Heiko Rauer

Calmodulin Mediates Calcium-dependent Activation of the Intermediate Conductance KCa Channel,IKCa1

Small and intermediate conductance Ca2+-activated K+ channels play a crucial role in hyperpolarizing the membrane potential of excitable and nonexcitable cells. These channels are exquisitely sensitive to cytoplasmic Ca2+, yet their protein-coding regions do not contain consensus Ca2+-binding motifs. We investigated the involvement of an accessory protein in the Ca2+-dependent gating of hIKCa1, a human intermediate conductance channel expressed in peripheral tissues. Cal- modulin was found to interact strongly with the cytoplasmic carboxyl (C)-tail of hIKCa1 in a yeast two-hybrid system. Deletion analyses defined a requirement for the first 62 amino acids of the C-tail, and the binding of calmodulin to this region did not require Ca2+. The C-tail ofhSKCa3, a human neuronal small conductance channel, also bound calmodulin, whereas that of a voltage-gated K+channel, mKv1.3, did not. Calmodulin co-precipitated with the channel in cell lines transfected with hIKCa1, but not with mKv1.3-transfected lines. A mutant calmodulin, defective in Ca2+ sensing but retaining binding to the channel, dramatically reduced current amplitudes when co-expressed withhIKCa1 in mammalian cells. Co-expression with varying amounts of wild-type and mutant calmodulin resulted in a dominant-negative suppression of current, consistent with four calmodulin molecules being associated with the channel. Taken together, our results suggest that Ca2+-calmodulin-induced conformational changes in all four subunits are necessary for the channel to open.

Calcium-activated Potassium Channels Sustain Calcium Signaling in T Lymphocytes SELECTIVE BLOCKERS AND MANIPULATED CHANNEL EXPRESSION LEVELS

To maintain Ca2+ entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca2+-activated K+ (KCa) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of KCa channels but not KV channels reduce Ca2+ entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native KCa channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca2+ influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca2+ entry despite pharmacological inhibition of the native small conductance KCa channel. Thus, KCachannels play a vital role in T cell Ca2+ signaling.

Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels

We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca2+-activated K+channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3and the Ca2+-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys32, which lies in close proximity to this cluster. These analogs block IKCa1with ∼20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 andKv1.6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-KCa channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K+channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K+channels. The availability of potent and selective inhibitors ofIKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.

Structural Conservation of the Pores of Calcium-activated and Voltage-gated Potassium Channels Determined by a Sea Anemone Toxin

The structurally defined sea anemone peptide toxins ShK and BgK potently block the intermediate conductance, Ca2+-activated potassium channel IKCa1, a well recognized therapeutic target present in erythrocytes, human T-lymphocytes, and the colon. The well characterized voltage-gatedKv1.3 channel in human T-lymphocytes is also blocked by both peptides, although ShK has a ∼1,000-fold greater affinity forKv1.3 than IKCa1. To gain insight into the architecture of the toxin receptor in IKCa1, we used alanine-scanning in combination with mutant cycle analyses to map the ShK-IKCa1 interface, and compared it with the ShK-Kv1.3 interaction surface. ShK uses the same five core residues, all clustered around the critical Lys22, to interact with IKCa1 and Kv1.3, although it relies on a larger number of contacts to stabilize its weaker interactions with IKCa1 than with Kv1.3. The toxin binds to IKCa1 in a region corresponding to the external vestibule of Kv1.3, and the turret and outer pore of the structurally defined bacterial potassium channel, KcsA. Based on the NMR structure of ShK, we deduce the toxin receptor inIKCa1 to have x-y dimensions of ∼22 Å, a diameter of ∼31 Å, and a depth of ∼8 Å; we estimate that the ion selectivity lies ∼13 Å below the outer lip of the toxin receptor. These dimensions are in good agreement with those of the KcsA channel determined from its crystal structure, and the inferred structure of Kv1.3 based on mapping with scorpion toxins. Thus, these distantly related channels exhibit architectural similarities in the outer pore region. This information could facilitate development of specific and potent modulators of the therapeutically important IKCa1 channel.

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.

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.

The effect of deep pore mutations on the action of phenylalkylamines on the Kv1.3 potassium channel.

We investigated the action of the phenylalkylamines verapamil and N‐methyl‐verapamil on the Kv1.3 potassium channel using the whole‐cell configuration of the patch‐clamp technique. Our goal was to identify their binding as a prerequisite for using the phenylalkylamines as small, well‐defined molecular probes, not only to expand the structural findings made with peptide toxins or by crystallization, but also to use them as lead compounds for the generation of more potent and therefore more specific K+ channel modulators. Competition experiments with charybdotoxin, known to interact with external residues of Kv1.3, showed no interaction with verapamil. The internal application of quarternary N‐methyl‐verapamil in combination with verapamil suggested competition for the same internal binding site. Verapamil affinity was decreased 6 fold by a mutation (M395V) in a region of the internal pore which forms part of the internal tetraethylammonium (TEA+) binding site, although mutations at neighbouring residues (T396 and T397) were without effect. Modification of C‐type inactivation by mutations in the internal pore suggest that this region participates in the inactivation process. The action of phenylalkylamines and local anaesthetics on L‐type Ca2+ channels and Na+ channels, respectively, and verapamil on Kv1.3 indicate very similar blocking mechanisms. This might allow the use of these compounds as molecular probes to map the internal vestibule of all three channel types.

Vanadate Induces Calcium Signaling, Ca2+ Release-Activated Ca2+ Channel Activation, and Gene Expression in T Lymphocytes and RBL-2H3 Mast Cells Via Thiol Oxidation

Using ratiometric Ca2+ imaging and patch-clamp measurement of Ca2+ channel activity, we investigated Ca2+ signaling induced by vanadium compounds in Jurkat T lymphocytes and rat basophilic leukemia cells. In the presence of external Ca2+, vanadium compounds produced sustained or oscillatory Ca2+ elevations; in nominally Ca2+-free medium, a transient Ca2+ rise was generated. Vanadate-induced Ca2+ signaling was blocked by heparin, a competitive inhibitor of the 1,4,5-inositol trisphosphate (IP3) receptor, suggesting that Ca2+ influx is secondary to depletion of IP3-sensitive Ca2+ stores. In Jurkat T cells, vanadate also activated the Ca2+-dependent transcription factor, NF-AT. Intracellular dialysis with vanadate activated Ca2+ influx through Ca2+ release-activated Ca2+ (CRAC) channels with kinetics comparable to those of dialysis with IP3. Neither phosphatase inhibitors nor nonhydrolyzable nucleotide analogues modified CRAC channel activation. The action of vanadate, but not IP3, was prevented by the thiol-reducing agent DTT. In addition, the activation of CRAC channels by vanadate was mimicked by the thiol-oxidizing agent chloramine T. These results suggest that vanadate enhances Ca2+ signaling via thiol oxidation of a proximal element in the signal transduction cascade.