthetic antibodies discriminate multiple functional states of the ion channel CorA.

CorA, the primary magnesium ion channel in prokaryotes and archaea, is a prototypical homopentameric ion channel that undergoes ion-dependent conformational transitions. CorA adopts five-fold symmetric non-conductive states in the presence of high concentrations of Mg 2+ , and highly asymmetric flexible states in its complete absence. However, the latter were of insufficient resolution to be thoroughly characterized. In order to gain additional insights into the relationship between asymmetry and channel activation, we exploited phage display selection strategies to generate conformation-specific synthetic antibodies (sABs) against CorA in the absence of Mg 2+ . Two sABs from these selections, C12 and C18, showed different degrees of Mg 2+ -sensitivity. Through structural, biochemical, and biophysical characterization, we found the sABs are both conformation-specific but probe different features of the channel under open-like conditions. C18 is highly specific to the Mg 2+ -depleted state of CorA and through negative-stain electron microscopy (ns-EM), we show sAB binding reflects the asymmetric arrangement of CorA protomers in Mg 2+ -depleted conditions. We used X-ray crystallography to determine a structure at 2.0 Å resolution of sAB C12 bound to the soluble N-terminal regulatory domain of CorA. The structure shows C12 is a competitive inhibitor of regulatory magnesium binding through its interaction with the divalent cation sensing site. We subsequently exploited this relationship to capture and visualize asymmetric CorA states in different [Mg 2+ ] using ns-EM. We additionally utilized these sABs to provide insights into the energy landscape that governs the ion-dependent conformational transitions of CorA.

Folding of Prestin's Anion-Binding Site and the Mechanism of Outer Hair Cell Electromotility.

Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin's voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl - anion at a conserved binding site formed by amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl - binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion-binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin's mechanical expansion. We observe helix fraying at prestin's anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin's fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and helps define prestin's unique voltage-sensing mechanism and electromotility.

State-specific morphological deformations of the lipid bilayer explain mechanosensitive gating of MscS ion channels.

The force-from-lipids hypothesis of cellular mechanosensation posits that membrane channels open and close in response to changes in the physical state of the lipid bilayer, induced for example by lateral tension. Here, we investigate the molecular basis for this transduction mechanism by studying the mechanosensitive ion channel MscS from Escherichia coli and its eukaryotic homolog MSL1 from Arabidopsis thaliana. First, we use single-particle cryo-electron microscopy to determine the structure of a novel open conformation of wild-type MscS, stabilized in a thinned lipid nanodisc. Compared with the closed state, the structure shows a reconfiguration of helices TM1, TM2, and TM3a, and widening of the central pore. Based on these structures, we examined how the morphology of the membrane is altered upon gating, using molecular dynamics simulations. The simulations reveal that closed-state MscS causes drastic protrusions in the inner leaflet of the lipid bilayer, both in the absence and presence of lateral tension, and for different lipid compositions. These deformations arise to provide adequate solvation to hydrophobic crevices under the TM1-TM2 hairpin, and clearly reflect a high-energy conformation for the membrane, particularly under tension. Strikingly, these protrusions are largely eradicated upon channel opening. An analogous computational study of open and closed MSL1 recapitulates these findings. The gating equilibrium of MscS channels thus appears to be dictated by opposing conformational preferences, namely those of the lipid membrane and of the protein structure. We propose a membrane deformation model of mechanosensation, which posits that tension shifts the gating equilibrium towards the conductive state not because it alters the mode in which channel and lipids interact, but because it increases the energetic cost of the morphological perturbations in the membrane required by the closed state.

Mechanism of voltage gating in the voltage-sensing phosphatase Ci-VSP.

Conformational changes in voltage-sensing domains (VSDs) are driven by the transmembrane electric field acting on the protein charges. Yet, the overall energetics and detailed mechanism of this process are not fully understood. Here, we determined free energy and displacement charge landscapes as well as the major conformations visited during a complete functional gating cycle in the isolated VSD of the phosphatase Ci-VSP (Ci-VSD) comprising four transmembrane helices (segments S1 to S4). Molecular dynamics simulations highlight the extent of S4 movements. In addition to the crystallographically determined activated "Up" and resting "Down" states, the simulations predict two Ci-VSD conformations: a deeper resting state ("down-minus") and an extended activated ("up-plus") state. These additional conformations were experimentally probed via systematic cysteine mutagenesis with metal-ion bridges and the engineering of proton conducting mutants at hyperpolarizing voltages. The present results show that these four states are visited sequentially in a stepwise manner during voltage activation, each step translocating one arginine or the equivalent of ∼1 e0 across the membrane electric field, yielding a transfer of ∼3 e0 charges in total for the complete process.

Mechanism of voltage sensing in Ca2+- and voltage-activated K+ (BK) channels.

In neurosecretion, allosteric communication between voltage sensors and Ca2+ binding in BK channels is crucially involved in damping excitatory stimuli. Nevertheless, the voltage-sensing mechanism of BK channels is still under debate. Here, based on gating current measurements, we demonstrate that two arginines in the transmembrane segment S4 (R210 and R213) function as the BK gating charges. Significantly, the energy landscape of the gating particles is electrostatically tuned by a network of salt bridges contained in the voltage sensor domain (VSD). Molecular dynamics simulations and proton transport experiments in the hyperpolarization-activated R210H mutant suggest that the electric field drops off within a narrow septum whose boundaries are defined by the gating charges. Unlike Kv channels, the charge movement in BK appears to be limited to a small displacement of the guanidinium moieties of R210 and R213, without significant movement of the S4.

Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative.

The human voltage-gated proton channel (hHv1) is important for control of intracellular pH. We designed C6, a specific peptide inhibitor of hHv1, to evaluate the roles of the channel in sperm capacitation and in the inflammatory immune response of neutrophils [R. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 115, E11847­E11856 (2018)]. One C6 binds with nanomolar affinity to each of the two S3­S4 voltage-sensor loops in hHv1 in cooperative fashion so that C6-bound channels require greater depolarization to open and do so more slowly. As depolarization drives hHv1 sensors outwardly, C6 affinity decreases, and inhibition is partial. Here, we identified residues essential to C6­hHv1 binding by scanning mutagenesis, five in the hHv1 S3­S4 loops and seven on C6. A structural model of the C6­hHv1 complex was then generated by molecular dynamics simulations and validated by mutant-cycle analysis. Guided by this model, we created a bivalent C6 peptide (C62) that binds simultaneously to both hHv1 subunits and fully inhibits current with picomolar affinity. The results help delineate the structural basis for C6 state-dependent inhibition, support an anionic lipid-mediated binding mechanism, and offer molecular insight into the effectiveness of engineered C6 as a therapeutic agent or lead.

The conformational cycle of prestin underlies outer-hair cell electromotility.

The voltage-dependent motor protein prestin (also known as SLC26A5) is responsible for the electromotive behaviour of outer-hair cells and underlies the cochlear amplifier1. Knockout or impairment of prestin causes severe hearing loss2-5. Despite the key role of prestin in hearing, the mechanism by which mammalian prestin senses voltage and transduces it into cellular-scale movements (electromotility) is poorly understood. Here we determined the structure of dolphin prestin in six distinct states using single-particle cryo-electron microscopy. Our structural and functional data suggest that prestin adopts a unique and complex set of states, tunable by the identity of bound anions (Cl- or SO42-). Salicylate, a drug that can cause reversible hearing loss, competes for the anion-binding site of prestin, and inhibits its function by immobilizing prestin in a new conformation. Our data suggest that the bound anion together with its coordinating charged residues and helical dipole act as a dynamic voltage sensor. An analysis of all of the anion-dependent conformations reveals how structural rearrangements in the voltage sensor are coupled to conformational transitions at the protein-membrane interface, suggesting a previously undescribed mechanism of area expansion. Visualization of the electromotility cycle of prestin distinguishes the protein from the closely related SLC26 anion transporters, highlighting the basis for evolutionary specialization of the mammalian cochlear amplifier at a high resolution.

Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel.

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-µs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

Direct activation of the proton channel by albumin leads to human sperm capacitation and sustained release of inflammatory mediators by neutrophils.

Human voltage-gated proton channels (hHv1) extrude protons from cells to compensate for charge and osmotic imbalances due metabolism, normalizing intracellular pH and regulating protein function. Human albumin (Alb), present at various levels throughout the body, regulates oncotic pressure and transports ligands. Here, we report Alb is required to activate hHv1 in sperm and neutrophils. Dose-response studies reveal the concentration of Alb in semen is too low to activate hHv1 in sperm whereas the higher level in uterine fluid yields proton efflux, allowing capacitation, the acrosomal reaction, and oocyte fertilization. Likewise, Alb activation of hHv1 in neutrophils is required to sustain production and release of reactive oxygen species during the immune respiratory burst. One Alb binds to both voltage sensor domains (VSDs) in hHv1, enhancing open probability and increasing proton current. A computational model of the Alb-hHv1 complex, validated by experiments, identifies two sites in Alb domain II that interact with the VSDs, suggesting an electrostatic gating modification mechanism favoring the active "up" sensor conformation. This report shows how sperm are triggered to fertilize, resolving how hHv1 opens at negative membrane potentials in sperm, and describes a role for Alb in physiology that will operate in the many tissues expressing hHv1.

Mechanism of C-type inactivation in the hERG potassium channel.

The fast C-type inactivation displayed by the voltage-activated potassium channel hERG plays a critical role in the repolarization of cardiac cells, and malfunction caused by nonspecific binding of drugs or naturally occurring missense mutations affecting inactivation can lead to pathologies. Because of its impact on human health, understanding the molecular mechanism of C-type inactivation in hERG represents an advance of paramount importance. Here, long-time scale molecular dynamics simulations, free energy landscape calculations, and electrophysiological experiments are combined to address the structural and functional impacts of several disease-associated mutations. Results suggest that C-type inactivation in hERG is associated with an asymmetrical constricted-like conformation of the selectivity filter, identifying F627 side-chain rotation and the hydrogen bond between Y616 and N629 as key determinants. Comparison of hERG with other K+ channels suggests that C-type inactivation depends on the degree of opening of the intracellular gate via the filter-gate allosteric coupling.

Electromechanical coupling in the hyperpolarization-activated K+ channel KAT1.

Voltage-gated potassium (Kv) channels coordinate electrical signalling and control cell volume by gating in response to membrane depolarization or hyperpolarization. However, although voltage-sensing domains transduce transmembrane electric field changes by a common mechanism involving the outward or inward translocation of gating charges1-3, the general determinants of channel gating polarity remain poorly understood4. Here we suggest a molecular mechanism for electromechanical coupling and gating polarity in non-domain-swapped Kv channels on the basis of the cryo-electron microscopy structure of KAT1, the hyperpolarization-activated Kv channel from Arabidopsis thaliana. KAT1 displays a depolarized voltage sensor, which interacts with a closed pore domain directly via two interfaces and indirectly via an intercalated phospholipid. Functional evaluation of KAT1 structure-guided mutants at the sensor-pore interfaces suggests a mechanism in which direct interaction between the sensor and the C-linker hairpin in the adjacent pore subunit is the primary determinant of gating polarity. We suggest that an inward motion of the S4 sensor helix of approximately 5-7 Å can underlie a direct-coupling mechanism, driving a conformational reorientation of the C-linker and ultimately opening the activation gate formed by the S6 intracellular bundle. This direct-coupling mechanism contrasts with allosteric mechanisms proposed for hyperpolarization-activated cyclic nucleotide-gated channels5, and may represent an unexpected link between depolarization- and hyperpolarization-activated channels.

Molecular basis of force-from-lipids gating in the mechanosensitive channel MscS.

Prokaryotic mechanosensitive (MS) channels open by sensing the physical state of the membrane. As such, lipid-protein interactions represent the defining molecular process underlying mechanotransduction. Here, we describe cryo-electron microscopy (cryo-EM) structures of the E. coli small-conductance mechanosensitive channel (MscS) in nanodiscs (ND). They reveal a novel membrane-anchoring fold that plays a significant role in channel activation and establish a new location for the lipid bilayer, shifted ~14 Å from previous consensus placements. Two types of lipid densities are explicitly observed. A phospholipid that 'hooks' the top of each TM2-TM3 hairpin and likely plays a role in force sensing, and a bundle of acyl chains occluding the permeation path above the L105 cuff. These observations reshape our understanding of force-from-lipids gating in MscS and highlight the key role of allosteric interactions between TM segments and phospholipids bound to key dynamic components of the channel.

Real time dynamics of Gating-Related conformational changes in CorA.

CorA, a divalent-selective channel in the metal ion transport superfamily, is the major Mg2+-influx pathway in prokaryotes. CorA structures in closed (Mg2+-bound), and open (Mg2+-free) states, together with functional data showed that Mg2+-influx inhibits further Mg2+-uptake completing a regulatory feedback loop. While the closed state structure is a symmetric pentamer, the open state displayed unexpected asymmetric architectures. Using high-speed atomic force microscopy (HS-AFM), we explored the Mg2+-dependent gating transition of single CorA channels: HS-AFM movies during Mg2+-depletion experiments revealed the channel's transition from a stable Mg2+-bound state over a highly mobile and dynamic state with fluctuating subunits to asymmetric structures with varying degree of protrusion heights from the membrane. Our data shows that at Mg2+-concentration below Kd, CorA adopts a dynamic (putatively open) state of multiple conformations that imply structural rearrangements through hinge-bending in TM1. We discuss how these structural dynamics define the functional behavior of this ligand-dependent channel.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Rapid constriction of the selectivity filter underlies C-type inactivation in the KcsA potassium channel.

C-type inactivation is a time-dependent process observed in many K+ channels whereby prolonged activation by an external stimulus leads to a reduction in ionic conduction. While C-type inactivation is thought to be a result of a constriction of the selectivity filter, the local dynamics of the process remain elusive. Here, we use molecular dynamics (MD) simulations of the KcsA channel to elucidate the nature of kinetically delayed activation/inactivation gating coupling. Microsecond-scale MD simulations based on the truncated form of the KcsA channel (C-terminal domain deleted) provide a first glimpse of the onset of C-type inactivation. We observe over multiple trajectories that the selectivity filter consistently undergoes a spontaneous and rapid (within 1-2 µs) transition to a constricted conformation when the intracellular activation gate is fully open, but remains in the conductive conformation when the activation gate is closed or partially open. Multidimensional umbrella sampling potential of mean force calculations and nonequilibrium voltage-driven simulations further confirm these observations. Electrophysiological measurements show that the truncated form of the KcsA channel inactivates faster and greater than full-length KcsA, which is consistent with truncated KcsA opening to a greater degree because of the absence of the C-terminal domain restraint. Together, these results imply that the observed kinetics underlying activation/inactivation gating reflect a rapid conductive-to-constricted transition of the selectivity filter that is allosterically controlled by the slow opening of the intracellular gate.

Never at rest: insights into the conformational dynamics of ion channels from cryo-electron microscopy.

The tightly regulated opening and closure of ion channels underlies the electrical signals that are vital for a wide range of physiological processes. Two decades ago the first atomic level view of ion channel structures led to a detailed understanding of ion selectivity and conduction. In recent years, spectacular developments in the field of cryo-electron microscopy have resulted in cryo-EM superseding crystallography as the technique of choice for determining near-atomic resolution structures of ion channels. Here, we will review the recent developments in cryo-EM and its specific application to the study of ion channel gating. We will highlight the advantages and disadvantages of the current technology and where the field is likely to head in the next few years.

Structural Dynamics of the MscL C-terminal Domain.

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.

The gating cycle of a K+ channel at atomic resolution.

C-type inactivation in potassium channels helps fine-tune long-term channel activity through conformational changes at the selectivity filter. Here, through the use of cross-linked constitutively open constructs, we determined the structures of KcsA's mutants that stabilize the selectivity filter in its conductive (E71A, at 2.25 Å) and deep C-type inactivated (Y82A at 2.4 Å) conformations. These structural snapshots represent KcsA's transient open-conductive (O/O) and the stable open deep C-type inactivated states (O/I), respectively. The present structures provide an unprecedented view of the selectivity filter backbone in its collapsed deep C-type inactivated conformation, highlighting the close interactions with structural waters and the local allosteric interactions that couple activation and inactivation gating. Together with the structures associated with the closed-inactivated state (C/I) and in the well-known closed conductive state (C/O), this work recapitulates, at atomic resolution, the key conformational changes of a potassium channel pore domain as it progresses along its gating cycle.