Selective block of human Kv1.1 channels and an epilepsy-associated gain-of-function mutation by AETX-K peptide.

Dysfunction of the human voltage-gated K+ channel Kv1.1 has been associated with epilepsy, multiple sclerosis, episodic ataxia, myokymia, and cardiorespiratory dysregulation. We report here that AETX-K, a sea anemone type I (SAK1) peptide toxin we isolated from a phage display library, blocks Kv1.1 with high affinity (Ki ~ 1.6 pM) and notable specificity, inhibiting other Kv channels we tested a million-fold less well. Nuclear magnetic resonance (NMR) was employed both to determine the three-dimensional structure of AETX-K, showing it to employ a classic SAK1 scaffold while exhibiting a unique electrostatic potential surface, and to visualize AETX-K bound to the Kv1.1 pore domain embedded in lipoprotein nanodiscs. Study of Kv1.1 in Xenopus oocytes with AETX-K and point variants using electrophysiology demonstrated the blocking mechanism to employ a toxin-channel configuration we have described before whereby AETX-K Lys23 , two positions away on the toxin interaction surface from the classical blocking residue, enters the pore deeply enough to interact with K+ ions traversing the pathway from the opposite side of the membrane. The mutant channel Kv1.1-L296 F is associated with pharmaco-resistant multifocal epilepsy in infants because it significantly increases K+ currents by facilitating opening and slowing closure of the channels. Consistent with the therapeutic potential of AETX-K for Kv1.1 gain-of-function-associated diseases, AETX-K at 4 pM decreased Kv1.1-L296 F currents to wild-type levels; further, populations of heteromeric channels formed by co-expression Kv1.1 and Kv1.2, as found in many neurons, showed a Ki of ~10 nM even though homomeric Kv1.2 channels were insensitive to the toxin (Ki > 2000 nM).

Molecular determinants of inhibition of the human proton channel hHv1 by the designer peptide C6 and a bivalent derivative.

The human voltage-gated proton channel (hHv1) is important for control of intracellular pH. We designed C6, a specific peptide inhibitor of hHv1, to evaluate the roles of the channel in sperm capacitation and in the inflammatory immune response of neutrophils [R. Zhao et al., Proc. Natl. Acad. Sci. U.S.A. 115, E11847­E11856 (2018)]. One C6 binds with nanomolar affinity to each of the two S3­S4 voltage-sensor loops in hHv1 in cooperative fashion so that C6-bound channels require greater depolarization to open and do so more slowly. As depolarization drives hHv1 sensors outwardly, C6 affinity decreases, and inhibition is partial. Here, we identified residues essential to C6­hHv1 binding by scanning mutagenesis, five in the hHv1 S3­S4 loops and seven on C6. A structural model of the C6­hHv1 complex was then generated by molecular dynamics simulations and validated by mutant-cycle analysis. Guided by this model, we created a bivalent C6 peptide (C62) that binds simultaneously to both hHv1 subunits and fully inhibits current with picomolar affinity. The results help delineate the structural basis for C6 state-dependent inhibition, support an anionic lipid-mediated binding mechanism, and offer molecular insight into the effectiveness of engineered C6 as a therapeutic agent or lead.

Tethered peptide toxins for ion channels.

In this method paper, we describe protocols for using membrane-tethered peptide toxins (T-toxins) to study the structure/function and biophysics of toxin-channel interactions with two-electrode voltage clamp (TEVC). Here, we show how T-toxins can be used to determine toxin equilibrium affinity, to quantify toxin surface level by enzyme-linked immunosorbent assay (ELISA) and/or single-molecule total internal reflection fluorescence (smTIRF) microscopy, to assess toxin association and dissociations rate, to identify toxin residues critical to binding via scanning mutagenesis, and to study of toxin blocking mechanism. The sea anemone type I (SAK1) toxin HmK and a potassium channel are used to demonstrate the strategies. T-toxins offer experimental flexibility that facilitates studies of toxin variants by mutation of the expression plasmid, avoiding the need to synthesize and purify individual peptides, speeding and reducing the cost of studies. T-toxins can be applied to peptide toxins that target pores or regulatory domains, that inhibit or activate, that are derived from different species, and that bind to different types of ion channels.

Role of human Hv1 channels in sperm capacitation and white blood cell respiratory burst established by a designed peptide inhibitor.

Using a de novo peptide inhibitor, Corza6 (C6), we demonstrate that the human voltage-gated proton channel (hHv1) is the main pathway for H+ efflux that allows capacitation in sperm and permits sustained reactive oxygen species (ROS) production in white blood cells (WBCs). C6 was identified by a phage-display strategy whereby ∼1 million novel peptides were fabricated on an inhibitor cysteine knot (ICK) scaffold and sorting on purified hHv1 protein. Two C6 peptides bind to each dimeric channel, one on the S3-S4 loop of each voltage sensor domain (VSD). Binding is cooperative with an equilibrium affinity (Kd) of ∼1 nM at -50 mV. As expected for a VSD-directed toxin, C6 inhibits by shifting hHv1 activation to more positive voltages, slowing opening and speeding closure, effects that diminish with membrane depolarization.

Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection.

In order to multiply and cause disease a virus must transport its genome from outside the cell into the cytosol, most commonly achieved through the endocytic network. Endosomes transport virus particles to specific cellular destinations and viruses exploit the changing environment of maturing endocytic vesicles as triggers to mediate genome release. Previously we demonstrated that several bunyaviruses, which comprise the largest family of negative sense RNA viruses, require the activity of cellular potassium (K+) channels to cause productive infection. Specifically, we demonstrated a surprising role for K+ channels during virus endosomal trafficking. In this study, we have used the prototype bunyavirus, Bunyamwera virus (BUNV), as a tool to understand why K+ channels are required for progression of these viruses through the endocytic network. We report three major findings: First, the production of a dual fluorescently labelled bunyavirus to visualize virus trafficking in live cells. Second, we show that BUNV traffics through endosomes containing high [K+] and that these K+ ions influence the infectivity of virions. Third, we show that K+ channel inhibition can alter the distribution of K+ across the endosomal system and arrest virus trafficking in endosomes. These data suggest high endosomal [K+] is a critical cue that is required for virus infection, and is controlled by cellular K+ channels resident within the endosome network. This highlights cellular K+ channels as druggable targets to impede virus entry, infection and disease.

Designer and natural peptide toxin blockers of the KcsA potassium channel identified by phage display.

Peptide neurotoxins are powerful tools for research, diagnosis, and treatment of disease. Limiting broader use, most receptors lack an identified toxin that binds with high affinity and specificity. This paper describes isolation of toxins for one such orphan target, KcsA, a potassium channel that has been fundamental to delineating the structural basis for ion channel function. A phage-display strategy is presented whereby ∼1.5 million novel and natural peptides are fabricated on the scaffold present in ShK, a sea anemone type I (SAK1) toxin stabilized by three disulfide bonds. We describe two toxins selected by sorting on purified KcsA, one novel (Hui1, 34 residues) and one natural (HmK, 35 residues). Hui1 is potent, blocking single KcsA channels in planar lipid bilayers half-maximally (Ki) at 1 nM. Hui1 is also specific, inhibiting KcsA-Shaker channels in Xenopus oocytes with a Ki of 0.5 nM whereas Shaker, Kv1.2, and Kv1.3 channels are blocked over 200-fold less well. HmK is potent but promiscuous, blocking KcsA-Shaker, Shaker, Kv1.2, and Kv1.3 channels with Ki of 1-4 nM. As anticipated, one Hui1 blocks the KcsA pore and two conserved toxin residues, Lys21 and Tyr22, are essential for high-affinity binding. Unexpectedly, potassium ions traversing the channel from the inside confer voltage sensitivity to the Hui1 off-rate via Arg23, indicating that Lys21 is not in the pore. The 3D structure of Hui1 reveals a SAK1 fold, rationalizes KcsA inhibition, and validates the scaffold-based approach for isolation of high-affinity toxins for orphan receptors.

A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics.

K(2P)Ø, the two-pore domain potassium background channel that determines cardiac rhythm in Drosophila melanogaster, and its homologues that establish excitable membrane activity in mammals are of unknown structure. K(2P) subunits have two pore domains flanked by transmembrane (TM) spans: TM1-P1-TM2-TM3-P2-TM4. To establish spatial relationships in K(2P)Ø, we identified pairs of sites that display electrostatic compensation. Channels silenced by the addition of a charge in pore loop 1 (P1) or P2 were restored to function by countercharges at specific second sites. A three-dimensional homology model was determined using the crystal structure of K(V)1.2, effects of K(2P)Ø mutations to establish alignment, and compensatory charge-charge pairs. The model was refined and validated by continuum electrostatic free energy calculations and covalent linkage of introduced cysteines. K(2P) channels use two subunits arranged so that the P1 and P2 loops contribute to one pore, identical P loops face each other diagonally across the pore, and the channel complex has bilateral symmetry with a fourfold symmetric selectivity filter.

Sumoylation silences the plasma membrane leak K+ channel K2P1.

Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage.

Pore- and state-dependent cadmium block of I(Ks) channels formed with MinK-55C and wild-type KCNQ1 subunits.

Human MinK and KCNQ1 subunits assemble to form I(Ks) channels. When MinK position 55 is mutated to cysteine (MinK-55C), I(Ks) channels can be blocked by external cadmium (Cd(2+)). We have supported a pore-associated location for MinK-55C because Cd(2+) block is sensitive to voltage, permeant ions on the opposite side of the membrane (trans-ions), and external tetraethylammonium (TEA), an I(Ks) pore-blocker. Two recent reports argue that MinK-55C is distant from the pore: one finds TEA does not affect Cd(2+) block if channels are formed with a KCNQ1 mutant (K318I, V319Y) that increases TEA affinity; the second proposes that Cd(2+) binds between MinK-55C and a cysteine in KCNQ1 that is posited to lie toward the channel periphery. Here, these discrepancies are considered. First, Cd(2+) block of MinK-55C channels formed with wild-type KCNQ1 is shown to depend not only on voltage and trans-ions but state (showing decreased on-rate with increased open time and blocker trapping on channel closure). Conversely, MinK-55C channels with K318I, V319Y KCNQ1 are found to demonstrate Cd(2+) block that is independent of voltage, trans-ions and state (and to have a lower unitary conductance): thus, the KCNQ1 mutations alter the process under study, yielding Cd(2+) inhibition that is pore-independent and, perforce, TEA-insensitive. Second, MinK-55C channels are found to remain sensitive to Cd(2+) despite mutation of any single native cysteine in KCNQ1 or all nine simultaneously; this suggests no KCNQ1 cysteine binds Cd(2+) and can serve to localize MinK-55C. Despite many concerns that are enumerated, we remain obliged to conclude that Cd(2+) enters and leaves the pore to reach MinK-55C, placing that residue in or near the pore.

Hyperpolarization moves S4 sensors inward to open MVP, a methanococcal voltage-gated potassium channel.

MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.

Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals.

Proteins with dibasic retention motifs are subject to retrograde transport to endoplasmic reticulum (ER) by COPI-coated vesicles. As forward transport requires escape from ER retention, general release mechanisms have been expected. Here, KCNK3 potassium channels are shown to bear two cytoplasmic trafficking motifs: an N-terminal dibasic site that binds beta-COP to hold channels in ER and a C-terminal "release" site that binds the ubiquitous intracellular regulator 14-3-3beta on a nonclassical motif in a phosphorylation-dependent fashion to suppress beta-COP binding and allow forward transport. The strategy appears to be common. The major histocompatibility antigen class II-associated invariant chain Iip35 exhibits dibasic retention, carries a release motif, and shows mutually exclusive binding of beta-COP and 14-3-3beta on adjacent N-terminal sites. Other retained proteins are demonstrated to carry functional 14-3-3beta release motifs.

Disease-associated mutations in KCNE potassium channel subunits (MiRPs) reveal promiscuous disruption of multiple currents and conservation of mechanism.

KCNE genes encode single transmembrane-domain subunits, the MinK-related peptides (MiRPs), which assemble with pore-forming alpha subunits to establish the attributes of potassium channels in vivo. To investigate whether MinK, MiRP1, and MiRP2 operate similarly with their known native alpha subunit partners (KCNQ1, HERG, and Kv3.4, respectively) two conserved residues associated with human disease and influential in channel function were evaluated. As MiRPs assemble with a variety of alpha subunits in experimental cells and may do so in vivo, each peptide was also assessed with the other two alpha subunits. Inherited mutation of aspartate to asparagine (D --> N) to yield D76N-MinK is linked to cardiac arrhythmia and deafness; the analogs D82N-MiRP1 and D90N-MiRP2 were studied. Mutation of arginine to histidine (R --> H) to yield R83H-MiRP2 is associated with periodic paralysis; the analogs K69H-MinK and K75H-MiRP1 were also studied. Macroscopic and single-channel currents showed that D --> N mutations suppressed a subset of functions whereas R/K --> H changes altered the activity of MinK, MiRP1, and MiRP2 with all three alpha subunits. The findings indicate that the KCNE peptides interact similarly with different alpha subunits and suggest a hypothesis: that clinical manifestations of inherited KCNE point mutations result from disruption of multiple native currents via promiscuous interactions.