Direct evidence of conformational changes associated with voltage gating in a voltage sensor protein by time-resolved X-ray/neutron interferometry.
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ABSTRACT:
SUBMITTER: Tronin AY
PROVIDER: S-EPMC4007984 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Available experimental techniques cannot determine high-resolution three-dimensional structures of membrane proteins under a transmembrane voltage. Hence, the mechanism by which voltage-gated cation channels couple conformational changes within the four voltage sensor domains, in response to either depolarizing or polarizing transmembrane voltages, to opening or closing of the pore domain's ion channel remains unresolved. Single-membrane specimens, composed of a phospholipid bilayer containing a vectorially oriented voltage-gated K+ channel protein at high in-plane density tethered to the surface of an inorganic multilayer substrate, were developed to allow the application of transmembrane voltages in an electrochemical cell. Time-resolved neutron reflectivity experiments, enhanced by interferometry enabled by the multilayer substrate, were employed to provide directly the low-resolution profile structures of the membrane containing the vectorially oriented voltage-gated K+ channel for the activated, open and deactivated, closed states of the channel under depolarizing and hyperpolarizing transmembrane voltages applied cyclically. The profile structures of these single membranes were dominated by the voltage-gated K+ channel protein because of the high in-plane density. Importantly, the use of neutrons allowed the determination of the voltage-dependent changes in both the profile structure of the membrane and the distribution of water within the profile structure. These two key experimental results were then compared to those predicted by three computational modeling approaches for the activated, open and deactivated, closed states of three different voltage-gated K+ channels in hydrated phospholipid bilayer membrane environments. Of the three modeling approaches investigated, only one state-of-the-art molecular dynamics simulation that directly predicted the response of a voltage-gated K+ channel within a phospholipid bilayer membrane to applied transmembrane voltages by utilizing very long trajectories was found to be in agreement with the two key experimental results provided by the time-resolved neutron interferometry experiments.
| S-EPMC6712512 | biostudies-literature
Project description:The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was investigated as a function of the applied transbilayer electric potential via time-resolved neutron reflectivity, enhanced by interferometry. Significant, and fully reversible structural changes were observed in the distal half (with respect to the substrate surface) of the hybrid bilayer comprised of a zwitterionic phospholipid in response to a +100mV potential with respect to 0mV. These arise presumably due to reorientation of the electric dipole present in the polar headgroup of the phospholipid and its resulting effect on the thickness of the phospholipid's hydrocarbon chain layer within the hybrid bilayer's profile structure. The profile structure of the voltage-sensor domain from a voltage-gated ion channel protein within a phospholipid bilayer membrane, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was also investigated as a function of the applied transmembrane electric potential via time-resolved X-ray reflectivity, enhanced by interferometry. Significant, fully-reversible, and different structural changes in the protein were detected in response to ±100mV potentials with respect to 0mV. The approach employed is that typical of transient spectroscopy, shown here to be applicable to both neutron and X-ray reflectivity of thin films.
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Project description:The voltage sensitivity of voltage-gated cation channels is primarily attributed to conformational changes of a four transmembrane segment voltage-sensing domain, conserved across many levels of biological complexity. We have identified a remarkable point mutation that confers significant voltage dependence to Kir6.2, a ligand-gated channel that lacks any canonical voltage-sensing domain. Similar to voltage-dependent Kv channels, the Kir6.2[L157E] mutant exhibits time-dependent activation upon membrane depolarization, resulting in an outwardly rectifying current-voltage relationship. This voltage dependence is convergent with the intrinsic ligand-dependent gating mechanisms of Kir6.2, since increasing the membrane PIP2 content saturates Po and eliminates voltage dependence, whereas voltage activation is more dramatic when channel Po is reduced by application of ATP or poly-lysine. These experiments thus demonstrate an inherent voltage dependence of gating in a "ligand-gated" K+ channel, and thereby provide a new view of voltage-dependent gating mechanisms in ion channels. Most interestingly, the voltage- and ligand-dependent gating of Kir6.2[L157E] is highly sensitive to intracellular [K+], indicating an interaction between ion permeation and gating. While these two key features of channel function are classically dealt with separately, the results provide a framework for understanding their interaction, which is likely to be a general, if latent, feature of the superfamily of cation channels.
| S-EPMC2826373 | biostudies-other
Project description:Biological functions frequently require protein-protein interactions that involve secondary and tertiary structural perturbation. Here we study protein-protein dissociation and reassociation dynamics in insulin, a model system for protein oligomerization. Insulin dimer dissociation into monomers was induced by a nanosecond temperature-jump (T-jump) of ∼8 °C in aqueous solution, and the resulting protein and solvent dynamics were tracked by time-resolved X-ray solution scattering (TRXSS) on time scales of 10 ns to 100 ms. The protein scattering signals revealed the formation of five distinguishable transient species during the association process that deviate from simple two-state kinetics. Our results show that the combination of T-jump pump coupled to TRXSS probe allows for direct tracking of structural dynamics in nonphotoactive proteins.
| S-EPMC5804350 | biostudies-literature
Project description:Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.
| S-EPMC4024920 | biostudies-literature
Project description:The voltage-sensor domain (VSD) is a modular four-helix bundle component that confers voltage sensitivity to voltage-gated cation channels in biological membranes. Despite extensive biophysical studies and the recent availability of X-ray crystal structures for a few voltage-gated potassium (Kv) channels and a voltage-gate sodium (Nav) channel, a complete understanding of the cooperative mechanism of electromechanical coupling, interconverting the closed-to-open states (i.e., nonconducting to cation conducting) remains undetermined. Moreover, the function of these domains is highly dependent on the physical-chemical properties of the surrounding lipid membrane environment. The basis for this work was provided by a recent structural study of the VSD from a prokaryotic Kv-channel vectorially oriented within a single phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)) membrane investigated by X-ray interferometry at the solid/moist He (or solid/vapor) and solid/liquid interfaces, thus achieving partial to full hydration, respectively (Gupta et al. Phys. Rev. E2011, 84, 031911-1-15). Here, we utilize neutron interferometry to characterize this system in substantially greater structural detail at the submolecular level, due to its inherent advantages arising from solvent contrast variation coupled with the deuteration of selected submolecular membrane components, especially important for the membrane at the solid/liquid interface. We demonstrate the unique vectorial orientation of the VSD and the retention of its molecular conformation manifest in the asymmetric profile structure of the protein within the profile structure of this single bilayer membrane system. We definitively characterize the asymmetric phospholipid bilayer solvating the lateral surfaces of the VSD protein within the membrane. The profile structures of both the VSD protein and phospholipid bilayer depend upon the hydration state of the membrane. We also determine the distribution of water and exchangeable hydrogen throughout the profile structure of both the VSD itself and the VSD:POPC membrane. These two experimentally determined water and exchangeable hydrogen distribution profiles are in good agreement with molecular dynamics simulations of the VSD protein vectorially oriented within a fully hydrated POPC bilayer membrane, supporting the existence of the VSD's water pore. This approach was extended to the full-length Kv-channel (KvAP) at a solid/liquid interface, providing the separate profile structures of the KvAP protein and the POPC bilayer within the reconstituted KvAP:POPC membrane.
| S-EPMC3406608 | biostudies-literature
Project description:The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K(+) channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.
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Project description:Voltage-driven activation of Kv channels results from conformational changes of four voltage sensor domains (VSDs) that surround the K(+) selective pore domain. How the VSD helices rearrange during gating is an area of active research. Luminescence resonance energy transfer (LRET) is a powerful spectroscopic ruler uniquely suitable for addressing the conformational trajectory of these helices. Using a geometric analysis of numerous LRET measurements, we were able to estimate LRET probe positions relative to existing structural models. The experimental movement of helix S4 does not support a large 15-20 A transmembrane "paddle-type" movement or a near-zero A vertical "transporter-type" model. Rather, our measurements demonstrate a moderate S4 displacement of 10 +/- 5 A, with a vertical component of 5 +/- 2 A. The S3 segment moves 2 +/- 1 A in the opposite direction and is therefore not moving as an S3-S4 rigid body.
| S-EPMC2603612 | biostudies-literature
Project description:The activation of voltage-dependent ion channels is associated with the movement of gating charges, which give rise to gating currents. Although gating currents from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents from a population of channels allows a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a rate model of channel activation and assuming sufficiently high bandwidth, is in accordance with the presence of a main step along the activation pathway carrying a charge of 2.3-2.4 e0. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we used a Brownian model of voltage-dependent gating based on atomic detail structure, that follows the laws of electrodynamics. The model predicts gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of the model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real channels the voltage sensor moves along a sequence of intermediate states separated by relatively low (<5 kT) energy barriers. As a consequence, crossings of successive gating charges through the gating pore become very frequent, and the corresponding current shots are often seen to overlap because of the relatively high filtering. Notably, this limited bandwidth effect is at the origin of the relatively high single-step charge experimentally detected.
| S-EPMC8510977 | biostudies-literature
Project description:The conformational ensembles of structured RNA's are crucial for biological function, but they remain difficult to elucidate experimentally. We demonstrate with HIV-1 TAR RNA that X-ray scattering interferometry (XSI) can be used to determine RNA conformational ensembles. X-ray scattering interferometry (XSI) is based on site-specifically labeling RNA with pairs of heavy atom probes, and precisely measuring the distribution of inter-probe distances that arise from a heterogeneous mixture of RNA solution structures. We show that the XSI-based model of the TAR RNA ensemble closely resembles an independent model derived from NMR-RDC data. Further, we show how the TAR RNA ensemble changes shape at different salt concentrations. Finally, we demonstrate that a single hybrid model of the TAR RNA ensemble simultaneously fits both the XSI and NMR-RDC data set and show that XSI can be combined with NMR-RDC to further improve the quality of the determined ensemble. The results suggest that XSI-RNA will be a powerful approach for characterizing the solution conformational ensembles of RNAs and RNA-protein complexes under diverse solution conditions.
| S-EPMC5416899 | biostudies-literature