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Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.

Cell blebbing is a key feature in apoptosis. Because blebbing dynamically alters cell volume and regulatory volume changes have been linked to chloride (Cl) channels, we evaluated an association between blebbing and Cl channels activity. We used scanning electron microscopy, confocal laser microscopy, and cell sorting to quantify cell volume and blebbing and whole-cell recording to characterize Cl(-) currents. We found that blockade of Cl channel activity as well as inhibition of adenylyl cyclase or protein kinase A (PKA) activity suppressed ammonia-induced blebbing in the microglia cell line, BV-2. In further experiments, we elucidated the common mechanism of Cl channel activity and cyclic adenosine 3',5'-monophosphate (cAMP)-dependent pathway on cell blebbing. These experiments indicated that perfusion of cells with cAMP or the catalytic subunit of PKA activated a Cl(-) current under normotonic conditions. The pharmacological profile (sensitivity to 5-nitro-2-(3-phenylpropylamino)benzoic acid [NPPB], flufenamic acid, and [(dihydroindenyl)oxy]alkanoic acid [DIOA]), outward rectification, and kinetic of the current were identical to the swelling-activated Cl channel. Superfusion of cells with ammonia elicited an outwardly rectifying current sensitive to Cl channel blockers. We propose that ammonia induces a PKA-dependent phosphorylation of Cl channels. Localized influx of Cl(-) is followed by influx of water, required for bleb expansion.

Background and purpose: Verapamil blocks current through the voltage-gated K+ channel Kv1.3 in the open and inactivated state of the channel but not the closed state. The binding site for verapamil was proposed to be close to the selectivity filter and the occupancy of the selectivity filter might therefore influence verapamil affinity. Experimental approach: We investigated the influence of intra- and extracellular K+ and Rb+ on the effect of verapamil by patch-clamp studies, in COS-7 cells transfected with hKv1.3 channels. Key results: Verapamil affinity was highest in high intracellular K+ concentrations ([K+]i) and lowest in low [Rb+]i, indicating an influence of intracellular cations on verapamil affinity. Experiments with a mutant channel (H399T), exhibiting a strongly reduced C-type inactivated state, demonstrated that part of this changed verapamil affinity in wild-type channels could be caused by altered C-type inactivation. External K+ and Rb+ could influence verapamil affinity by a voltage-dependent entry into the channel thereby modifying the verapamil off-rate and in addition causing a voltage-dependent verapamil off-rate. Conclusions and implications: Recovery from verapamil block was mainly due to the voltage-dependent closing of channels (state-dependent block), implying a second open state of the channel. This hypothesis was confirmed by the dependency of the tail current time course on duration of the prepulse. We conclude that the wild-type hKv1.3 channel undergoes at least two different conformational changes before finally closing with a low verapamil affinity in one open state and a high verapamil affinity in the other open state.

New structural classes of K(V)1.3 and IK-1 ion channel blockers have been identified based on a virtual high throughput screening approach using a homology model of KcsA. These compounds display inhibitory effects on T-cell and/or keratinocyte proliferation and immunosuppressant activity within a DTH animal model.

Proliferation of astrocytes plays an essential role during ontogeny and in the adult brain, where it occurs following trauma and in inflammation and neurodegenerative diseases as well as in normal, healthy mammals. The cellular mechanisms underlying glial proliferation remain poorly understood. As dopamine is known to modulate proliferation in different cell populations, we investigated the effects of dopamine on the proliferation of striatal astrocytes in vitro. We found that dopamine reduced proliferation. As proliferation involves, among other things, a change in cell volume, which normally comes with water movement across the membrane, water channels might represent a molecular target of the dopamine effect. Therefore we studied the effect of dopamine on aquaporin 4 (AQP4) expression, the main aquaporin subtype expressed in glial cells, and observed a down-regulation of the AQP4-M23 isoform. This down-regulation was the cause of the dopamine-induced decrease in proliferation as knockdown of AQP4 using siRNA techniques mimicked the effects of dopamine on proliferation. Furthermore, stimulation of glial proliferation by basic fibroblast growth factor was also abolished by knocking down AQP4. In addition, blocking of AQP4 with 10 mum tetraethylammonium inhibited osmotically induced cell swelling and stimulation of glial cell proliferation by basic fibroblast growth factor. These results demonstrate a clear-cut involvement of AQP4 in the regulation of proliferation and implicate that modulation of AQP4 could be used therapeutically in the treatment of neurodegenerative diseases as well as in the regulation of reactive astrogliosis by preventing or reducing the glia scar formation, thus improving regeneration following ischemia or other trauma.

Throughout the CNS, small conductance Ca(2+)-activated potassium (SK) channels modulate firing frequency and neuronal excitability. We have identified a novel, shorter isoform of standard SK2 (SK2-std) in mouse brain which we named SK2-sh. SK2-sh is alternatively spliced at exon 3 and therefore lacks 140 amino acids, which include transmembrane domains S3, S4 and S5, compared with SK2-std. Western blot analysis of mouse hippocampal tissue revealed a 47 kDa protein product as predicted for SK2-sh along with a 64 kDa band representing the standard SK2 isoform. Electrophysiological recordings from transiently expressed SK2-sh revealed no functional channel activity or interaction with SK2-std. With the help of real-time PCR, we found significantly higher expression levels of SK2-sh mRNA in cortical tissue from AD cases when compared with age-matched controls. A similar increase in SK2-sh expression was induced in cortical neurons from mice by cytokine exposure. Substantial clinical evidence suggests that excess cytokines are centrally involved in the pathogenesis of Alzheimer's disease. Thus, SK2-sh as a downstream target of cytokines, provide a promising target for additional investigation regarding potential therapeutic intervention.

Agitoxin 2 (AgTx2) is a 38-residues toxin that has been isolated from the venom of leiurus quinquestriatus hebraeus scorpion (Garcia et al. 1994). This toxin was shown to reversibly block voltage-gated K+ channels (Shaker B, Kv1.1, Kv1.2, and Kv1.3) (Hidalgo and MacKinnon 1995; Lipkind and Fozzard 1997; Gao and Garcia 2003). MTX is another small toxin isolated from the venom of the scorpion Scorpio maurus palmatus (Kharrat et al. 1997). It is a basic, C-terminal amidated, 34-mer peptide cross-linked by four disulfide bridges. The solid-phase technique has been used to obtain synthetic MTX (sMTX), and it was found that both the natural and synthetic MTX are equally lethal to mice by ICV inoculation (LD50 of 80 ng/mouse). sMTX has been shown to be active in the nanomolar range on both voltage-gated Kv1.2 and IKCa channels (Fajloun et al. 2000a). AgTx2 is cross-linked according to C1–C4, C2–C5, and C3–C6, while MTX possesses C1–C5, C2–C6, C3–C4, and C7–C8 disulfide bridge organization (vs. C1–C5, C2–C6, C3–C7, and C4–C8 for other K+ channel-selective four-disulfide-bridged scorpion toxins) (Bontems et al. 1991; Darbon et al. 1999). MTX is the sole toxin that exhibits an uncommon disulfide bridge organization among the four-disulfide-bridged scorpion toxins (e.g., Pi1, Pi4, Pi7, and HsTX1) (Olamendi-Portugal et al. 1996; Rogowski et al. 1996; Lebrun et al. 1997; Savarin et al. 1999; Fajloun et al. 2000b,c; M'Barek et al. 2003a), but yet it adopts the classical α/β scaffold (Blanc et al. 1997; M'Barek et al. 2003b). This motif, from which arises the wide functional diversity of scorpion toxins, is mainly composed of a short α-helix connected to a β-sheet by two disulfide bridges. However, some equally Cys-stabilized α/β-motifs composed of three antiparallel β-strands and one α-helix are found in insect and plant defensins (Spelbrink et al. 2004) that acts on the Ca2+ channel. Here, we used MTX as a molecular template to design and chemically synthesize a scorpion toxin-derived chimera cross-linked by four disulfide bridges, AgTx2-MTX. This peptide encompasses residues 1–5 of AgTx2 at its N terminus, and the sequence of MTX at its C terminus. The N-terminal portion of AgTx2 was chosen to form the N-terminal extremity of AgTx2-MTX, because this domain is structured as being part of the three-stranded β-sheet that could be observed in the 3D structure of AgTx2 in solution (Krezel et al. 1995). In this contribution, we describe the synthesis by an optimized Fmoc/t-butyl strategy (Merrifield 1986), disulfide bridge organization, 3D structure in solution by 1H-NMR means, and electrophysiological activity on both the Ca2+-activated intermediate conductance and voltage-gated K+ channels subtypes (IKCa and Kv1.2, respectively). Because AgTx2-MTX was found to be highly potent in blocking the Kv1.2 channel, we detailed the AgTx2-MTX to the mammalian Kv1.2 channel interaction by computed docking simulations using the HADDOCK program (High Ambiguity Driven DOCKing program) in order to better understand the structural features responsible for this difference in bioactivity.

Lamellipodium extension and retraction is the driving force for cell migration. Although several studies document that activation of chloride channels are essential in cell migration, little is known about their contribution in lamellipodium formation. To address this question, we characterized chloride channels and transporters by whole cell recording and RT-PCR, respectively, as well as quantified lamellipodium formation in murine primary microglial cells as well as the microglial cell-line, BV-2, using time-lapse microscopy. The repertoire of chloride conducting pathways in BV-2 cells included, swelling-activated chloride channels as well as the KCl cotransporters, KCC1, KCC2, KCC3, and KCC4. Swelling-activated chloride channels were either activated by a hypoosmotic solution or by a high KCl saline, which promotes K(+) and Cl(-) influx instead of efflux by KCCs. Conductance through swelling-activated chloride channels was completely blocked by flufenamic acid (200 microM), SITS (1 mM) and DIOA (10 microM). By exposing primary microglial cells or BV-2 cells to a high KCl saline, we observed a local swelling, which developed into a prominent lamellipodium. Blockade of chloride influx by flufenamic acid (200 microM) or DIOA (10 microM) as well as incubation of cells in a chloride-free high K(+) saline suppressed formation of a lamellipodium. We assume that local swellings, established by an increase in chloride influx, are a general principle in formation of lamellipodia in eukaryotic cells.

The calcium-activated potassium channels can be divided in two functionally different groups. The first group belongs to the family of potassium channels that consists of one pore region (1P) with six putative transmembrane segments (6T) per alpha subunit (1P6T). These channels are potassium selective and are opened by an increase in [Ca++]i via calmodulin. The opening of the channel is independent from the applied voltage. The single channel conductance of these calcium-activated potassium channels range from small (SK channels; ∼ 5 pS) to intermediate (IK channels; 1060 pS) conductances …

Potassium channels play fundamental roles in physiology. Chemically diverse drugs bind in the pore region of K+ channels. Here, we homology-modeled voltage- and Ca2+-gated K+ channel BK and voltage-gated Kv1.3 using the X-ray structures of MthK and Kv1.2, respectively, and simulated the binding of d-tubocurarine in the inner pore of the channels. Monte Carlo minimization predicted that d-tubocurarine can bind in the open pore of both channels with its long axis parallel to the pore axis. The cationic groups of d-tubocurarine can displace K+ from the ion dehydration site at the selectivity filter. The predicted binding energy of d-tubocurarine in Kv1.3 is less preferable than in BK. To test this prediction, the currents through Kv1.3 and BK channels were measured in the absence and presence of d-tubocurarine. Results show that d-tubocurarine blocks current through Kv1.3 when applied from either side of the membrane only in millimolar concentrations (Kd= 1 mM), whereas half-blocking concentrations of the internally applied d-tubocurarine to BK are as low as approximately 8 microM. This indicates that the affinities of both external and internal d-tubocurarine to Kv1.3 are much lower than those to BK channels. Our study reveals the K+ dehydration site as a determinant of the d-tubocurarine receptor, predicts binding modes of d-tubocurarine in K+ channels, and suggests that the open pore in BK is wider than in Kv1.3. The results imply that MthK can be used for homology modeling of the pore region of channels activated by forces applied to the inner helices.

International audience; Scorpion toxins interact with their target ion channels through multiple molecular contacts. Because a "gain of function" approach has never been described to evaluate the importance of the molecular contacts in defining toxin affinity, we experimentally examined whether increasing the molecular contacts between a toxin and an ion channel directly impacts toxin affinity. For this purpose, we focused on two scorpion peptides, the well-characterized maurotoxin with its variant Pi1-like disulfide bridging (MTX(Pi1)), used as a molecular template, and butantoxin (BuTX), used as an N-terminal domain provider. BuTX is found to be 60-fold less potent than MTX(Pi1) in blocking Kv1.2 (IC(50) values of 165 nM for BuTX versus 2.8 nM for MTX(Pi1)). Removal of its N-terminal domain (nine residues) further decreases BuTX affinity for Kv1.2 by 5.6-fold, which is in agreement with docking simulation data showing the importance of this domain in BuTX-Kv1.2 interaction. Transfer of the BuTX N-terminal domain to MTX(Pi1) results in a chimera with five disulfide bridges (BuTX-MTX(Pi1)) that exhibits 22-fold greater affinity for Kv1.2 than MTX(Pi1) itself, in spite of the lower affinity of BuTX as compared to MTX(Pi1). Docking experiments performed with the 3-D structure of BuTX-MTX(Pi1) in solution, as solved by (1)H-NMR, reveal that the N-terminal domain of BuTX participates in the increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicate that acting on molecular contacts between a toxin and a channel is an efficient strategy to modulate toxin affinity.

Maurotoxin (MTX) is a potent blocker of human voltage-activated Kv1.2 and intermediate-conductance calcium-activated potassium channels, hIKCa1. Because its blocking affinity on both channels is similar, although the pore region of these channels show only few conserved amino acids, we aimed to characterize the binding sites of MTX in these channels. Investigating the pH(o) dependence of MTX block on current through hKv1.2 channels, we concluded that the block is less pH(o) - sensitive than for hIKCa1 channels. Using mutant cycle analysis and computer docking, we tried to identify the amino acids through which MTX binds to hKv1.2 and hIKCa1 channels. We report that MTX interacts with hKv1.2 mainly through six strong interactions. Lys(23) from MTX protrudes into the channel pore interacting with the GYGD motif, whereas Tyr(32) and Lys(7) interact with Val(381), Asp(363), and Glu(355), stabilizing the toxin onto the channel pore. Because only Val(381), Asp(363), and the GYGD motif are conserved in hIKCa1 channels, and the replacement of His(399) from hKv1.3 channels with a threonine makes this channel MTX-sensitive, we concluded that MTX binds to all three channels through the same amino acids. Glu(355), although important, is not essential in MTX recognition. A negatively charged amino acid in this position could better stabilize the toxin-channel interaction and could explain the pH(o) sensitivity of MTX block on current through hIKCa1 versus hKv1.2 channels.

The lymphocyte potassium channel Kv1.3 is widely regarded as a promising new target for immunosuppression. To identify a potent small-molecule Kv1.3 blocker, we synthesized a series of 5-phenylalkoxypsoralens and tested them by whole-cell patch clamp. The most potent compound of this series, 5-(4-phenylbutoxy)psoralen (Psora-4), blocked Kv1.3 in a use-dependent manner, with a Hill coefficient of 2 and an EC50 value of 3 nM, by preferentially binding to the C-type inactivated state of the channel. Psora-4 is the most potent small-molecule Kv1.3 blocker known. It exhibited 17- to 70-fold selectivity for Kv1.3 over closely related Kv1-family channels (Kv1.1, Kv1.2, Kv1.4, and Kv1.7) with the exception of Kv1.5 (EC50, 7.7 nM) and showed no effect on human ether-a-go-go-related channel, Kv3.1, the calcium-activated K+ channels (IKCa1, SK1-SK3, and BKCa), or the neuronal NaV1.2 channel. In a test of in vivo toxicity in rats, Psora-4 did not display any signs of acute toxicity after five daily subcutaneous injections at 33 mg/kg body weight. Psora-4 selectively suppressed the proliferation of human and rat myelin-specific effector memory T cells with EC50 values of 25 and 60 nM, respectively, without persistently suppressing peripheral blood naive and central memory T cells. Because autoantigen-specific effector memory T cells contribute to the pathogenesis of T cell-mediated autoimmune diseases such as multiple sclerosis, Psora-4 and other Kv1.3 blockers may be useful as immunomodulators for the therapy of autoimmune disorders.

Ion channels are important in controlling cell cycle progression and proliferation in a variety of cell types. Using the whole-cell recording mode of the patch-clamp technique, functional ion channels were electrophysiologically characterized in PANC-1 (K-ras G12D (+/-), p53 R273C, Deltap16), BxPC-3 (smad4-, p53 Y220C, Deltap16), and MiaPaCa-2 [transforming growth factor-beta receptor type II defect, K-ras G12C(-/-), p53 R248W, Deltap16] human pancreatic cancer cell lines. In BxPC-3 and the MiaPaCa-2 cells, we could identify approximately 600 or approximately 1200 functional Ca2+-activated K+ channels (IK) per cell, respectively, whereas PANC-1 cells expressed approximately 200 functional IK channels per cell. These channels were observed by using pipette solutions buffering [Ca2+]i to 1 microM. The channels were voltage-independent, blocked by charybdotoxin, clotrimazole, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34), and blocked by Ba2+ in a voltage-dependent manner. In the presence of 10 microM clotrimazole or TRAM-34, proliferation of the BxPC-3 as well as the MiaPaCa-2 cells was completely stopped. In contrast, proliferation of PANC-1 cells was hardly affected by clotrimazole or TRAM-34. Proliferation in all three cell lines could be inhibited in the presence of the Ca2+ channel antagonists verapamil, diltiazem, and nifedipine. By quantitative RT-PCR, we could show that MiaPaCa-2 cells exhibit a 2.8-fold and BxPC3 cells a more than 8-fold elevated level of IK mRNA level compared with PANC-1 cells. Interestingly, in primary pancreatic tumors we found a tremendous up-regulation of IK mRNA. In eight of nine (or 89%) primary pancreatic tumor tissues, we found a 6- to 66-fold increase in IK mRNA. Our findings suggest that a certain amount of functional IK channels is crucial for the proliferation of some pancreatic cancer types. The blockade of IK channels may ultimately prove useful as a therapeutic option for some patients with ductal adenocarcinoma of the pancreas with an up-regulated IK channel expression.

We have isolated an hSK3 isoform from a human embryonic cDNA library that we have named hSK3_ex4. This isoform contains a 15 amino acid insertion within the S5 to P-loop segment. Transcripts encoding hSK3_ex4 are coexpressed at lower levels with hSK3 in neuronal as well as in non-neuronal tissues. To investigate the pharmacokinetic properties of hSK3_ex4, we expressed the isoforms hSK3 and hSK3_ex4 in tsA cells. Both isoforms were similarly activated by cytosolic Ca2+ (hSK3, EC50=0.91 +/- 0.4 microM; hSK3_ex4, EC50=0.78 +/- 0.2 microM) and by 1-ethyl-2-benzimidazolinone (hSK3, EC50=0.17 mM; hSK3_ex4, 0.19 mM). They were both blocked by tetraethylammonium (hSK3, Kd=2.2 mM; hSK3_ex4, 2.6 mM) and showed similar permeabilities relative to K+ for Cs+ (hSK3, 0.17 +/- 0.04, n=3; hSK3_ex4, 0.17 +/- 0.05, n=3) and Rb+ (hSK3, 0.79 +/- 0.04, n=3; hSK3_ex4, 0.8 +/- 0.07, n=3). Ba2+ blocked both isoforms, and in both cases, the block was strongest at hyperpolarizing membrane potentials. However, the voltage-dependence of hSK3 was stronger than that of hSK3_ex4. The most obvious distinguishing feature of this new isoform was that whereas hSK3 was blocked by apamin (Kd=0.8 nM), scyllatoxin (Kd=2.1 nM), and d-tubocurarine (Kd=33.4 microM), hSK3_ex4 was not affected by apamin up to 100 nM, scyllatoxin up to 500 nM, and d-tubocurarine up to 500 microM. So far, isoform hSK3_ex4 forms the only small-conductance calcium-activated potassium (SK) channels, which are insensitive to the classic SK blockers.

International audience; CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.

International audience; 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.

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/.

Using human intermediate-conductance calcium-activated potassium (hIKCa1) channels as a model we aimed to characterize structural differences between maurotoxin (MTX) and charybdotoxin (CTX) and to gain new insights into the molecular determinants that define the interaction of these pore-blocking peptides with hIKCa1 channel. We report here that the block of MTX, but not of CTX on current through hIKCa1 channels is pH0 dependent. The replacement of histidine 236 from hIKCa1 channel with a smaller amino acid, cystein, did not change MTX binding affinity, however, partially affected the pH0 dependency of its block at low pH0. In contrast, CTX binding affinity to the hIKCa1_H236C channel mutant was increased suggesting that His236 might play a role in the binding of CTX, but has only a weak influence in the binding of MTX to hIKCa1 channels.

We have identified a hSK3-transcript, hSK3_ex1c, which is generated by alternative splicing. Isoform hSK3_ex1c lacks the cytosolic N-terminus and the first transmembrane helix and is exclusively expressed in non-neuronal tissues. hSK3 transfected tsA cells showed a Ca2+-activated K+ current in patch-clamp experiments, whereas hSK3_ex1c transfected cells and cells co-transfected with both isoforms did not. We fused both isoforms to fluorescence proteins and observed hSK3 localization predominantly in the plasma membrane. The co-expression of hSK3 + hSK3_ex1c resulted in their cytoplasmic co-localization. Thus, hSK3_ex1c has a dominant-negative effect on hSK3 by preventing its transport into the plasma membrane.

The whole cell recording mode of the patch-clamp technique was used to study the effect of hypotonic NaCl or isotonic high-KCl solution on membrane currents in a human osteoblast-like cell line, C1. Both hypotonic NaCl or isotonic high-KCl solution activated Cl–channels expressed in these cells as described previously. The reversal potential of the induced Cl–current is more negative when activated through hypotonic NaCl solution (–47 ± 5 mV; n = 6) compared with activation through isotonic high-KCl solution (–35 ± 3 mV; n = 8). This difference can be explained by an increase in intracellular [Cl–] through the activity of a K-Cl cotransporter. Potassium aspartate was unable to activate the current, and furosemide or DIOA suppressed the increase in Cl–current induced by isotonic high-KCl solution. In addition, we used the polymerase chain reaction to demonstrate the presence of KCC1–KCC4 mRNA in the osteoblast-like cell line. From these results, we conclude that human osteoblasts express functional K-Cl cotransporters in their cell membrane that seem to be able to induce the indirect activation of volume-sensitive Cl–channels by KCl through an increase in the intracellular ion concentration followed by water influx and cell swelling.

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 Lys(25) and Tyr(26). These interactions are (i) electrostatic interactions between the extremity of Lys(25) 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 Tyr(26) and Phe(6) and Kv1.1 residue Tyr(379) 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.

Using the whole-cell recording mode of the patch-clamp technique we studied the effects of removal of extracellular potassium, [K + ] o , on a mammalian Shaker -related K + channel, h Kv1.5. In the absence of [K + ] o , current through h Kv1.5 was similar to currents obtained in the presence of 4.5 mM [K + ] o . This observation was not expected as earlier results had suggested that either positively charged residues or the presence of a nitrogen-containing residue at the external TEA + binding site (R487 in h Kv1.5) caused current loss upon removal of [K + ] o . However, the current loss in h Kv1.5 was observed when the extracellular pH, pH o , was reduced from 7.4 to 6.0, a behavior similar to that observed previously for current through m Kv1.3 with a histidine at the equivalent position (H404). These observations suggested that the charge at R487 in h Kv1.5 channels was influenced by other amino acids in the vicinity. Replacement of a histidine at position 463 in h Kv1.5 by glycine confirmed this hypothesis making this H463G mutant channel sensitive to removal of [K + ] o even at pH o 7.4. We conclude that the protonation of H463 at pH 7.4 might induce a p K a shift of R487 that influences the effective charge at this position leading to a not fully protonated arginine. Furthermore, we assume that the charge at position 487 in h Kv1.5 can directly or indirectly disturb the occupation of a K + binding site within the channel pore possibly by electrostatic interaction. This in turn might interfere with the concerted transition of K + ions resulting in a loss of K + conduction.

Using a peptide toxin, kaliotoxin (KTX), we gained new insight into the topology of the pore region of a voltage-gated potassium channel, mKv1.1. In order to find new interactions between mKv1.1 and KTX, we investigated the pH dependence of KTX block which was stronger at pH(o) 6.2 compared with pH(o) 7.4. Using site-directed mutagenesis on the channel and the toxin, we found that protonation of His(34) in KTX caused the pH(o) dependence of KTX block. Glu(350) and Glu(353) in mKv1.1, which interact with His(34) in KTX, were calculated to be 4 and 7 A away from His(34)/KTX, respectively. Docking of KTX into a homology model of mKv1.1 based on the KcsA crystal structure using this and other known interactions as constraints showed structural differences between mKv1.1 and KcsA within the turret (amino acids 348-357). To satisfy our data, we would have to modify the KcsA crystal structure for the mKv1.1 channel orienting Glu(350) 7 A and Glu(353) 4 A more toward the center of the pore compared with KcsA. This would place Glu(350) 15 A and Glu(353) 11 A away from the center of the pore instead of the distances for the equivalent KcsA residues with 22 A for Gly(53) and 15 A for Gly(56), respectively. Bacterial and mammalian potassium channels may have structural differences regarding the turret of the outer pore vestibule. This topological difference between both channel types may have substantial influence on structure-guided development of new drugs for mammalian potassium channels by rational drug design.

Using the patch-clamp technique, we have characterized membrane currents in single detrusor smooth muscle cells from rat and human urinary bladder. From the voltage- and Ca(2+)-dependence of the current as well as the single channel conductance we conclude that rat and human urinary bladder smooth muscle cells express MaxiK channels. In smooth muscle cells from rat urinary bladder we tested the action of NS1608 on current through these MaxiK channels. Application of 10 microm NS1608 increased the amplitude of the current and this increase could be explained by a shift in the activation voltage of the MaxiK channels approximately 100 mV towards more negative potentials. Charybdotoxin as well as paxilline, well known blockers of MaxiK channels, were able to reduce current through MaxiK channels in our cell preparation. In addition, application of 10 microm NS1608 hyperpolarized the membrane potential of the investigated cells. This hyperpolarization could be antagonized by the application of paxilline. We conclude that application of NS1608 results in the opening of MaxiK channels under physiological conditions that leads to a hyperpolarization of the cells. This hyperpolarization in turn could relax urinary bladder smooth muscle cells. MaxiK channels in these cells could therefore play a role in directly controlling muscle tone by regulating the membrane potential. This opens up the possibility of MaxiK channels being targets for the treatment of urge incontinence.

Episodic ataxia type 1 (EA-1) is a rare neurological disorder and was the first ionic channel disease to be associated with defects in a potassium channel. Until now 10 different point mutations in the KCNA1-gene have been reported to cause this disorder. We have investigated the functional consequences of two mutations leading to amino acid substitutions in the first and sixth transmembrane segments of a Kv1.1 channel subunit, by means of the patch-clamp technique; we injected cRNA coding for, respectively, F184C and V408A mutant Kv1.1 channels into mammalian cells and compared the resulting currents with those in the wild-type. The expression levels of F184C and V408A mutant channels relative to that of the wild-type was 38 and 68%, respectively. Since the single-channel conductance of the F184C mutant was similar to that of the wild-type (12 pS) without an apparent change in the maximum open probability, we conclude that the lower expression level in the F184C mutant channels is due to a reduced number of functional channels on the cell surface. F184C activated slower, and at more depolarized potentials, and deactivated faster compared with the wild-type. V408A channels deactivated and inactivated faster compared with the wild-type. Studies with different extracellular cations and tetraethylammonium gave no indication that the pore structure was changed in the mutant channels. Acetazolamide, that is helpful in some patients suffering from EA-1, was without effect on Kv1.1 wild-type or mutant channels. This study confirms and extends earlier studies on the functional consequences of Kv1.1 mutations associated with EA-1, in an attempt to understand the pathophysiology of the disease.

1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellular K+ and Rb+ on membrane currents in human osteoblasts, in a human osteoblast-like cell line, and in the Jurkat human leukaemic T cell line. 2. Increasing the extracellular concentration of K+ increased the membrane conductance of the cells in a concentration-dependent manner. This increase in membrane conductance was due to the activation of a Cl- conductance. Rb+ also induced this conductance, but conductance was less than half that seen in K+. 3. The Cl- channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanatostilbene 2,2'-disulphonic acid (SITS) blocked the K(+)-induced Cl- current in a voltage-dependent manner. The degree of blockade increased with membrane depolarization to a maximum level at 40 mV. At potentials above this value the block appeared to decrease. 4. Both tonicity and K+ were required for maximal activation of the Cl- conductance since the K(+)-induced Cl- conductance could be inhibited by hypertonic solutions and the activation of a volume-sensitive Cl- conductance by hypotonic solutions could be enhanced by extracellular K+. 5. We conclude that an outwardly rectifying Cl- conductance can be activated either upon osmotic swelling or by an increase in extracellular K+. Both activation pathways may be involved in cell volume regulation and seem to apply to volume-sensitive Cl- channels in general since we observe this phenomenon in two different cell types, in human osteoblasts as well as in human leukaemic T lymphocytes.

1. The whole-cell recording mode of the patch-clamp technique was used to study the effect of extracellularly applied ions, toxins and drugs on voltage-independent, apamin-sensitive Ca(2+)-activated K+ channels, K(Ca), expressed in the Jurkat human leukaemic T cell line. 2. Extracellular Ba2+ and Sr+ produced a voltage-dependent block. The equilibrium dissociation constant of the Ba2+/K(Ca) channel complex increased e-fold for a 20 mV change of potential. Ba2+ block of Jurkat K(Ca) channels is therefore as steep as expected from the movement of a single divalent cation about half-way into the electric field of the membrane from the outside. 3. We determined the ion selectivity as well as the conductance of these channels. Calculated permeability ratios, PX/PK, for these K(Ca) channels were 1.0, 0.96, 0.26 and 0.53 for K+, Rb+, Cs+ and NH4+, respectively. Conductance ratios, gX/gK, for the same ions were 1.0, 1.0, 0.67 and 0.11, respectively. Most strikingly this channel can also carry significant current with Cs+ as current carrier. 4. Scyllatoxin (ScTX), a thirty-one amino acid peptide toxin, reduced current through these K(Ca) channels with a half-blocking concentration of approximately 0.3 nM independent of the pH. Other drugs that were able to reduce current through these channels include the classical calcium antagonists diltiazem and verapamil. In contrast, nifedipine, clotrimazole and kaliotoxin (100 nM) were unable to block current through these channels in Jurkat T cells.

The voltage-gated hKv1.3 channel, member of the Shaker-related potassium channels, is involved in T-cell activation and is characterized by its typical C-type inactivation. To characterize the three-dimensional structure of the C-type inactivated state a cysteine was introduced at position 417 (Shaker position 467) in the hKv1.3 channel and a putative involvement in C-type inactivation was determined using MTS-reagents. MTSEA application led, in contrast to wildtype channels, to a fast and irreversible current reduction through hKv1.3_V417C channels in the open or inactivated state indicating that a modification of both states was possible. This modification could be prevented by verapamil. In contrast, the closed state of this mutant channel could not be modified by MTSEA. Furthermore a current reduction was observed only when the positively charged MTSET was applied intracellularly and not when applied extracellulary to hKv1.3_V417C channels. These experiments indicated that the binding site for MTS-reagents is intracellular and that a modification of the cysteine at position 417 in the hKv1.3_V417C mutant channel was possible in the open and also inactivated state of the channel. In addition, the fact that the inactivated state of the hKv1.3_V417C mutant channel could be modified by MTSEA indicated also that the activation gate must be open during inactivation, the side chain of the cysteine at position 417 does not move during inactivation in a way that it is not available for modification any more and the channel is, using the model by Cuello et al. (2010, Nature 466:203), in the open-inactivated state.This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Gr 848/14-1) and by the International Graduate School in Molecular Medicine Ulm.

Current through wt hKv1.3channels is characterized by the typical C-type inactivation and the high affinity block by scorpion peptide toxins. These toxins belong to a short peptide toxin family with high similarities in 3D shape and sequence and are thought to block current through hKv1.3 channels by interacting with the outer vestibule of the channel thereby physically occluding the channel pore. In this study we measured current through hKv1.3_V388C mutant channels, which inactivated faster compared to the wt channels and recovery from inactivation was also slower. In our experiments we examined the effect of CTX, NTX, KTX, AgTX2 and MgTX, each at a concentration of 100 nM, on current through mutant channels. KTX and AgTX2 did not or hardly block peak current through the mutant channel at 100 nM, respectively, indicating a loss of affinity by a factor of >100 due to the mutation in the channel. In addition, KTX and AgTX2 did not change the inactivation time course of the current through the mutant channels. In contrast, MgTX, even at a concentration of 10 nM, almost completely blocked peak current, indicating similar affinities of MgTX to mutant and wt channels. Interestingly, CTX and NTX did not block peak current through the mutant channels, however, almost completely abolished inactivation. This is different from the effect of TEA that is known to prevent inactivation while blocking current through the channel. We conclude that CTX and NTX bind to the external vestibule of the channel thereby preventing structural rearrangements of the outer vestibule that normally occur when channels inactivate.This work was supported by a grant from the Deutsche Forschugsgemeinschaft (Gr848/14-1).

Publisher Summary In the nervous system, voltage-gated Na + and K + channels effect the upstroke and repolarizing phases, respectively, of the action potential. Cells of the immune system do not generate action potentials. Depolarization that induces Ca 2+ signals in electrically excitable nerve and muscle cells inhibits Ca 2+ signaling and the subsequent cascade of cell-activation events in lymphocytes. Several distinct types of voltage-gated and second-messenger-operated K + and C1 − channels exist in T and B lymphocytes, including channel types also expressed in the nervous system, as well as novel channels not described in other cell types. In electrically inexcitable cells, ion channels mediate cellular functions, involving intracellular biochemical signaling. The presence of K + channels is apparently required for several basic functions in T lymphocytes. There is diversity of ion channels within cells of the hematopoietic lineage. In T lymphocytes, at least eight distinct types of K + , C1 − , and Ca 2+ channels have been characterized. Investigators have also recorded from bone marrow cells, thymocytes, B lymphocytes, monocytes, macrophages, neutrophils, red blood cells, eosinophils, and platelets, as well as numerous related cell lines, including a human T-leukemia line, a mouse T-lymphoma line (S49), a variety of antibody-secreting hybridomas, and a mast-cell line derived from a rat basophilic leukemia (RBL). These studies have revealed transient or T-type voltage-gated Ca 2+ channels (hybridomas), inward-rectifying K + channels (macrophages, RBL cells), large-conductance Ca 2+ -activated K + channels (macrophages), Ca 2+ -activated C1 − current (mast cells), and G-protein-activated K + channels (RBL cells). In this chapter, K + channels in T lymphocytes also containing a summary of the properties of known voltage- and Ca 2+ -activated K + channels in lymphocytes and their molecular characteristics, functions, and expression during normal development, mitogen activation, and in autoimmune disorders are discussed. In addition, the chapter describes the coordinated activity of Ca 2+ and K + channels that underlies the mitogen-stimulated Ca 2+ signaling.

We recently isolated a family of three closely related mouse K+ channel genes (MK1, MK2, and MK3) with coding regions contained in single uninterrupted exons. Here we have used patch-clamp recordings from Xenopus oocytes injected with mRNA to show that MK3 encodes a channel with biophysical and pharmacological properties indistinguishable from those of voltage-gated type n K+ channels in T cells. In addition, we used the polymerase chain reaction to demonstrate the presence of MK3 mRNA in T cells. These data suggest that MK3 may encode the T-cell voltage-gated type n K+ channel. We also show that MK3 and MK2 are localized on human chromosomes 13 and 12, respectively.

Using the patch-clamp technique in combination with fluorescence microscopy we have found an abnormality in voltage-gated K+ channel expression in T cells that represents the first molecular marker linking three disparate autoimmune diseases in mice. CD4-CD8-Thy-1.2+ (double-negative or DN) lymphocytes from every known murine model for systemic lupus erythematosus, type-1 diabetes mellitus and experimental allergic encephalomyelitis exhibit abnormally high numbers of an unusual K+ channel, termed type l compared to their phenotypic counterparts in normal mice. Other T cell subsets from these diseased mice retain their normal pattern of K+ channel expression. The unique K+ channel phenotype of DN T cells arises in parallel with the onset of autoimmunity. Although mitogen-activated T cells and rapidly proliferating thymocytes exhibit large numbers of K+ channels, these channels are of an electrophysiologically distinct type called n. Thus, abundant expression of type l K+ channels appears to be a useful marker for DN T cells associated with autoimmunity and may provide a valuable tool for delineating the role of DN T cells in the pathogenesis of autoimmune diseases.

The whole-cell recording mode of the patch-clamp technique was used to study the effect of external tetraethylammonium ([TEA+]o) on the inactivating, voltage-dependent K+ channels of human T lymphocytes. TEA+ reduced the peak amplitude and slowed the time course of the K+ current decay during a depolarizing pulse, resulting in a crossover of the current records in the presence and absence of TEA+. In solutions with different [TEA+]o both the peak K+ current amplitude, lKpeak, and the time constant of the decay of the K+ current, tau d, were reduced in a dose-dependent manner, both with apparent binding constants, KD, of 12 mM. The integral of K+ current during a prolonged depolarizing pulse was unaltered in solutions with different [TEA+]o. The concentration dependence of [TEA+]o on lKpeak, tau d, and the unchanged current integral can be explained with a kinetic scheme in which open channels blocked by TEA+ cannot inactivate.

Using the patch-clamp whole-cell recording technique, we investigated the influence of external Ca2+, Ba2+, K+, Rb+, and internal Ca2+ on the rate of K+ channel inactivation in the human T lymphocyte-derived cell line, Jurkat E6-1. Raising external Ca2+ or Ba2+, or reducing external K+, accelerated the rate of the K+ current decay during a depolarizing voltage pulse. External Ba2+ also produced a use-dependent block of the K+ channels by entering the open channel and becoming trapped inside. Raising internal Ca2+ accelerated inactivation at lower concentrations than external Ca2+, but increasing the Ca2+ buffering with BAPTA did not affect inactivation. Raising [K+]0 or adding Rb+ slowed inactivation by competing with divalent ions. External Rb+ also produced a use-dependent removal of block of K+ channels loaded with Ba2+ or Ca2+. From the removal of this block we found that under normal conditions ~25% of the channels were loaded with Ca2+, whereas under conditions with 10 MM internal Ca2+ the proportion of channels loaded with Ca2+ increased to -50%. Removing all the divalent cations from the external and internal solution resulted in the induction of a non-selective, voltage-independent conductance. We conclude that Ca2+ ions from the outside or the inside can bind to a site at the K+ channel and thereby block the channel or accelerate inactivation. © 1989, Rockefeller University Press., All rights reserved.

1. Voltage-clamp experiments were performed on single frog internodes after acute demyelination with lysolecithin. The action of lysolecithin was stopped by washing out the lysolecithin with normal Ringer solution containing bovine albumin when the first delayed current was observed. After washing, the temperature was lowered from 25 to 15 degrees C. These procedures greatly prolonged the survival of the demyelinated internode up to 1 h. 2. External tetraethylammonium chloride (TEA+, 110 mM) reduced the K+ current in the internode only to 11% of the control value. 110 mM-TEA+ increased the time constant tau n of K+ activation by a factor of two in the node and by a factor of four in the internode. 120 mM-CsCl at the cut ends of the fibre also reduced the delayed outward current recorded at 60 mV in the internode to 11% of the control value, hardly changing the time constant tau n. 3. After a depolarization, the K+ tail current decayed in two phases, suggesting that the K+ conductance of the internodal membrane may be composed of at least two components, a slow one (gKs) and a fast one (gKf). As in the node, the fast K+ conductance of the internode can be further decomposed into two components (gKf1 and gKf2) with different activation potential ranges. The fast phase of the tail current was blocked by external application of 1 mM-4-aminopyridine (4-AP). The slow phase was almost unaltered by 1 mM-4-AP. The extrapolated slow tail current was 33% of the total tail current in the internode and 15% at the node, i.e. the proportion of slow K+ channels is larger in the internode than in the node. 4. Tetrodotoxin (TTX)-sensitive transient inward currents could be measured in the demyelinated internode, provided the large K+ currents were blocked by internal Cs+. The time course, TTX sensitivity, reversal potential and steady-state inactivation of the transient early inward current indicate that this current is caused mainly by Na+ passing through Na+ channels. 5. The density of K+ and Na+ channels in the demyelinated internode is estimated from the size of the K+ and Na+ current, respectively, and the capacity of the demyelinated segment. The K+ channel density of the internode seems to be about 20 times smaller than in the node, whereas the Na+ channel density in the internode appears to be about 500 times smaller than in the node.

The loop between transmembrane regions S5 and S6 (P-region) of voltage-gated K+ channels has been proposed to form the ion-conducting pore, and the internal part of this segment is reported to be responsible for ion permeation and internal tetraethylammonium (TEA) binding. The two T-cell K+ channels, Kv3.1 and Kv1.3, with widely divergent pore properties, differ by a single residue in this internal P-region, leucine 401 in Kv3.1 corresponding to valine 398 in Kv1.3. The L401V mutation in Kv3.1 was created with the anticipation that the mutant channel would exhibit Kv1.3-like deep-pore properties. Surprisingly, this mutation did not alter single channel conductance and only moderately enhanced internal TEA sensitivity, indicating that residues outside the P-region influence these properties. Our search for additional residues was guided by the model of Durell and Guy, which predicted that the C-terminal end of S6 formed part of the K+ conduction pathway. In this segment, the two channels diverge at only one position, Kv3.1 containing M430 in place of leucine in Kv1.3. The M430L mutant of Kv3.1 exhibited permeant ion- and voltage-dependent flickery outward single channel currents, with no obvious changes in other pore properties. Modification of one or more ion-binding sites located in the electric field and possibly within the channel pore could give rise to this type of channel flicker.

The replacement of the amino acid valine with the smaller cysteine at position 388 (Shaker position 438) in the mutant voltage-gated potassium channel hKv1.3_V388C opened a new pathway (σ-pore) behind the central α-pore. At potentials more negative than -100 mV, where the central α-pore of the hKv1.3_wt would normally be closed, a large inward current through the σ-pore appeared being carried by different cations like Na+, Li+, Cs+, and NH4+ (Prütting et al. 2011; JBC 286:20031-20042) whereas the current through the α-pore in Na+ solution showed faster inactivation. For the pharmacological characterization of the σ-pore we performed measurements with the patch-clamp technique in the whole cell recording mode. For each experiment drugs were added to the external bath solution. To simultaneously measure current through both α- and σ-pores and determine the blocking effect we used depolarizing pulses from the holding potential of -120 mV to +40 mV for 100 ms to show currents through the α-pore, followed by hyperpolarizing pulses to -180 mV to show currents through the σ-pore. α-pore-blocking peptide toxins acting at the external vestibule of the channel such as CTX, KTX, and AgTX blocked the α-pore with five-times lower affinity than hKv1.3_wt channels and showed no blocking effect on the σ-pore of the mutant channel. PAP-1 blocked current through both α- and σ-pore with high affinity similar to wildtype (KD ~ 2 nM). The phenylalkylamine blocker verapamil and its derivate norverapamil acting from the intracellular side of the channel reduced currents through both pores with the same affinity, similar to wildtype. Methoxyverapamil and acyanoverapamil could also reduce both currents, however with somewhat different affinities for the σ-pore compared to current through the α-pore indicating a possibility to distinguish the location of the α-pore- and σ-pore-exit into the internal vestibule.