Potassium Channel Voltage Sensor Research Articles

Tracking the movement of discrete gating charges in a voltage-gated potassium channel.

Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel

Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.

Structure and dynamics of spin label side chains in KvAP voltage-sensor domain: an all-atom MD simulation study

Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy has become a useful approach to investigate the structural and dynamical properties of biomolecules, especially membrane protein. In this method, spin label side chains due to their motion highly depending on the local environment were employed as external molecules to probe the structure and dynamics of the membrane protein. The close relationships between the dynamics of the spin label side chain and protein structure have been studied for T4 lysozyme. Besides, molecular dynamics (MD) simulations were widely applied as a powerful tool to study the microscopic behavior of the proteins, completing experimental approaches. Here, we presented an all-atom MD simulation study of the nitroxide spin label (MTSSL) in the X-ray structure of a potassium channel voltage-sensor domain (VSD) from Aeropyrum Pernix (KvAP).

Nano-Positioning System for Structural Analysis of Functional Homomeric Proteins in Multiple Conformations

Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target-site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a positioning method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states, with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one per subunit), relative to a single fluorescent acceptor placed in a static site. As first application, the method is used to determine the position of a functional voltage-gated potassium channel's voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.

Microscopic Origin of Gating Current Fluctuations in a Potassium Channel Voltage Sensor

Voltage-dependent ion channels open and close in response to changes in membrane electrical potential due to the motion of their voltage-sensing domains (VSDs). VSD charge displacements within the membrane electric field are observed in electrophysiology experiments as gating currents preceding ionic conduction. The elementary charge motions that give rise to the gating current cannot be observed directly, but appear as discrete current pulses that generate fluctuations in gating current measurements. Here we report direct observation of gating-charge displacements in an atomistic molecular dynamics simulation of the isolated VSD from the KvAP channel in a hydrated lipid bilayer on the timescale (10-μs) expected for elementary gating charge transitions. The results reveal that gating-charge displacements are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1–S3 transmembrane segments in the hydrated interior of the VSD.

Microscopic Origin of Gating Current Fluctuations in a Potassium Channel Voltage Sensor

Voltage-sensing domains (VSDs) regulate the opening of voltage-dependent ion channels in response to changes in membrane electrical potential. They are comprised of four transmembrane segments (S1 through S4) and their overall architecture is conserved from Archaea to mammals. VSD charge displacements within the membrane electric field after a sudden change in membrane potential are measured in electrophysiology experiments as capacitive currents, termed gating currents, which precede ion conduction. The elementary charge motions that give rise to the gating current appear as shot-like events in high-bandwidth recordings of gating current fluctuations, but cannot be observed directly due to limitations of present recording technology. To characterize the conformational dynamics of the VSD under an applied membrane potential, we performed all-atom MD simulations of the isolated Kv VSD from Aeropyrum pernix (KvAP) in a 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer in excess water under a constant electric field on the timescale (10-μs) expected for elementary gating charge transitions. The results suggest that shot-like events of gating current are associated with the water-catalyzed rearrangement of salt bridges between the S4 arginines and a set of conserved acidic side chains on the S1-S3 transmembrane segments in the hydrated interior of the VSD. Research supported by NIH grants GM74637 and GM86685 and NSF grant CHE-0750175. The computations were carried out on the Anton computer at the National Resource for Biomedical Supercomputing at the Pittsburgh Supercomputing Center, which is funded by NIH grant RC2GM093307.

The Pore of the Voltage-Gated Proton Channel

In classical tetrameric voltage-gated ion channels four voltage-sensing domains (VSDs), one from each subunit, control one ion permeation pathway formed by four pore domains. The human Hv1 proton channel has a different architecture, containinga VSD, but lacking a pore domain. Since its location is not known, we searched for the Hv permeation pathway. We find that mutation of the S4 segment's third arginine R211 (R3) compromises proton selectivity, enabling conduction of a metal cation and even of the large organic cation guanidinium, reminiscent of Shaker's omega pore. In the open state, R3 appears to interact with an aspartate (D112) that is situated in the middle of S1 and is unique to Hv channels. The double mutation of both residues further compromises cation selectivity. We propose that membrane depolarization reversibly positions R3 next to D112 inthe transmembrane VSD to form the ion selectivity filter in the channel's open conformation.

Initial Response of the Potassium Channel Voltage Sensor to a Transmembrane Potential

Early transition events of the voltage sensor (VS) of Kv1.2 potassium channel embedded in a lipid membrane are triggered using full atomistic molecular dynamics (MD) simulations. When subject to an applied hyperpolarized transmembrane (TM) voltage, the VS undergoes conformational changes and reaches a stable kinetic intermediate state, beta', within 20 ns. The gating charge ( approximately 2e) associated with this fast transition results mainly from salt-bridge rearrangements involving negative charges in S2 and S3 and all but the two top residues R(294) and R(297) of S4. Interactions of the latter with phosphomoieties of the lipid head groups appear to stabilize the kinetic state beta'.

The structure of the lipid‐embedded potassium channel voltage sensor determined by double‐electron–electron resonance spectroscopy

A four-pulse electron paramagnetic resonance experiment was used to measure long-range inter-subunit distances in reconstituted KvAP, a voltage-dependent potassium (Kv) channel. The measurements have allowed us to reach the following five conclusions about the native structure of the voltage sensor of KvAP. First, the S1 helix of the voltage sensor engages in a helix packing interaction with the pore domain. Second, the crystallographically observed antiparallel helix-turn-helix motif of the voltage-sensing paddle is retained in the membrane-embedded voltage sensor. Third, the paddle is oriented in such a way as to expose one face to the pore domain and the opposite face to the membrane. Fourth, the paddle and the pore domain appear to be separated by a gap that is sufficiently wide for lipids to penetrate between the two domains. Fifth, the critical voltage-sensing arginine residues on the paddle appear to be lipid exposed. These results demonstrate the importance of the membrane for the native structure of Kv channels, suggest that lipids are an integral part of their native structure, and place the voltage-sensing machinery into a complex lipid environment near the pore domain.

Structure prediction for the down state of a potassium channel voltage sensor

Voltage-gated potassium (Kv) channels, essential for regulating potassium uptake and cell volume in plants and electrical excitability in animals, switch between conducting and non-conducting states as a result of conformational changes in the four voltage-sensing domains (VSDs) that surround the channel pore. This process, known as gating, is initiated by a cluster of positively charged residues on the fourth transmembrane segment (S4) of each VSD, which drives the VSD into a 'down state' at negative voltages and an 'up state' at more positive voltages. The crystal structure of Kv1.2 probably corresponds to the up state, but the local environment of S4 in the down state and its motion in voltage gating remains unresolved. Here we employed several conditional lethal/second-site suppressor yeast screens to determine the transmembrane packing of the VSD in the down state. This screen relies on the ability of KAT1, a eukaryotic Kv channel, to conduct potassium when its VSDs are in the down state, thereby rescuing potassium-transport-deficient yeast. Starting with KAT1 channels bearing conditional lethal mutations, we identified second-site suppressor mutations throughout the VSD that recover yeast growth. We then constructed a down state model of the channel using six pairs of interacting residues as structural constraints and verified this model by engineering suppressor mutations on the basis of spatial considerations. A comparison of this down state model with the up state Kv1.2 structure suggests that the VSDs undergo large rearrangements during gating, whereas the S4 segment remains positioned between the central pore and the remainder of the VSD in both states.

Focused Electric Field across the Voltage Sensor of Potassium Channels

Voltage-gated ion channels respond to changes in membrane potential by movement of their voltage sensors across the electric field between cytoplasmic and extracellular solutions. The principal voltage sensors in these proteins are positively charged S4 segments. The absolute magnitude of S4 movement discriminates two competing classes of gating models. In one class, the movement is <10 Angstrom due to the fact that the electric field is focused by aqueous crevices in the channel protein. In an alternative model, based in part on the crystal structure of a potassium channel, the side chains of S4 arginines move their charges across the bilayer's electric field, a distance of >25 Angstrom. Here, using tethered charges attached to an S4 segment, we provide evidence that the electric field falls across a distance of <4 Angstrom, supporting a model in which the relative movement between S4 and the electric field is very small.

Voltage-Regulated Phosphatase

A survey of the genome of the sea squirt Ciona intestinalis turned up an unusual molecule with sequence similarity to both ion channels and phosphatases, dubbed Ci-VSP, short for C. intestinalis voltage sensor-containing phosphatase. Murata et al. characterized the protein and showed that it is in fact a transmembrane phosphatase and that its enzymatic activity can be controlled by changes in voltage across the plasma membrane. The sequence similarity of Ci-VSP to channels includes similarity to the voltage-sensing portions of voltage-regulated potassium channels. The catalytic region of Ci-VSP resembles that of the lipid phosphatase PTEN, and the authors showed that Ci-VSP dephosphorylated phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] in vitro. Murata et al. expressed inwardly rectifying potassium channels [which are regulated by PtdIns(4,5)P 2 ] as sensors for lipid phosphatase activity in Xenopus oocytes that also expressed Ci-VSP. These experiments revealed voltage-dependent regulation of Ci-VSP activity. A great deal of effort has gone into understanding how movements of the voltage sensor of potassium channels regulate channel opening. The results of Murata et al. open a new area for investigation into the mechanisms by which similar segments may be used to control enzymatic activity. Y. Murata, H. Iwasaki, M. Sasaki, K. Inaba, Y. Okamura, Phosphoinositide phosphatase activity coupled to an intrinsic voltage sensor. Nature 435 , 1239-1243 (2005). [PubMed]

A Proton Pore in the K Channel Voltage Sensor

Several models have been proposed for how the movement of the voltage-sensing S4 domain of K channels from the "in" conformation to the "out" conformation (in and out refer to the position of the S4 domain relative to the membrane) results in channel opening. The S5 and S6 domains of each of the four subunits that form a functional channel contribute to the ion-selective pore of the channel. Starace and Bezanilla demonstrate that mutation of a specific arginine residue in the S4 domain of the Aeropyrum pernix K channel results in the formation of a proton pore. They replaced the first arginine in the S4 domain with histidine (R362H) and used electrophysiological recordings of oocytes expressing this mutated channel to determine that the channel can conduct K + ions through the channel pore and protons through the voltage sensor region. The R362H channel only conducted protons when the S4 domain was in the "in" conformation. Proton flux persisted if the pore for K + flux was blocked with agitoxin, thus suggesting that protons were flowing through the sensor region and not the K + pore. Blaustein and Miller discuss the implications for these results in understanding channel gating and refining the various proposed models. D. M. Starace, F. Bezanilla, A proton pore in a potassium channel voltage sensor reveals a focused electric field. Nature 427 , 548-553 (2004). [Online Journal] R. O. Blaustein, C. Miller, Shake, rattle or roll? Nature 427 , 499-500 (2004). [Online Journal]

Specificity of Charge-carrying Residues in the Voltage Sensor of Potassium Channels

Positively charged voltage sensors of sodium and potassium channels are driven outward through the membrane's electric field upon depolarization. This movement is coupled to channel opening. A recent model based on studies of the KvAP channel proposes that the positively charged voltage sensor, christened the “voltage-sensor paddle”, is a peripheral domain that shuttles its charged cargo through membrane lipid like a hydrophobic cation. We tested this idea by attaching charged adducts to cysteines introduced into the putative voltage-sensor paddle of Shaker potassium channels and measuring fractional changes in the total gating charge from gating currents. The only residues capable of translocating attached charges through the membrane-electric field are those that serve this function in the native channel. This remarkable specificity indicates that charge movement involves highly specialized interactions between the voltage sensor and other regions of the protein, a mechanism inconsistent with the paddle model.

A proton pore in a potassium channel voltage sensor reveals a focused electric field.

Voltage-dependent potassium channels are essential for the generation of nerve impulses. Voltage sensitivity is conferred by charged residues located mainly in the fourth transmembrane segment (S4) of each of the four identical subunits that make up the channel. These charged segments relocate when the potential difference across the membrane changes, controlling the ability of the pore to conduct ions. In the crystal structure of the Aeropyrum pernix potassium channel KvAP, the S4 and part of the third (S3B) transmembrane alpha-helices are connected by a hairpin turn in an arrangement termed the 'voltage-sensor paddle'. This structure was proposed to move through the lipid bilayer during channel activation, transporting positive charges across a large fraction of the membrane. Here we show that replacing the first S4 arginine by histidine in the Shaker potassium channel creates a proton pore when the cell is hyperpolarized. Formation of this pore does not support the paddle model, as protons would not have access to a lipid-buried histidine. We conclude that, at hyperpolarized potentials, water and protons from the internal and external solutions must be separated by a narrow barrier in the channel protein that focuses the electric field to a small voltage-sensitive region.

The dipole moments of membrane proteins: potassium channel proteins. II. T1 assembly. Search for the voltage sensor

The dipole moments of potassium channel protein (Kcsa) and β-subunits were discussed in the previous paper of this series [Takashima, Biophys. Chem. 94 (2001) 209–218]. While the dipole moment of β-subunits was found to be very large, the dipole moment of Kcsa turned out to be somewhat smaller than β-subunits. As the continuation of this work, the discussion of the present paper is focussed on the dipole moment of T1 assembly, another component of the K-channel. As discussed later, the calculation using the X-ray crystallographic data by MacKinnon et al., revealed an astoundingly large dipole moment for T1 assembly. The dipole moment of T1 assembly combined with the likewise large dipole moment of β-subunits amounts to a sufficient value to play an essential role as a voltage sensor of potassium channel.

Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence.

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.

S15-4 - Elements of the voltage sensor of Potassium Channels

S15-4 - Elements of the voltage sensor of Potassium Channels