Stephan Grissmer

International Union of Pharmacology. LIII. Nomenclature and Molecular Relationships of Voltage-Gated Potassium Channels

Potassium-selective channels are the largest and most diverse group of ion channels, represented by some 70 known loci in the mammalian genome. The first cloned potassium channel gene was the Drosophila voltage-gated shaker channel, and this was rapidly followed by the identification of other

International Union of Pharmacology. XLI. Compendium of Voltage-Gated Ion Channels: Potassium Channels

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels.1 The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom

OSK1 (alpha-KTx3.7) is a 38-residue toxin cross-linked by three disulphide bridges that was initially isolated from the venom of the Asian scorpion Orthochirus scrobiculosus. OSK1 and several structural analogues were produced by solid-phase chemical synthesis, and were tested for lethality in mice and for their efficacy in blocking a series of 14 voltage-gated and Ca2+-activated K+ channels in vitro. In the present paper, we report that OSK1 is lethal in mice by intracerebroventricular injection, with a LD50 (50% lethal dose) value of 2 microg/kg. OSK1 blocks K(v)1.1, K(v)1.2, K(v)1.3 channels potently and K(Ca)3.1 channel moderately, with IC50 values of 0.6, 5.4, 0.014 and 225 nM respectively. Structural analogues of OSK1, in which we mutated positions 16 (Glu16-->Lys) and/or 20 (Lys20-->Asp) to amino acid residues that are conserved in all other members of the alpha-KTx3 toxin family except OSK1, were also produced and tested. Among the OSK1 analogues, [K16,D20]-OSK1 (OSK1 with Glu16-->Lys and Lys20-->Asp mutations) shows an increased potency on K(v)1.3 channel, with an IC50 value of 0.003 nM, without loss of activity on K(Ca)3.1 channel. These data suggest that OSK1 or [K16,D20]-OSK1 could serve as leads for the design and production of new immunosuppressive drugs.

UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation

UK‐78,282, a novel piperidine blocker of the T lymphocyte voltage‐gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high‐throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of ∼200 nM and 1 : 1 stoichiometry. A closely related compound, CP‐190,325, containing a benzyl moiety in place of the benzhydryl in UK‐78,282, is significantly less potent. Three lines of evidence indicate that UK‐78,282 inhibits Kv1.3 in a use‐dependent manner by preferentially blocking and binding to the C‐type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at −50 mV enhances the channels sensitivity to UK‐78,282. Decreasing the number of inactivated channels by exposure to ∼160 mM external K+ decreases the sensitivity to UK‐78,282. Mutations that alter the rate of C‐type inactivation also change the channels sensitivity to UK‐78,282 and there is a direct correlation between τh and IC50 values. Competition experiments suggest that UK‐78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK‐78,282s action on the channel. UK‐78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage‐gated K+ channel, Kv1.4. UK‐78,282 effectively suppresses human T‐lymphocyte activation.

Selective blockage of Kv1.3 and Kv3.1 channels increases neural progenitor cell proliferation

The modulation of cell proliferation in neural progenitor cells (NPCs) is believed to play a role in neuronal regeneration. Recent studies showed that K+ channel activity influenced cell proliferation. Therefore, we examined NPCs for K+ channels and tested whether NPC self renewing can be modulated by synthetic K+ channel modulators. The whole‐cell K+ current was partly K+ dependent and showed a cumulative inactivating component. Two tetra‐ethyl‐ammonium ion (TEA)‐sensitve K+ currents with different voltage dependencies ( = 65 μm, E50 = −0.3 ± 1.3 mV and  = 8 mm, E50 = −15.2 ± 2.8 mV) and an almost TEA‐insensitive current were identified. Kaliotoxin blocked approximately 50% of the entire K+ currents (IC50 = 0.25 nm). These properties resembled functional characteristics of Kv1.4, Kv1.3 and Kv3.1 channels. Transcripts for these channels, as well as proteins for Kv1.3 and Kv3.1, were identified. Immunocytochemical staining revealed Kv1.3 and Kv3.1 K+ channel expression in almost all NPCs. The blockage of Kv3.1 by low concentrations of TEA, as well as the blockage of Kv1.3 by Psora‐4, increased NPC proliferation. These findings underline the regulatory role of K+ channels on the cell cycle and imply that K+ channel modulators might be used therapeutically to activate endogenous NPCs.

The 'functional' dyad of scorpion toxin Pi1 is not itself a prerequisite for toxin binding to the voltage-gated Kv1.2 potassium channels.

Pi1 is a 35-residue scorpion toxin cross-linked by four disulphide bridges that acts potently on both small-conductance Ca2+-activated (SK) and voltage-gated (Kv) K+ channel subtypes. Two approaches were used to investigate the relative contribution of the Pi1 functional dyad (Tyr-33 and Lys-24) to the toxin action: (i) the chemical synthesis of a [A24,A33]-Pi1 analogue, lacking the functional dyad, and (ii) the production of a Pi1 analogue that is phosphorylated on Tyr-33 (P-Pi1). According to molecular modelling, this phosphorylation is expected to selectively impact the two amino acid residues belonging to the functional dyad without altering the nature and three-dimensional positioning of other residues. P-Pi1 was directly produced by peptide synthesis to rule out any possibility of trace contamination by the unphosphorylated product. Both Pi1 analogues were compared with synthetic Pi1 for bioactivity. In vivo, [A24,A33]-Pi1 and P-Pi1 are lethal by intracerebroventricular injection in mice (LD50 values of 100 and 40 microg/mouse, respectively). In vitro, [A24,A33]-Pi1 and P-Pi1 compete with 125I-apamin for binding to SK channels of rat brain synaptosomes (IC50 values of 30 and 10 nM, respectively) and block rat voltage-gated Kv1.2 channels expressed in Xenopus laevis oocytes (IC50 values of 22 microM and 75 nM, respectively), whereas they are inactive on Kv1.1 or Kv1.3 channels at micromolar concentrations. Therefore, although both analogues are less active than Pi1 both in vivo and in vitro, the integrity of the Pi1 functional dyad does not appear to be a prerequisite for the recognition and binding of the toxin to the Kv1.2 channels, thereby highlighting the crucial role of other toxin residues with regard to Pi1 action on these channels. The computed simulations detailing the docking of Pi1 peptides on to the Kv1.2 channels support an unexpected key role of specific basic amino acid residues, which form a basic ring (Arg-5, Arg-12, Arg-28 and Lys-31 residues), in toxin binding.

Structure of the BgK-Kv1.1 complex based on distance restraints identified by double mutant cycles. Molecular basis for convergent evolution of Kv1 channel blockers.

A structural model of BgK, a sea anemone toxin, complexed with the S5-S6 region of Kv1.1, a voltage-gated potassium channel, was determined by flexible docking under distance restraints identified by a double mutant cycles approach. This structure provides the molecular basis for identifying the major determinants of the BgK-Kv1.1 channel interactions involving the BgK dyad residues Lys25 and Tyr26. These interactions are (i) electrostatic interactions between the extremity of Lys25 side chain and carbonyl oxygen atoms of residues from the channel selectivity filter that may be strengthened by solvent exclusion provided by (ii) hydrophobic interactions involving BgK residues Tyr26 and Phe6 and Kv1.1 residue Tyr379 whose side chain protrudes in the channel vestibule. In other Kv1 channel-BgK complexes, these interactions are likely to be conserved, implicating both conserved and variable residues from the channels. The data suggest that the conservation in sea anemone and scorpion potassium channel blockers of a functional dyad composed of a lysine, and a hydrophobic residue reflects their use of convergent binding solutions based on a crucial interplay between these important conserved interactions.

Regulation of mammalian Shaker‐related K+ channels: evidence for non‐conducting closed and non‐conducting inactivated states

1 Using the whole‐cell recording mode we have characterized two non‐conducting states in mammalian Shaker‐related voltage‐gated K+ channels induced by the removal of extracellular potassium, K+o. 2 In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3 To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mm K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4 The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C‐type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild‐type Kv1.3 channel. 5 CP‐339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6 We conclude that removal of K+o induces the Kv1.3 channel to transition to a non‐conducting ‘closed’ state which can switch into a non‐conducting ‘inactivated’ state upon depolarization.

Pharmacological Profiling of Orthochirus scrobiculosus Toxin 1 Analogs with a Trimmed N-Terminal Domain

OSK1, a toxin from the venom of the Asian scorpion Orthochirus scrobiculosus, is a 38-residue peptide cross-linked by three disulfide bridges. A structural analog of OSK1, [Lys16,Asp20]-OSK1, was found previously to be one of the most potent blockers of the voltage-gated K+ channel Kv1.3 hitherto characterized. Here, we demonstrate that progressive trimming of the N-terminal domain of [Lys16,Asp20]-OSK1 results in marked changes in its pharmacological profile, in terms of both K+ channel affinity and selectivity. Whereas the affinity to Kv1.1 and Kv1.3 did not change significantly, the affinity to Kv1.2 and KCa3.1 was drastically reduced with the truncations. It is surprising that a striking gain in potency was observed for Kv3.2. In contrast, a truncation of the C-terminal domain, expected to partially disrupt the toxin β-sheet structure, resulted in a significant decrease or a complete loss of activity on all channel types tested. These data highlight the value of structure-function studies on the extended N-terminal domain of [Lys16,Asp20]-OSK1 to identify new analogs with unique pharmacological properties.

SK2 encodes the apamin-sensitive Ca2+-activated K+ channels in the human leukemic T cell line, Jurkat

T cells express two different types of voltage‐independent Ca2+‐activated K+ channels with small (SK) and intermediate (IK) conductance that serve important roles in the activation of T lymphocytes. In contrast to the IK channels from T lymphocytes which are upregulated upon mitogen stimulation, SK channels of Jurkat T cells, a human leukemic T cell line, are constitutively expressed even in the absence of mitogenic stimulation. We have used patch‐clamp recordings from transfected or injected mammalian cells to show that the cloned SK2 channel demonstrates the biophysical and pharmacological properties of the majority of K(Ca) channels in Jurkat T cells. The cloned and native channels are voltage‐independent, Ca2+‐activated, apamin‐sensitive, show an equivalent voltage‐dependent Ba2+ block and possess a similar ion selectivity. In addition, we used the polymerase chain reaction to demonstrate the presence of SK2 mRNA in Jurkat T cells, whereas SK3 transcripts encoding the other cloned apamin‐sensitive SK channel were not detected. These data suggest that the voltage‐independent apamin‐sensitive K(Ca) channel in Jurkat T cells represents the recently cloned SK2 channel.