Shaker Potassium Channel Research Articles

Random insertion of split-cans of the fluorescent protein venus into Shaker channels yields voltage sensitive probes with improved membrane localization in mammalian cells

FlaSh-YFP, a fluorescent protein (FP) voltage sensor that is a fusion of the Shaker potassium channel with yellow fluorescent protein (YFP), is primarily expressed in the endoplasmic reticulum (ER) of mammalian cells, possibly due to misfolded monomers. In an effort to improve plasma membrane expression, the FP was split into two non-fluorescent halves. Each half was randomly inserted into Shaker monomers via a transposon reaction. Shaker subunits containing the 5′ half were co-expressed with Shaker subunits containing the 3′ half. Tetramerization of Shaker subunits is required for re-conjugation of the FP. The misfolded monomers trapped in ER are unlikely to tetramerize and reconstitute the beta-can structure, and thus intracellular fluorescence might be reduced. This split-can transposon approach yielded 56 fluorescent probes, 30 (54%) of which were expressed at the plasma membrane and were capable of optically reporting changes in membrane potential. The largest signal from these novel FP-sensors was a −1.4% in Δ F/ F for a 100 mV depolarization, with on time constants of about 15 ms and off time constants of about 200 ms. This split-can transposon approach has the potential to improve other multimeric probes.

The Phenylalanine in the Gating Charge Transfer Center is Critical for Modulating the Rate of Voltage Sensor Movement in Shaker Potassium Channels

Voltage-gated potassium channels contribute to cellular excitability by selectively gating the transmembrane passage of potassium ions. The homologous residue to Phe290 in Shaker potassium channels is invariably an aromatic side chain in voltage-gated ion channels. Interestingly, a recent study has suggested that aromaticity at this position is not required for WT-like function. Furthermore, tryptophan substitutions, together with lysine substitutions of the charge-carrying arginines in S4 can dramatically affect voltage-sensor movement. However, it remains unclear how individual mutations at position 290 alone affect voltage-sensor movements. Given the close physical proximity of Phe290 and the voltage sensor, we hypothesized that mutations at Phe290 should affect the rate of voltage sensor movement. To test this hypothesis we replaced Phe290 by the smaller, hydrophobic side chains, Ala and Leu, and monitored voltage sensor movement with gating current recordings in HEK293 cells and voltage-clamp fluorometry experiments in Xenopus oocytes. Surprisingly, our results show that hydrophobic, non-cyclic side chain substitutions at position 290 do not change the voltage dependence of the voltage sensor movement (i.e. QV and FV). However, the ON component of gating currents and fluorescence readout from reporter groups on the top of S4 are substantially slowed. As voltage-gated sodium channels display dramatically faster gating kinetics than potassium channels we next sought to investigate if aromaticity is required at the homologous positions in one or more of the four domains of the skeletal sodium channel. We employed in vivo non-sense suppression to incorporate fluorinated Phe derivatives in all four domains. However, fluorination had only minor effects on the voltage-dependence of activation and steady-state inactivation, suggesting that, similar to Shaker potassium channels, aromaticity is not absolutely required in these positions in sodium channels.

A shaker Potassium Channel with a Miniature Engineered Voltage Sensor

Voltage-gated ion channels sense transmembrane voltage changes via a paddle-shaped motif that includes the C-terminal part of the third transmembrane segment (S3b) and the N-terminal part of the fourth segment (S4) that harbors voltage-sensing arginines. Here, we find that residue triplets in S3b and NTS4 can be deleted individually, or even in some combinations, without compromising the channels’ basic voltage-gating capability. Thus, a high degree of complementarity between these S3b and NTS4 regions is not required for basic voltage gating per se. Remarkably, the voltage-gated Shaker K+ channel remains voltage gated after a 43 residue paddle sequence is replaced by a glycine triplet. Therefore, the paddle motif comprises a minimal core that suffices to confer voltage gating in the physiological voltage range, and a larger, modulatory part. Our study also shows that the hydrophobic residues between the voltage-sensing arginines help set the sensor's characteristic chemical equilibrium between activated and deactivated states.

Molecular Rearrangements During Slow Inactivation of the Shaker-Ir Potassium Channel

Crosstalk between the activation and slow inactivation gates in Shaker potassium channels is now well-established. The activation gate perceives the conformation of the inactivation gate (Panyi and Deutsch, 2006, 2007). Closure of the inactivation gate speeds opening and slows closing of the activation gate, i.e., stabilizing the gate in the open configuration. If this coupling involves movement of the S6 transmembrane segment, then we predict state-dependent changes in accessibility of residues lining the channel cavity.

Fluorinated General Anesthetics Modulate Shaker Potassium Channels at Clinically Relevant Concentrations

Recent studies demonstrated that selective intra-thalamic block of the Kv1.2 potassium channels rapidly reverses the unconsciousness of inhalational anesthesia (Alkire et al., 2009). Furthermore, intra-thalamic block of Kv1.3 with selective blocker, ShK, reverses the unconsciousness effect of sevoflurane. Both findings suggestthat Shaker-related potassium channels might be key targets for anesthetics. We tested this hypothesis using patch clamp recordings of potassium currents in cell lines stably expressing homomeric human Shaker family channels. We found that at low voltages, sub-clinical and clinical doses of sevoflurane irreversibly potentiate potassium current through Kv1.3>Kv1.2>Kv1.5>Kv1.1. channels, by increasing the peak current amplitude and accelerating the current activation kinetics. At higher voltages, clinical doses of sevoflurane inhibit Kv1.4>Kv1.5.>Kv1.3 and potentiate Kv1.2>Kv1.1. Sevoflurane had no effect on lipid bilayer conductance, suggesting direct interaction of the anesthetic with potassium channel subunits. Thus, Shaker potassium channel modulation may contribute to the clinical effects of general anesthesia. Supported by the Hillblom Foundation and NIH 1P01AG032131.

Ion Conduction in a Shaker Potassium Channel Mutant Having an Unusually High Single Channel Conductance

K-channels are endowed of a highly K+ selective pore but are very diverse in single channel conductance, ranging from 3 to 300 pS. Because the selectivity filter is so conserved among K-channels, the origin of this diversity must be searched for in other parts of the pore. We studied the single-channel behavior of the Shaker K-channel with the P475D point mutation located at its internal entrance. In 100 mM K+ solution, this variant has a unitary conductance 8-10 fold larger than wild type (Sukhareva et al.

Voltage Sensor Immobilization in Kv1.2 Channels

The voltage gated potassium channel family contains several functionally distinct isoforms which serve to shape the duration, frequency and timing of action potential firing in electrically excitable cells. Several mechanistic elements that contribute to the function of the voltage sensing apparatus are not yet fully described, especially in channels other than the prototypical Shaker potassium channel. We have studied voltage sensor rearrangements of Kv1.2 channels using two-electrode voltage clamp fluorometry and gating current recordings in mammalian cells. Fluorescence measurements reporting on voltage sensor movement revealed a transition into a relaxed state upon prolonged depolarization, causing a left shift in the fluorescence-voltage relationship. Gating current measurements of wild type Kv1.2 channels recorded in permeant ion free solutions (NMDG+int//TEA+ext) also displayed a left shifted Q-V after depolarizing pre-pulses, reflecting a stabilization of the activated state of the voltage sensor. To enable examination of the effects of different cations and processes of inactivation in Kv1.2 a non-conducting double mutant channel mimicking the permanently slow inactivated and non-conducting Shaker W434F, Kv1.2(W366F, V381T) was created. Off-gating currents recorded from this channel in the presence of permeant ions displayed less voltage sensor stabilization in the activated state than the wild type channel. These data suggest that cations play a specific role in regulating voltage sensor dynamics in the Kv1.2 channel.

Position and motions of the S4 helix during opening of the Shaker potassium channel

The four voltage sensors in voltage-gated potassium (Kv) channels activate upon membrane depolarization and open the pore. The location and motion of the voltage-sensing S4 helix during the early activation steps and the final opening transition are unresolved. We studied Zn2+ bridges between two introduced His residues in Shaker Kv channels: one in the R1 position at the outer end of the S4 helix (R362H), and another in the S5 helix of the pore domain (A419H or F416H). Zn2+ bridges readily form between R362H and A419H in open channels after the S4 helix has undergone its final motion. In contrast, a distinct bridge forms between R362H and F416H after early S4 activation, but before the final S4 motion. Both bridges form rapidly, providing constraints on the average position of S4 relative to the pore. These results demonstrate that the outer ends of S4 and S5 remain in close proximity during the final opening transition, with the S4 helix translating a significant distance normal to the membrane plane.

State-dependent sculpting of olfactory sensory neurons is attributed to sensory enrichment, odor deprivation, and aging

Gene-targeted deletion of the predominant Shaker potassium channel, Kv1.3, in the mitral cells of the olfactory bulb, decreases the number of presynaptic, odorant receptor (OR)-identified olfactory sensory neurons (OSNs) in the main olfactory epithelium (MOE) and alters the nature of their postsynaptic connections to mitral cell targets. The current study examined whether OSN density was state-dependent by examining the impact of (1) odor enrichment, (2) sensory deprivation, and (3) aging upon the number of P2- or M72-expressing neurons. Histological approaches were used to quantify the number of OSNs across entire epithelia for wildtype (WT) vs. Kv1.3-null (KO) mice bred onto an ORtauLacZ reporter background. Following either odor enrichment or early unilateral naris-occlusion, the number of M72-expressing OSNs was significantly decreased in WT mice, but was unchanged in KO animals. Following naris-occlusion, the number of P2-expressing OSNs was decreased regardless of genotype. Animals that were reared to 2 years of age demonstrated loss of both P2- and M72-expressing OSNs in WT mice and a concomitant loss of only M72-expressing neurons in KO mice. These findings suggest that voltage-gated activity of the mitral cells is important for OSN plasticity, and can prevent neuronal loss via sensory- and OR-dependent mechanisms.

Structure of the full-length Shaker potassium channel Kv1.2 by normal-mode-based X-ray crystallographic refinement

Voltage-dependent potassium channels (Kv) are homotetramers composed of four voltage sensors and one pore domain. Because of high-level structural flexibility, the first mammalian Kv structure, Kv1.2 at 2.9 A, has about 37% molecular mass of the transmembrane portion not resolved. In this study, by applying a novel normal-mode-based X-ray crystallographic refinement method to the original diffraction data and structural model, we established the structure of full-length Kv1.2 in its native form. This structure offers mechanistic insights into voltage sensing. Particularly, it shows a hydrophobic layer of about 10 A at the midpoint of the membrane bilayer, which is likely the molecular basis for the observed "focused electric field" of Kv1.2 between the internal and external solutions. This work also demonstrated the potential of the refinement method in bringing up large chunks of missing densities, thus beneficial to structural refinement of many difficult systems.

Calculation of the Gating Charge for the Kv1.2 Voltage-Activated Potassium Channel

The atomic models of the Kv1.2 potassium channel in the active and resting state, originally presented elsewhere, are here refined using molecular dynamics simulations in an explicit membrane-solvent environment. With a minor adjustment of the orientation of the first arginine along the S4 segment, the total gating charge of the channel determined from >0.5 μs of molecular dynamics simulation is ∼12–12.7 e, in good accord with experimental estimates for the Shaker potassium channel, indicating that the final models offer a realistic depiction of voltage-gating. In the resting state of Kv1.2, the S4 segment in the voltage-sensing domain (VSD) spontaneously converts into a 310 helix over a stretch of 10 residues. The 310 helical conformation orients the gating arginines on S4 toward a water-filled crevice within the VSD and allows salt-bridge interactions with negatively charged residues along S2 and S3. Free energy calculations of the fractional transmembrane potential, acting upon key charged residues of the VSD, reveals that the applied field varies rapidly over a narrow region of 10–15 Å corresponding to the outer leaflet of the bilayer. The focused field allows the transfer of a large gating charge without translocation of S4 across the membrane.

Modulation of the Frequency Response of Shaker Potassium Channels by the Quiver Peptide Suggesting a Novel Extracellular Interaction Mechanism

:Recent studies have indicated that the Shaker potassium channel regulates sleep in Drosophila. The Drosophila quiver (qvr) gene encodes a novel potassium channel subunit that modulates the Shaker potassium channel. The Qvr peptide contains a signal sequence for extracellular localization and may regulate a unique feature of the Shaker IA current that confers special neuronal excitability patterns. Thus, studies of the Shaker channel properties in the qvr mutant background should provide an opportunity to uncover a new form of physiologic modulation of potassium channels. We have begun to investigate the impact of qvr protein on the Shaker channel properties and its implications in synaptic function in vivo. We studied synaptic transmission at the larval neuromuscular junction and characterized the transient potassium current IA in larval muscles. We identified two different functional states of IA in qvr larval muscles, as reflected by two distinct components, IAF and IAS, differing in their kinetics of recovery from inactivation and sensitivity to a K+ channel blocker. Correspondingly, qvr mutant larvae exhibit multiple synaptic discharges following individual nerve stimuli during repetitive activity.

Double gaps along Shaker S4 demonstrate omega currents at three different closed states

The aim of the present study was to investigate in detail how the voltage sensor in the Shaker potassium channel moves during the gating process. After the publication of the open channel structure from the crystallized KvAP channel in 2003, an alternative so-called "paddle" model was put forward in contrast to the existing helical screw model. The voltage sensor S4 contains 4 arginine residues relevant for gating, R1(362), R2(365), R3(368) and R4(371), each separated by 2 neutral residues. These charged residues coil as one of three threads on the S4-α-helix. Based on a previous finding that the mutation R1S leads to the so-called omega leak current through a "gating-pore" in the closed state, we introduced gaps systematically along the arginine thread substituting long arginines by short serines. Mutations R2S or R3S did neither create transient nor steady leaks. The fact that the native residue A359, which is located three amino acids in front of R1, is a short one, motivated us to check its role. Mutation of A359 to arginine blocked the omega current in R1S mutant indicating that the omega pore is occupied by A359 and R1. Introducing further double gaps (RR to SS) at sequential positions (0+1, 1+2, 2+3), produced clear leak currents which were remarkably stable over a wide voltage range. These leaks contradict that S4 would swing together with S3 in lipid according to the paddle hypothesis. Rather, our results show that during gating the S4 segment moves in 3 helical steps through a fixed pore formed by the channel protein.

Biophysical Properties of Three Omega Gaps Along the Voltage Sensor S4 of Shaker Potassium Channel

Omega current is a cation-selective current conducted through the voltage sensor domain of ion channel when the first arginine R1 is replaced by a short residue. We were able to show Omega current for three different gaps along the voltage sensor S4 in Shaker potassium channel. These omega currents appear when two successive arginines were mutated to short amino acids (serines) creating a gap of short residues in between the long residues. The omega current starts to show at different negative potentials according to the position of the mutated arginines and down to approximately −200 mV. While the classical mutant which has Ala359 and R362S occupying the pore show an onset of omega current at −70 mV, the two other mutants with gaps at R362S/R365S and R365S/R368S showed omega onsets at −50 and −30 mV respectively (Gamal El-Din et al. Biophys. J. 96(3) pp. 381a 2009). The Omega current in the three constructs were conducted down to −200 mV and voltage-dependent closing of the gaps seems to occur at potentials less than −200 mV. Fluorescence of EGFP-bound ion channels was used as a measure of number of expressed ion channels and thus to quantify the omega currents. The biophysical properties of these different omega pores (current-voltage, conductance and gating charge-voltage correlations) are presented in this work. Gating charge for the different constructs is correlated with our proposed model of the gating steps of the voltage sensor S4.

Two Structurally Distinct Pathways for the Voltage-Sensing S4 Helices

In voltage-dependent ion channels, the movement of the voltage-sensing S4 helices produces gating currents. The charge displaced as a function of the membrane potential (Q-V) is well described by a sequential two-state Boltzmann relation, indicating that there are at least two steps of gating charge movement from their Resting state to the Active state. In addition, it has been shown that at a maintained positive potential, the S4 helices of voltage-gated Na, Ca and K channels and the voltage sensitive phosphatase Ci-VSP, undergo a slower secondary conformational transition stabilizing the sensor in a Relaxed (inactivated) state. From the Relaxed state, the Q-V relation exhibits a strong shift towards negative potentials when compared to the Q-V relation measured from the resting state. We engineered gating perturbations in the Shaker potassium channel, by substituting specific aromatic residues in positions spatially close to the S4. One of these mutants, in position I241 of S1, part of the hydrophobic plug of the voltage sensor, when mutated to tryptophan (I241W), produces a strong split in the Q-V when measured from the resting state. By labeling M356C with TMRM we also find the same split in the fluorescence-voltage curve. We propose that the presence of the tryptophan in the 241 position favors an interaction with one of the positively-charged arginines along the S4, thus stabilizing a fleeting intermediate state in the gating pathway. However, in the I241W mutant, the split in the Q-V almost disappears when the gating currents are measured from the relaxed state and the same result is seen with the fluorescence-voltage curve. This result and the effect of other tryptophan perturbations near the S4 segment strongly support the existence of two structurally distinct gating pathways for the movement of the S4 helices. Supported: NIHGM030376.

Charge Reversion to Charge Carrying Positions of S4 in Voltage Gated Shaker K-Channels

Voltage gated Shaker potassium channels increases open probability (Po) by 20-fold with a ∼6 mV depolarization. Such high voltage sensitivity is mostly due to the electrophoretic transmembrane relocation of four arginines residues in each of their four voltage sensing protein domains (VSD). These arginines movement across the electric field make possible channel opening upon membrane depolarization. We tested if the positions occupied by the voltage sensing arginines could carry acidic residues. We mutated three of these positions to aspartate: Arg362, Arg365, and Arg368 on an N-type inactivation removed background Shaker channel. All mutations were introduced with the use of the QuikChange kit and the mutation was verified by sequencing. Heterologous expressed in Xenopus oocytes, all mutants showed levels of expression comparable to that of the native channel. To determine voltage sensitivity of these charge reverting modified channels, we measured the voltage dependency of Po at voltages negative enough to observe only sporadic single channel openings in membrane patches containing hundreds of channels. From the exponential relation between Po and voltage we estimated the effective valence of opening in the range of Po ∼10-6. All charge mutants showed an effective valence ∼50% of that of the native Shaker. These results together with the comparable level of channel expression in oocytes are consistent with the idea the voltage sensitive positions in S4 are not specific for basic residues.VGP was a doctoral Conicyt fellow and DN was financed by Fondecyt #1090493

Refinement and Validation of Atomic Models of the Kv1.2 Potassium Channel Through Molecular Dynamics and Gating Charge Calculations

Voltage-gated potassium (Kv) channels are membrane proteins that respond to changes in transmembrane potential through voltage-sensing domains (VSD). These domains are composed of highly charged transmembrane segments that move in response to changes in electric potential and control opening of the ion conduction gate. Complete atomistic models of Kv1.2, in the open and closed states, have been constructed using the structure prediction program Rosetta and available crystallographic structures. By means of molecular dynamics simulations, the two models are refined in the presence of an external voltage bias leading to stable conformations of the channel in an explicit membrane-solvent environment. Salt-bridge interactions stabilizing the VSD are identified within the VSD and between the charged residues of the VSD and lipid head groups. Conformational changes in the VSD result in the transfer of electric charge across the membrane, that can be measured as a gating current. The magnitude of the gating charge in Kv1.2 potassium channel is calculated from more than 1microsecond of all-atom molecular dynamics simulation. Free energy calculations are performed to determine the individual contribution of several (nine) charged residues of the VSD to the gating charge. The total gating charge obtained for the refined models of the channel is ∼10.5e, indicating that the refined model of the closed resting state most likely represents an intermediate conformation that precedes closing of the channel. Through steered molecular dynamics (SMD) simulations we identify a closed conformation of the channel, corresponding to a gating charge of 12.7e, in accord with experimental values obtained for the Shaker potassium channel.

Probing Energetic Contributions of Aromatic Residues at the Intracellular Gate of Shaker Potassium Channels

Voltage-gated potassium channels contribute to cellular excitability by selectively gating the transmembrane passage of potassium ions. The intracellular bundle crossing of these channels is populated by aromatic phenylalanine and tyrosine residues that have been proposed to form a hydrophobic seal in the closed conformation of the channel. One such residue, Phe481, has resisted previous study by traditional site-directed mutagenesis because non-aromatic substitutions at this site fail to produce functional channels.

SLEEPLESS, a Ly-6/neurotoxin family member, regulates the levels, localization and activity of Shaker

Sleep is a whole–organism phenomenon accompanied by global changes in neural activity. We previously identified SLEEPLESS (SSS) as a novel glycosylphosphatidyl–inositol–anchored protein required for sleep in Drosophila. Here, we demonstrate a critical role for SSS in regulating the sleep–modulating potassium channel, Shaker. SSS and Shaker exhibit similar expression patterns in the brain and specifically affect each other’s expression levels. sss mutants exhibit altered Shaker localization, reduced Shaker current density, and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescues defects in Shaker expression and activity cell–autonomously and also suggests that SSS functions in wake–promoting, cholinergic neurons. Importantly, in heterologous cells, SSS accelerates kinetics of Shaker currents and can be co–immunoprecipitated with Shaker, suggesting that SSS interacts with Shaker and modulates its activity. SSS is predicted to belong to the Ly–6/neurotoxin superfamily, suggesting a novel mechanism for regulation of neuronal excitability by endogenous toxin–like molecules.

Use of voltage clamp fluorimetry in understanding potassium channel gating: a review of Shaker fluorescence data

Voltage clamp fluorimetry (VCF) utilizes fluorescent probes that covalently bind to cysteine residues introduced into proteins and emit light as a function of their environment. Measurement of this emitted light during membrane depolarization reveals changes in the emission level as the environment of the labelled residue changes. This allows for the correlation of channel gating events with movement of specific protein moieties, at nanosecond time resolution. Since the pioneering use of this technique to investigate Shaker potassium channel activation movements, VCF has become an invaluable technique used to understand ion channel gating. This review summarizes the theory and some of the data on the application of the VCF technique. Although its usage has expanded beyond voltage-gated potassium channels and VCF is now used in a number of other voltage- and ligand-gated channels, we will focus on studies conducted in Shaker potassium channels, and what they have told us about channel activation and inactivation gating.