Voltage Sensors Of Ion Channels Research Articles

Molecular Insights from Conformational Ensembles via Machine Learning

Biomolecular simulations are intrinsically high dimensional and generate noisy data sets of ever-increasing size. Extracting important features from the data is crucial for understanding the biophysical properties of molecular processes, but remains a big challenge. Machine learning (ML) provides powerful dimensionality reduction tools. However, such methods are often criticized as resembling black boxes with limited human-interpretable insight. We use methods from supervised and unsupervised ML to efficiently create interpretable maps of important features from molecular simulations. We benchmark the performance of several methods, including neural networks, random forests, and principal component analysis, using a toy model with properties reminiscent of macromolecular behavior. We then analyze three diverse biological processes: conformational changes within the soluble protein calmodulin, ligand binding to a G protein-coupled receptor, and activation of an ion channel voltage-sensor domain, unraveling features critical for signal transduction, ligand binding, and voltage sensing. This work demonstrates the usefulness of ML in understanding biomolecular states and demystifying complex simulations.

Main-chain mutagenesis reveals intrahelical coupling in an ion channel voltage-sensor

Membrane proteins are universal signal decoders. The helical transmembrane segments of these proteins play central roles in sensory transduction, yet the mechanistic contributions of secondary structure remain unresolved. To investigate the role of main-chain hydrogen bonding on transmembrane function, we encoded amide-to-ester substitutions at sites throughout the S4 voltage-sensing segment of Shaker potassium channels, a region that undergoes rapid, voltage-driven movement during channel gating. Functional measurements of ester-harboring channels highlight a transitional region between α-helical and 310 segments where hydrogen bond removal is particularly disruptive to voltage-gating. Simulations of an active voltage sensor reveal that this region features a dynamic hydrogen bonding pattern and that its helical structure is reliant upon amide support. Overall, the data highlight the specialized role of main-chain chemistry in the mechanism of voltage-sensing; other catalytic transmembrane segments may enlist similar strategies in signal transduction mechanisms.

Exploring Structural Interactions of Tarantula Toxins with Lipid Membranes using Rosetta and Molecular Dynamics Simulation

Tarantula venom toxins belong to a class of cystine knot peptide toxins that have three disulfide bridges stabilizing the inner core of the toxin structure. These toxins target a wide range of ion channels and modulate physiological activities of channels upon binding. Experimental data indicates that tarantula toxins which target voltage sensors of ion channels interact with cell membrane bilayers and potentially form interactions with channels at the membrane-protein interface. However, how these peptide toxins partition and orient in the membrane are not fully understood. We used Rosetta structural modeling and molecular dynamics simulation, to embed three tarantula toxins, VsTx1, GxTx-1E and SgTx1, into lipid membrane environments. Our results suggest that the three tarantula toxins prefer a similar orientation and depth in the membrane bilayer. We observe that hydrophobic residues on the toxins embed deeper in the hydrophobic lipid core region, basic residues prefer interactions with lipid head groups, and acidic residues are exposed to water environment. This unique orientation of peptide toxins exposes residues critical for interaction with the voltage sensors within the lipid environment, suggesting that toxins may utilize this embedding and orientation to interact favorably with voltage sensors of ion channels.

Protein-fluctuation-induced water-pore formation in ion channel voltage-sensor translocation across a lipid bilayer membrane.

We have applied a combined fluorescence microscopy and single-ion-channel electric current recording approach, correlating with molecular dynamics (MD) simulations, to study the mechanism of voltage-sensor domain translocation across a lipid bilayer. We use the colicin Ia ion channel as a model system, and our experimental and simulation results show the following: (1) The open-close activity of an activated colicin Ia is not necessarily sensitive to the amplitude of the applied cross-membrane voltage when the cross-membrane voltage is around the resting potential of excitable membranes; and (2) there is a significant probability that the activation of colicin Ia occurs by forming a transient and fluctuating water pore of ∼15 Å diameter in the lipid bilayer membrane. The location of the water-pore formation is nonrandom and highly specific, right at the insertion site of colicin Ia charged residues in the lipid bilayer membrane, and the formation is intrinsically associated with the polypeptide conformational fluctuations and solvation dynamics. Our results suggest an interesting mechanistic pathway for voltage-sensitive ion channel activation, and specifically for translocation of charged polypeptide chains across the lipid membrane under a transmembrane electric field: the charged polypeptide domain facilitates the formation of hydrophilic water pore in the membrane and diffuses through the hydrophilic pathway across the membrane; i.e., the charged polypeptide chain can cross a lipid membrane without entering into the hydrophobic core of the lipid membrane but entirely through the aqueous and hydrophilic environment to achieve a cross-membrane translocation. This mechanism sheds light on the intensive and fundamental debate on how a hydrophilic and charged peptide domain diffuses across the biologically inaccessible high-energy barrier of the hydrophobic core of a lipid bilayer: The peptide domain does not need to cross the hydrophobic core to move across a lipid bilayer.

Voltage-dependent gating and gating charge measurements in the Kv1.2 potassium channel.

Much has been learned about the voltage sensors of ion channels since the x-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. High resolution structural data of a Kv channel enabled the structural interpretation of numerous electrophysiological findings collected in various ion channels, most notably Shaker, and permitted the development of meticulous computational simulations of the activation mechanism. The fundamental premise for the structural interpretation of functional measurements from Shaker is that this channel and Kv1.2 have the same characteristics, such that correlation of data from both channels would be a trivial task. We tested these assumptions by measuring Kv1.2 voltage-dependent gating and charge per channel. We found that the Kv1.2 gating charge is near 10 elementary charges (eo), ∼25% less than the well-established 13-14 eo in Shaker. Next, we neutralized positive residues in the Kv1.2 S4 transmembrane segment to investigate the cause of the reduction of the gating charge and found that, whereas replacing R1 with glutamine decreased voltage sensitivity to ∼50% of the wild-type channel value, mutation of the subsequent arginines had a much smaller effect. These data are in marked contrast to the effects of charge neutralization in Shaker, where removal of the first four basic residues reduces the gating charge by roughly the same amount. In light of these differences, we propose that the voltage-sensing domains (VSDs) of Kv1.2 and Shaker might undergo the same physical movement, but the septum that separates the aqueous crevices in the VSD of Kv1.2 might be thicker than Shaker's, accounting for the smaller Kv1.2 gating charge.

The Gating Charge of Kv1.2 is Less than Expected from its Similarity to Shaker

Much has been learned about the voltage sensors of ion channels since the X-ray structure of the mammalian voltage-gated potassium channel Kv1.2 was published in 2005. The availability of high-resolution structural data of a Kv channel paved the way for the structural interpretation of numerous electrophysiological findings collected over many years in a variety of ion channels, most notably Shaker, and allowed the development of meticulous computational simulations of the activation mechanism of Kv1.2. The fundamental premise of the validity of the interpretation of functional measurements from Shaker using the structure of Kv1.2 is that both channels are related closely enough such that correlation of their data is a trivial task. We set out to confirm these assumptions by measuring the voltage sensitivity of the channel using the limiting slope method, followed by the determination of the gating charge through gating current recordings. We found that the gating charge, as measured by both techniques, is 10 e0, ∼25% less than the well-established 13-14 e0 in Shaker. Next, we neutralized each of the six positive residues in S4 of Kv1.2 to probe the cause of the reduction of the gating charge, and found that while replacing R1 with glutamine decreased voltage sensitivity to just about 50% of the wild-type channel value, mutation of the subsequent arginines did not have an effect nearly as large. These data stand out as different to Shaker's, where removal of the first four positive residues reduces the gating charge by roughly the same amount of elementary charges. We propose that the septum that separates the aqueous crevices in the VSD of Kv1.2 is thicker than Shaker's, and that this accounts for a smaller gating charge in Kv1.2.

Tethering Dimers of Voltage Sensor Toxins can Selectively Amplify their Affinity for Kv Channels

Potent and selective modulators are needed to elucidate the individual roles of ion channel subtypes in physiological systems. Many cystine knot peptides derived from tarantula venom allosterically modulate voltage sensors of ion channels. Although tarantula peptides have varying affinities for different channels, all affect multiple subtypes. Theoretically, a peptide's selectivity between channel types could be amplified by summing binding energies, if a multimeric version of the peptide could bind multiple subunits simultaneously. We have amplified the inherent specificity of a voltage sensor peptide, guangxitoxin-1E (GxTX), by tethering two peptides together to form a dimer coupled by a flexible polyethylene glycol linker. We synthesized GxTX mutants functionalized with artificial amino acids for chemoselective coupling and tethered them with homobifunctional linkers using azide-alkyne cycloaddition chemistry. The GxTX dimers inhibited Kv2.1 with higher affinity than GxTX monomers. Similar to GxTX monomers, these tethered dimers shifted channel opening to positive voltages. Association rates were reduced for the GxTX dimers versus monomers, indicating that increased affinity for Kv2.1 channels was entirely due to the extremely slow dissociation of GxTX dimers. The strong voltage dependence of dimer dissociation was consistent with a two step binding model, where the kinetics of the second GxTX binding domain determines the amplification of affinity by the dimer. This mechanism predicts that dimerization will selectively amplify the affinity for channels with slow toxin dissociation rates. Experimentally, dimerization amplified GxTX specificity for Kv2.1 over other channel subtypes. We conclude that tethered multimers can increase the pharmacological selectivity of voltage sensor modulators.

Voltage sensor of ion channels and enzymes.

Placed in the cell membrane (a two-dimensional environment), ion channels and enzymes are able to sense voltage. How these proteins are able to detect the difference in the voltage across membranes has attracted much attention, and at times, heated debate during the last few years. Sodium, Ca2+ and K+ voltage-dependent channels have a conserved positively charged transmembrane (S4) segment that moves in response to changes in membrane voltage. In voltage-dependent channels, S4 forms part of a domain that crystallizes as a well-defined structure consisting of the first four transmembrane (S1-S4) segments of the channel-forming protein, which is defined as the voltage sensor domain (VSD). The VSD is tied to a pore domain and VSD movements are allosterically coupled to the pore opening to various degrees, depending on the type of channel. How many charges are moved during channel activation, how much they move, and which are the molecular determinants that mediate the electromechanical coupling between the VSD and the pore domains are some of the questions that we discuss here. The VSD can function, however, as a bona fide proton channel itself, and, furthermore, the VSD can also be a functional part of a voltage-dependent phosphatase.

Inhibition of Sodium Channel Gating by Trapping the Domain II Voltage Sensor with Protoxin II

ProTx-II, an inhibitory cysteine knot toxin from the tarantula Thrixopelma pruriens, inhibits voltage-gated sodium channels. Using the cut-open oocyte preparation for electrophysiological recording, we show here that ProTx-II impedes movement of the gating charges of the sodium channel voltage sensors and reduces maximum activation of sodium conductance. At a concentration of 1 microM, the toxin inhibits 65.3 +/- 4.1% of the sodium conductance and 24.6 +/- 6.8% of the gating current of brain Na(v)1.2a channels, with a specific effect on rapidly moving gating charge. Strong positive prepulses can reverse the inhibitory effect of ProTx-II, indicating voltage-dependent dissociation of the toxin. Voltage-dependent reversal of the ProTx-II effect is more rapid for cardiac Na(v)1.5 channels, suggesting subtype-specific action of this toxin. Voltage-dependent binding and block of gating current are hallmarks of gating modifier toxins, which act by binding to the extracellular end of the S4 voltage sensors of ion channels. The mutation L833C in the S3-S4 linker in domain II reduces affinity for ProTx-II, and mutation of the outermost two gating-charge-carrying arginine residues in the IIS4 voltage sensor to glutamine abolishes voltage-dependent reversal of toxin action and toxin block of gating current. Our results support a voltage-sensor-trapping model for ProTx-II action in which the bound toxin impedes the normal outward gating movement of the IIS4 transmembrane segment, traps the domain II voltage sensor module in its resting state, and thereby inhibits channel activation.

The voltage sensor of ion channels revealed

Voltage-gated ion channels control the excitability of nerve, muscle, endocrine and other cell types, thereby regulating a broad spectrum of cellular processes. How these channels sense changes in the potential across the membrane has remained a mystery for decades. The long-awaited crystal structure of a voltage-gated ion channel was recently published by Jiang et al., revealing an unexpected and surprisingly simple mechanism of voltage sensing and gating in these channels.

Slow photo-cross-linking kinetics of benzophenone-labeled voltage sensors of ion channels.

Voltage-gated ion channels have voltage sensors that move in response to changes in membrane potential. This movement regulates the gates that control access of ions to the permeation pathway. To study the coupling between voltage sensors and gates, we immobilize the voltage sensors, using a bifunctional photo-cross-linking reagent that can be attached to an introduced cysteine, and observe the consequences for gate movement [Horn, R., Ding, S., and Gruber, H. J. (2000) J. Gen. Physiol. 116, 461-475]. UV irradiation of the benzophenone adduct attached to the cysteine residue immobilizes the voltage sensors, S4 segments, of both Na(+) and Shaker K(+) channels. Here we examine the kinetics of S4 immobilization after a brief UV flash. Immobilization has an exponential time course with time constants of >200 ms for Shaker and 17 ms for Na(+) channels, whereas the triplet excited state lifetime of the benzophenone adduct is <1 ms. This result suggests that H-atom abstraction by benzophenone is rapid and that the rate-limiting step in immobilization is the recombination of alkyl and ketyl free radicals generated by H-abstraction. H-Abstraction is also 2.7-fold more efficient at a hyperpolarized voltage than at a depolarized membrane potential in Shaker S4 segments. S4 immobilization after a UV flash can be prevented by depolarization of Shaker channels, suggesting that movement in the activation pathway is capable of separating the ketyl and alkyl free radicals. Exploiting the unique charge movement and gating properties of the L382V mutant of Shaker, we show that free radical separation follows S4 movement itself and is relatively independent of the movement of activation gates.

Primary hepatic cryptococcosis

ProTx-II, an inhibitory cysteine knot toxin from the tarantula <i>Thrixopelma pruriens</i>, inhibits voltage-gated sodium channels. Using the cut-open oocyte preparation for electrophysiological recording, we show here that ProTx-II impedes movement of the gating charges of the sodium channel voltage sensors and reduces maximum activation of sodium conductance. At a concentration of 1 μM, the toxin inhibits 65.3 ± 4.1% of the sodium conductance and 24.6 ± 6.8% of the gating current of brain Na_v1.2a channels, with a specific effect on rapidly moving gating charge. Strong positive prepulses can reverse the inhibitory effect of ProTx-II, indicating voltage-dependent dissociation of the toxin. Voltage-dependent reversal of the ProTx-II effect is more rapid for cardiac Na_v1.5 channels, suggesting subtype-specific action of this toxin. Voltage-dependent binding and block of gating current are hallmarks of gating modifier toxins, which act by binding to the extracellular end of the S4 voltage sensors of ion channels. The mutation L833C in the S3-S4 linker in domain II reduces affinity for ProTx-II, and mutation of the outermost two gating-charge-carrying arginine residues in the IIS4 voltage sensor to glutamine abolishes voltage-dependent reversal of toxin action and toxin block of gating current. Our results support a voltage-sensor-trapping model for ProTx-II action in which the bound toxin impedes the normal outward gating movement of the IIS4 transmembrane segment, traps the domain II voltage sensor module in its resting state, and thereby inhibits channel activation.