Sudha Chakrapani

Structural basis for the coupling between activation and inactivation gates in K(+) channels.

The coupled interplay between activation and inactivation gating is a functional hallmark of K+ channels. This coupling has been experimentally demonstrated through ion interaction effects and cysteine accessibility, and is associated with a well defined boundary of energetically coupled residues. The structure of the K+ channel KcsA in its fully open conformation, in addition to four other partial channel openings, richly illustrates the structural basis of activation–inactivation gating. Here, we identify the mechanistic principles by which movements on the inner bundle gate trigger conformational changes at the selectivity filter, leading to the non-conductive C-type inactivated state. Analysis of a series of KcsA open structures suggests that, as a consequence of the hinge-bending and rotation of the TM2 helix, the aromatic ring of Phe 103 tilts towards residues Thr 74 and Thr 75 in the pore-helix and towards Ile 100 in the neighbouring subunit. This allows the network of hydrogen bonds among residues Trp 67, Glu 71 and Asp 80 to destabilize the selectivity filter, allowing entry to its non-conductive conformation. Mutations at position 103 have a size-dependent effect on gating kinetics: small side-chain substitutions F103A and F103C severely impair inactivation kinetics, whereas larger side chains such as F103W have more subtle effects. This suggests that the allosteric coupling between the inner helical bundle and the selectivity filter might rely on straightforward mechanical deformation propagated through a network of steric contacts. Average interactions calculated from molecular dynamics simulations show favourable open-state interaction-energies between Phe 103 and the surrounding residues. We probed similar interactions in the Shaker K+ channel where inactivation was impaired in the mutant I470A. We propose that side-chain rearrangements at position 103 mechanically couple activation and inactivation in KcsA and a variety of other K+ channels.

Recovery from slow inactivation in K+ channels is controlled by water molecules

Application of a specific stimulus opens the intracellular gate of a K+ channel (activation), yielding a transient period of ion conduction until the selectivity filter spontaneously undergoes a conformational change towards a non-conductive state (inactivation). Removal of the stimulus closes the gate and allows the selectivity filter to interconvert back to its conductive conformation (recovery). Given that the structural differences between the conductive and inactivated filter are very small, it is unclear why the recovery process can take up to several seconds. The bacterial K+ channel KcsA from Streptomyces lividans can be used to help elucidate questions about channel inactivation and recovery at the atomic level. Although KcsA contains only a pore domain, without voltage-sensing machinery, it has the structural elements necessary for ion conduction, activation and inactivation. Here we reveal, by means of a series of long molecular dynamics simulations, how the selectivity filter is sterically locked in the inactive conformation by buried water molecules bound behind the selectivity filter. Potential of mean force calculations show how the recovery process is affected by the buried water molecules and the rebinding of an external K+ ion. A kinetic model deduced from the simulations shows how releasing the buried water molecules can stretch the timescale of recovery to seconds. This leads to the prediction that reducing the occupancy of the buried water molecules by imposing a high osmotic stress should accelerate the rate of recovery, which was verified experimentally by measuring the recovery rate in the presence of a 2-molar sucrose concentration.

A quantitative description of KcsA gating I: Macroscopic currents

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

Structural dynamics of an isolated voltage-sensor domain in a lipid bilayer.

A strong interplay between the voltage-sensor domain (VSD) and the pore domain (PD) underlies voltage-gated channel functions. In a few voltage-sensitive proteins, the VSD has been shown to function without a canonical PD, although its structure and oligomeric state remain unknown. Here, using EPR spectroscopy, we show that the isolated VSD of KvAP can remain monomeric in a reconstituted bilayer and retain a transmembrane conformation. We find that water-filled crevices extending deep into the membrane around S3, a scaffold conducive to transport of protons/cations, are intrinsic to the VSD. Differences in solvent accessibility in comparison to the full-length KvAP allowed us to define an interacting footprint of the PD on the VSD. This interaction is centered around S1 and S2 and suggests a rotation of 70 degrees -100 degrees relative to Kv1.2-Kv2.1 chimera. Sequence-conservation patterns in Kv channels, Hv channels, and voltage-sensitive phosphatases reveal several near-universal features suggesting a common molecular architecture for all VSDs.

A quantitative description of KcsA gating II: Single-channel currents

The kinetic transitions of proton-activated WT KcsA and the noninactivating E71A mutant were studied at the single-channel level in purified, liposome-reconstituted preparations. Single-channel currents were recorded using patch-clamp techniques under nonstationary and steady-state conditions. Maximum-likelihood analyses reveal that the key influence of acidic pH is to increase the frequency of bursting without an effect on the intraburst open and closed dwell times, consistent with the finding from macroscopic currents that protons promote activation without a significant effect on inactivation. However, in steady-conditions of pH, voltage not only alters the burst frequency but also affects their properties, such as the frequency of the flickers and the dwell times of the closed and open states. This is to be expected if voltage modulates pathways connecting open and inactivated states. Upon opening, KcsA can enter at least two closed states that are not part of the activation pathway. The frequency and duration of these closed states was found to be voltage dependent and therefore these are likely to represent short-lived inactivated states. Single-channel recordings of WT KcsA also show varying propensity for the presence of subconductance states. The probability of occurrence of these states did not show clear modulation by voltage or pH and their origin remains unclear and a focus for further investigation. A kinetic model is proposed to describe the gating events in KcsA that recapitulates its macroscopic and single-channel behavior. The model has been constrained by the single-channel analyses presented in this work along with data from macroscopic currents in the preceding paper.

A Multipoint Hydrogen-Bond Network Underlying KcsA C-Type Inactivation

In the prokaryotic potassium channel KcsA activation gating at the inner bundle gate is followed by C-type inactivation at the selectivity filter. Entry into the C-type inactivated state has been directly linked to the strength of the H-bond interaction between residues Glu-71 and Asp-80 behind the filter, and is allosterically triggered by the rearrangement of the inner bundle gate. Here, we show that H-bond pairing between residues Trp-67 and Asp-80, conserved in most K⁺ channels, constitutes another critical interaction that determines the rate and extent of KcsA C-type inactivation. Disruption of the equivalent interaction in Shaker (Trp-434-Asp-447) and Kv1.2 (Trp-366-Asp-379) leads also to modulation of the inactivation process, suggesting that these residues also play an analogous role in the inactivation gating of Kv channels. The present results show that in KcsA C-type inactivation gating is governed by a multipoint hydrogen-bond network formed by the triad Trp-67-Glu71-Asp-80. This triad exerts a critical role in the dynamics and conformational stability of the selectivity filter and might serve as a general modulator of selectivity filter gating in other members of the K⁺ channel family.

On The Structural Basis of Modal Gating Behavior in K+Channels

Modal-gating shifts represent an effective regulatory mechanism by which ion channels control the extent and time course of ionic fluxes. Under steady-state conditions, the K+ channel KcsA shows three distinct gating modes, high-Po, low-Po and a high-frequency flicker mode, each with about an order of magnitude difference in their mean open times. Here we show that in the absence of C-type inactivation, mutations at the pore-helix position Glu71 unmask a series of kinetically distinct modes of gating in a side chain–specific way. These gating modes mirror those seen in wild-type channels and suggest that specific interactions in the side chain network surrounding the selectivity filter, in concert with ion occupancy, alter the relative stability of pre-existing conformational states of the pore. The present results highlight the key role of the selectivity filter in regulating modal gating behavior in K+ channels.

Desensitization Mechanism in Prokaryotic Ligand-gated Ion Channel

Background: GLIC, a prokaryotic homologue of pentameric ligand-gated ion channel (LGIC), is activated by protons, and crystal structures suggest a putative open conformation. Results: GLIC function characterized in proteoliposomes reveals rapid activation and slow desensitization. Desensitization is modulated by voltage, blockers, and membrane cholesterol. Conclusion: GLIC desensitization shows many hallmark features of the mechanism in LGIC. Significance: Understanding GLIC desensitization is an important step toward structure-function characterization of LGIC. Crystal structures of Gloeobacter violaceus ligand-gated ion channel (GLIC), a proton-gated prokaryotic homologue of pentameric ligand-gated ion channel (LGIC) from G. violaceus, have provided high-resolution models of the channel architecture and its role in selective ion conduction and drug binding. However, it is still unclear which functional states of the LGIC gating scheme these crystal structures represent. Much of this uncertainty arises from a lack of thorough understanding of the functional properties of these prokaryotic channels. To elucidate the molecular events that constitute gating, we have carried out an extensive characterization of GLIC function and dynamics in reconstituted proteoliposomes by patch clamp measurements and EPR spectroscopy. We find that GLIC channels show rapid activation upon jumps to acidic pH followed by a time-dependent loss of conductance because of desensitization. GLIC desensitization is strongly coupled to activation and is modulated by voltage, permeant ions, pore-blocking drugs, and membrane cholesterol. Many of these properties are parallel to functions observed in members of eukaryotic LGIC. Conformational changes in loop C, measured by site-directed spin labeling and EPR spectroscopy, reveal immobilization during desensitization analogous to changes in LGIC and acetylcholine binding protein. Together, our studies suggest conservation of mechanistic aspects of desensitization among LGICs of prokaryotic and eukaryotic origin.

Conformational Transitions Underlying Pore Opening and Desensitization in Membrane-embedded Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Background: GLIC, a prokaryotic homologue of pentameric ligand-gated ion channels (LGIC), is activated by protons, and crystal structures suggest a putative open conformation. Results: EPR-spectroscopic studies in the pore-lining segment reveals major conformational changes during activation and desensitization. Conclusion: Gating mechanism in GLIC involves distinct activation and desensitization gates. Significance: These studies provide insights into the role of structural dynamics in the functioning of LGIC. Direct structural insight into the mechanisms underlying activation and desensitization remain unavailable for the pentameric ligand-gated channel family. Here, we report the structural rearrangements underlying gating transitions in membrane-embedded GLIC, a prokaryotic homologue, using site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We particularly probed the conformation of pore-lining second transmembrane segment (M2) under conditions that favor the closed and the ligand-bound desensitized states. The spin label mobility, intersubunit spin-spin proximity, and the solvent-accessibility parameters in the two states clearly delineate the underlying protein motions within M2. Our results show that during activation the extracellular hydrophobic region undergoes major changes involving an outward translational movement, away from the pore axis, leading to an increase in the pore diameter, whereas the lower end of M2 remains relatively immobile. Most notably, during desensitization, the intervening polar residues in the middle of M2 move closer to form a solvent-occluded barrier and thereby reveal the location of a distinct desensitization gate. In comparison with the crystal structure of GLIC, the structural dynamics of the channel in a membrane environment suggest a more loosely packed conformation with water-accessible intrasubunit vestibules penetrating from the extracellular end all the way to the middle of M2 in the closed state. These regions have been implicated to play a major role in alcohol and drug modulation. Overall, these findings represent a key step toward understanding the fundamentals of gating mechanisms in this class of channels.

Structural Insight into the Assembly of TRPV Channels

Transient receptor potential (TRP) proteins are a large family of polymodal nonselective cation channels. The TRP vanilloid (TRPV) subfamily consists of six homologous members with diverse functions. TRPV1-TRPV4 are nonselective cation channels proposed to play a role in nociception, while TRPV5 and TRPV6 are involved in epithelial Ca²⁺ homeostasis. Here we present the cryo-electron microscopy (cryo-EM) structure of functional, full-length TRPV2 at 13.6 Å resolution. The map reveals that the TRPV2 cytoplasmic domain displays a 4-fold petal-like shape in which high-resolution N-terminal ankyrin repeat domain (ARD) structures can be unambiguously fitted. Fitting of the available ARD structures for other TRPV subfamily members into the TRPV2 EM map suggests that TRPV subfamily members have highly homologous structural topologies. These results allowed us to postulate a structural explanation for the functional diversity among TRPV channels and their differential regulation by proteins and ligands.