Chimeras of KcsA and Kv1 as a bioengineering tool to study voltage-gated potassium channels and their ligands.

Chimeric potassium channels KcsA-Kv1, which are among the most intensively studied hybrid membrane proteins to date, were constructed by replacing a part of the pore domain of bacterial potassium channel KcsA (K channel of streptomyces A) with corresponding regions of the mammalian voltage-gated potassium channels belonging to the Kv1 subfamily. In this way, the pore blocker binding site of Kv1 channels was transferred to KcsA, opening up possibility to use the obtained hybrids as receptors of Kv1-channel pore blockers of different origin. In this review the recent progress in KcsA-Kv1 channel design and applications is discussed with a focus on the development of new assays for studying interactions of pore blockers with the channels. A summary of experimental data is presented demonstrating that hybrid channels reproduce the blocker-binding profiles of parental Kv1 channels. It is overviewed how the KcsA-Kv1 chimeras are used to get new insight into the structure of potassium channels, to determine molecular basis for high affinity and selectivity of binding of peptide blockers to Kv1 channels, as well as to identify new peptide ligands.

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.

Binding of κ-Conotoxin-PVIIA to Open and Closed Shaker K-Channels Are Differentially Affected by the Ionic Strength.

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.

Cryo-EM structure of the KvAP channel reveals a non-domain-swapped voltage sensor topology.

Conductance in voltage-gated ion channels is regulated by membrane voltage through structural domains known as voltage sensors. A single structural class of voltage sensor domain exists, but two different modes of voltage sensor attachment to the pore occur in nature: domain-swapped and non-domain-swapped. Since the more thoroughly studied Kv1-7, Nav and Cav channels have domain-swapped voltage sensors, much less is known about non-domain-swapped voltage-gated ion channels. In this paper, using cryo-EM, we show that KvAP from Aeropyrum pernix has non-domain-swapped voltage sensors as well as other unusual features. The new structure, together with previous functional data, suggests that KvAP and the Shaker channel, to which KvAP is most often compared, probably undergo rather different voltage-dependent conformational changes when they open.

Simulation of Gating Currents of the Shaker K Channel Using a Brownian Model of the Voltage Sensor.

The physical mechanism underlying the voltage-dependent gating of K channels is usually addressed theoretically using molecular dynamics simulations. However, besides being computationally very expensive, this approach is presently unable to fully predict the behavior of fundamental variables of channel gating such as the macroscopic gating current, and hence, it is presently unable to validate the model. To fill this gap, here we propose a voltage-gating model that treats the S4 segment as a Brownian particle moving through a gating channel pore and adjacent internal and external vestibules. In our model, charges on the S4 segment are screened by charged residues localized on neighboring segments of the channel protein and by ions present in the vestibules, whose dynamics are assessed using a flux conservation equation. The electrostatic voltage spatial profile is consistently assessed by applying the Poisson equation to all the charges present in the system. The treatment of the S4 segment as a Brownian particle allows description of the dynamics of a single S4 segment using the Langevin stochastic differential equation or the behavior of a population of S4 segments-useful for assessing the macroscopic gating current-using the Fokker-Planck equation. The proposed model confirms the gating charge transfer hypothesis with the movement of the S4 segment among five different stable positions where the gating charges interact in succession with the negatively charged residues on the channel protein. This behavior produces macroscopic gating currents quite similar to those experimentally found.

NMR Structural Analysis of Isolated Shaker Voltage-Sensing Domain in LPPG Micelles.

The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. To probe dynamics with site-specific resolution, we utilized NMR spectroscopy to characterize the VSD derived from Shaker potassium channel in 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPPG) micelles. The backbone dihedral angles predicted based on secondary chemical shifts using torsion angle likeliness obtained from shift (TALOS+) showed that the Shaker-VSD shares many structural features with the homologous Kv1.2/2.1 chimera, including a transition from α-helix to 310 helix in the C-terminal portion of the fourth transmembrane helix. Nevertheless, there are clear differences between the Shaker-VSD and Kv1.2/2.1 chimera in the S2-S3 linker and S3 transmembrane region, where the organization of secondary structure elements in Shaker-VSD appears to more closely resemble the KvAP-VSD. Comparison of microsecond-long molecular dynamics simulations of Kv 1.2-VSD in LPPG micelles and a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer showed that LPPG micelles do not induce significant structural distortion in the isolated voltage sensor. To assess the integrity of the tertiary fold, we directly probed the binding of BrMT analog 2-[2-({[3-(2-amino-ethyl)-6-bromo-1H-indol-2-yl]methoxy}k7methyl)-6-bromo-1H-indol-3-yl]ethan-1-amine (BrET), a gating modifier toxin, and identified the location of the putative binding site. Our results suggest that the Shaker-VSD in LPPG micelles is in a native-like fold and is likely to provide valuable insights into the dynamics of voltage-gating and its regulation.

In Silico Analysis of the Subtype Selective Blockage of KCNA Ion Channels through the µ-Conotoxins PIIIA, SIIIA, and GIIIA.

Understanding subtype specific ion channel pore blockage by natural peptide-based toxins is crucial for developing such compounds into promising drug candidates. Herein, docking and molecular dynamics simulations were employed in order to understand the dynamics and binding states of the µ-conotoxins, PIIIA, SIIIA, and GIIIA, at the voltage-gated potassium channels of the KV1 family, and they were correlated with their experimental activities recently reported by Leipold et al. Their different activities can only adequately be understood when dynamic information about the toxin-channel systems is available. For all of the channel-bound toxins investigated herein, a certain conformational flexibility was observed during the molecular dynamic simulations, which corresponds to their bioactivity. Our data suggest a similar binding mode of µ-PIIIA at KV1.6 and KV1.1, in which a plethora of hydrogen bonds are formed by the Arg and Lys residues within the α-helical core region of µ-PIIIA, with the central pore residues of the channel. Furthermore, the contribution of the K+ channel's outer and inner pore loops with respect to the toxin binding. and how the subtype specificity is induced, were proposed.

Conotoxin κM-RIIIJ, a tool targeting asymmetric heteromeric Kv1 channels.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

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.

Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels.

Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1-R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum.

S4-S5 linker movement during activation and inactivation in voltage-gated K+ channels.

The S4-S5 linker physically links voltage sensor and pore domain in voltage-gated ion channels and is essential for electromechanical coupling between both domains. Little dynamic information is available on the movement of the cytosolic S4-S5 linker due to lack of a direct electrical or optical readout. To understand the movements of the gating machinery during activation and inactivation, we incorporated fluorescent unnatural amino acids at four positions along the linker of the Shaker KV channel. Using two-color voltage-clamp fluorometry, we compared S4-S5 linker movements with charge displacement, S4 movement, and pore opening. We found that the proximal S4-S5 linker moves with the S4 helix throughout the gating process, whereas the distal portion undergoes a separate motion related to late gating transitions. Both pore and S4-S5 linker undergo rearrangements during C-type inactivation. In presence of accelerated C-type inactivation, the energetic coupling between movement of the distal S4-S5 linker and pore opening disappears.

Replacing voltage sensor arginines with citrulline provides mechanistic insight into charge versus shape.

Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.

Deglycosylation of Shaker KV channels affects voltage sensing and the open-closed transition.

Most membrane proteins are subject to posttranslational glycosylation, which influences protein function, folding, solubility, stability, and trafficking. This modification has been proposed to protect proteins from proteolysis and modify protein-protein interactions. Voltage-activated ion channels are heavily glycosylated, which can result in up to 30% of the mature molecular mass being contributed by glycans. Normally, the functional consequences of glycosylation are assessed by comparing the function of fully glycosylated proteins with those in which glycosylation sites have been mutated or by expressing proteins in model cells lacking glycosylation enzymes. Here, we study the functional consequences of deglycosylation by PNGase F within the same population of voltage-activated potassium (KV) channels. We find that removal of sugar moieties has a small, but direct, influence on the voltage-sensing properties and final opening-closing transition of Shaker KV channels. Yet, we observe that the interactions of various ligands with different domains of the protein are not affected by deglycosylation. These results imply that the sugar mass attached to the voltage sensor neither represents a cargo for the dynamics of this domain nor imposes obstacles to the access of interacting molecules.

Atom-by-atom tuning of the electrostatic potassium-channel modulator dehydroabietic acid.

Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel's positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel's voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug.

Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K+ channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K+ channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K+ depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters (V0, the voltage-dependent transition from the resting to intermediate state and V1, from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter (V1/2), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V0, V1, and V1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V0 is affected by the hydrophobicity of the AA in position 361, whereas V1 and V1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V1 and V1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics.

Crystal structure of an inactivated mutant mammalian voltage-gated K+ channel.

C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion-selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl-oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark of C-type inactivation.

Atomistic Modeling of Ion Conduction through the Voltage-Sensing Domain of the Shaker K+ Ion Channel.

Voltage-sensing domains (VSDs) sense changes in the membrane electrostatic potential and, through conformational changes, regulate a specific function. The VSDs of wild-type voltage-dependent K+, Na+, and Ca2+ channels do not conduct ions, but they can become ion-permeable through pathological mutations in the VSD. Relatively little is known about the underlying mechanisms of conduction through VSDs. The most detailed studies have been performed on Shaker K+ channel variants in which ion conduction through the VSD is manifested in electrophysiology experiments as a voltage-dependent inward current, the so-called omega current, which appears when the VSDs are in their resting state conformation. Only monovalent cations appear to permeate the Shaker VSD via a pathway that is believed to be, at least in part, the same as that followed by the S4 basic side chains during voltage-dependent activation. We performed µs-time scale atomistic molecular dynamics simulations of a cation-conducting variant of the Shaker VSD under applied electric fields in an experimentally validated resting-state conformation, embedded in a lipid bilayer surrounded by solutions containing guanidinium chloride or potassium chloride. Our simulations provide insights into the Shaker VSD permeation pathway, the protein-ion interactions that control permeation kinetics, and the mechanism of voltage-dependent activation of voltage-gated ion channels.

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel.

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

Complexes of Peptide Blockers with Kv1.6 Pore Domain: Molecular Modeling and Studies with KcsA-Kv1.6 Channel.

Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K+-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

Intramolecular interactions that control voltage sensitivity in the jShak1 potassium channel from Polyorchis penicillatus.

Voltage-gated potassium ion (Kv) channel proteins respond to changes in membrane potential by changing the probability of K+ flux through an ion-selective pore. Kv channels from different paralogous and orthologous families have widely varying V50 values. The voltage-sensing transmembrane helices (S4) of different channels contain four to seven basic residues that are responsible for transducing changes in transmembrane potential into the energy required to shift the equilibrium between the open- and closed-channel conformations. These residues also form electrostatic interaction networks with acidic residues in the S2 and S3 helices that stabilize the open and the closed states to different extents. The length and composition of the extracellular loop connecting the S3 and S4 helices (S3-S4 loop) also shape the voltage response. We describe mutagenesis experiments on the jellyfish (Polyorchis penicillatus) Kv1 family jShak1 channel to evaluate how variants of the S3-S4 loop affect the voltage sensitivity of this channel. In combination with changes in the length and composition of the S3-S4 linker, we mutated a residue on the S2 helix (N227) that in most Kv1 family channels is glutamate (E226 in mouse Kv1.2, E283 in D. melanogaster Shaker). Some individual loop replacement mutants cause major changes in voltage sensitivity, depending on a combination of length and composition. Pairwise combinations of the loop mutations and the S2 mutations interact to yield quantitatively distinct, non-additive changes in voltage sensitivity. We conclude that the S3-S4 loop interacts energetically with the residue at position N227 during the transitions between open and closed states of the channel.