-
[show abstract]
[hide abstract]
ABSTRACT: Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.
Journal of Biological Chemistry 06/2011; 286(22):20031-42. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: hKv1.3 channels in lymphocytes are targets for the chemotherapy treatment of autoimmune diseases. Phenylalkylamines block Kv1.3 channels by poorly understood mechanisms. In the inactivation-reduced mutant H399T, the second mutation A413C in S6 substantially decreases the potency of phenylalkylamines with a para-methoxy group at the phenylethylamine end, whereas potency of phenylalkylamines lacking this group is less affected. Intriguingly, completely demethoxylated emopamil blocks mutant H399T/A413C with a 2:1 stoichiometry. Here, we generated a triple mutant, H399T/C412A/A413C, and found that its emopamil-binding properties are similar to those of the double mutant. These data rule out disulfide bonding Cys412-Cys413, which would substantially deform the inner helix, suggest a clash of Cys413 with the para-methoxy group, and provide a distance constraint to dock phenylalkylamines in a Kv1.2-based homology model. Monte Carlo minimizations predict that the verapamil ammonium group donates an H-bond to the backbone carbonyl of Thr391 at the P-loop turn, the pentanenitrilephenyl moiety occludes the pore, whereas the phenylethylamine meta- and para-methoxy substituents approach, respectively, the side chains of Met390 and Ala413. In the double-mutant model, the Cys413 side chains accept H-bonds from two emopamil molecules whose phenyl rings fit in the hydrophobic intersubunit interfaces, whereas the pentanenitrilephenyl moieties occlude the pore. Because these interfaces are unattractive for a methoxylated phenyl ring, the ammonium group of respective phenylalkylamines cannot approach the Cys413 side chain and binds at the focus of P-helices, whereas the para-methoxy group clashes with Cys413. Our study proposes an atomistic mechanism of Kv1.3 block by phenylalkylamines and highlights the intra- and intersubunit interfaces as ligand binding loci.
Molecular pharmacology 01/2011; 79(4):681-91. · 4.53 Impact Factor
-
Stefano Pegoraro,
Martin Lang,
Tobias Dreker,
Jürgen Kraus,
Svetlana Hamm,
Cathal Meere,
Juliane Feurle,
Stefan Tasler,
Sylvia Prütting,
Zerrin Kuras,
Violeta Visan, Stephan Grissmer
[show abstract]
[hide abstract]
ABSTRACT: 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.
Bioorganic & medicinal chemistry letters 05/2009; 19(8):2299-304. · 2.65 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cellular Physiology and Biochemistry 02/2009; 24(1-2):53-64. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Throughout the CNS, small conductance Ca2+-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.
Journal of Neurochemistry 08/2008; 106(6):2312 - 2321. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.
Protein Science 02/2008; 17(1):107-18. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Cellular Physiology and Biochemistry 02/2008; 21(1-3):55-62. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ion channels are potent modulators for developmental processes in progenitor cells. In a screening approach for different ion channels in neural progenitor cells (NPCs) we observed a 1-ethyl-2-benzimidazolinone (1-EBIO) activated inward current, which could be blocked by scyllatoxin (ScTX, IC50=2+/- 0.3 nmol/L). This initial evidence for the expression of the small conductance Ca2+ activated K+-channel SK3 was confirmed by the detection of SK3 transcripts and protein in NPCs. Interestingly, SK3 proteins were highly expressed in non-differentiated NPCs with a focused localization in lamellipodia as well as filopodial structures. The activation of SK3 channels using 1-EBIO lead to an immediate filopodial sprouting and the translocation of the protein into these novel filopodial protrusions. Both effects could be prevented by the pre-incubation of NPCs with ScTX. Our study gives first evidence that the formation and prolongation of filopodia in NPCs is, at least in part, effectively induced and regulated by SK3 channels.
Journal of Neurochemistry 07/2007; 101(5):1338-1350. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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)-sensitive K(+) currents with different voltage dependencies ( = 65 microm, E(50) = -0.3 +/- 1.3 mV and = 8 mm, E(50) = -15.2 +/- 2.8 mV) and an almost TEA-insensitive current were identified. Kaliotoxin blocked approximately 50% of the entire K(+) currents (IC(50) = 0.25 nm). These properties resembled functional characteristics of K(v)1.4, K(v)1.3 and K(v)3.1 channels. Transcripts for these channels, as well as proteins for K(v)1.3 and K(v)3.1, were identified. Immunocytochemical staining revealed K(v)1.3 and K(v)3.1 K(+) channel expression in almost all NPCs. The blockage of K(v)3.1 by low concentrations of TEA, as well as the blockage of K(v)1.3 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.
Journal of Neurochemistry 11/2006; 99(2):426-37. · 4.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Molecular Pharmacology 05/2006; 69(4):1356-65. · 4.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Phenothiazines can be used as psychopharmaceutical agents and are known to cause many side effects during treatment since they interfere with many different cellular systems. Recently, phenothiazines were reported to block Ca(2+)-activated potassium channels of the SK type. Therefore we investigated their effect on the functionally related class of Ca(2+)-activated potassium channels of the IK type. The representative phenothiazine derivative promethazine (PTZ) blocked IK channels almost independently from the extracellular pH(o) with an IC(50) of 49 +/- 0.2 microM (pH(o) 7.4, n = 5) and 32 +/- 0.2 microM (pH(o) 6.2, n = 5) in whole cell experiments. The extracellularly applied membrane impermeable PTZ analogue methyl-promethazine (M-PTZ) had a strongly reduced blocking potency compared to PTZ. In contrast, intracellularly applied PTZ and M-PTZ had the same blocking potency on IK channels in excised inside out patch clamp experiments (K(d) = 9.3 +/- 0.5 microM for PTZ, n = 7 and 6.7 +/- 0.4 microM for M-PTZ, n = 5). The voltage dependency of the PTZ and M-PTZ block was investigated in excised inside out patch clamp experiments at a concentration of 100 microM. For both compounds the block was more pronounced at positive membrane potentials. The steepness of the voltage dependency was found to be 70 +/- 10 mV (for PTZ) and 61 +/- 6 mV (for M-PTZ) indicating that both compounds sensed approximately 40% of the entire membrane spanning electrical field from the inside. We conclude that PTZ and M-PTZ bind to a side in IK channels, which is located within the electrical field and is accessible from the intracellular side.
Neuropharmacology 04/2006; 50(4):458-67. · 4.81 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Ca(2+) activated K(+) channels modulate the afterhyperpolarization in neurons. Using a variety of different techniques we obtained information about the function of N- and C-terminal parts of the Ca(2+)-activated K(+) channel, SK3. By means of the yeast two hybrid technique we found an interaction between N-C and N-N- terminal parts of SK3. The strong N-C and N-N interaction was specific for SK3 and could not be observed for SK1 and SK2. Possibly a homotetrameric assembly of SK3 is favored in tissues were all SK channels are expressed. In addition, the interaction in SK3 was independent of the length of the polymorphic glutamine repeat in the N-terminus of SK3. Electrophysiological investigations showed that expression of amino acids 1-299 of SK3 (SK3N_299) modified the 1-EBIO pharmacology of endogenous SK3 channels in PC12 cells without affecting the Ca(2+)-sensitvity. The activation by 0.5 mM 1-EBIO in cells expressing amino acids 1-299 of SK3 was reduced by 32% in comparison to control experiments. Considering the N-C interaction in yeast, we conclude that the sensitivity of SK3 channels to 1-EBIO was modified by N-C interactions with SK3N_299. Therefore we conclude that N-C interactions influence SK3 channel function.
Cellular Physiology and Biochemistry 02/2006; 18(4-5):165-76. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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, [Lys(16),Asp(20)]-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 [Lys(16),Asp(20)]-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 K(Ca)3.1 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 beta-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 [Lys(16),Asp(20)]-OSK1 to identify new analogs with unique pharmacological properties.
Molecular Pharmacology 02/2006; 69(1):354-62. · 4.88 Impact Factor
-
George A Gutman,
K George Chandy, Stephan Grissmer,
Michel Lazdunski,
David McKinnon,
Luis A Pardo,
Gail A Robertson,
Bernardo Rudy,
Michael C Sanguinetti,
Walter Stühmer,
Xiaoliang Wang
Pharmacological Reviews 01/2006; 57(4):473-508. · 20.23 Impact Factor
-
Pharmacological Reviews 01/2006; 57(4):463-72. · 20.23 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To screen for residues of hKv1.3 important for current block by the phenylalkylamine verapamil, the inactivated-state-reduced H399T mutant was used as a background for mutagenesis studies. This approach was applied mainly to abolish the accumulation in the inactivated blocked state, recovery from which in the wild type is normally slow. Substitution of amino acids in the S6 transmembrane helix indicated a heavy disruption of verapamil block by the A413C mutation, reducing the IC(50) from 2.4 to 267 microM. Subsequent scanning for verapamil moieties essential for current block was performed by application of derivatives with altered side groups. Neither the removal of the nitrile or the methyl group nor the addition of a methoxy group resulted in major variations of IC(50) values for hKv1.3 (H399T) current block. However, disruption of current block by A413C was 4- to 10-fold less pronounced for derivatives lacking the 4-methoxy group of the (3,4-dimethoxyphenyl)ethylmethyl-amino part (devapamil) or all four methoxy groups (emopamil), respectively. Emopamil displayed a Hill coefficient of 2 for hKv1.3 (H399T/A413C) instead of 1 for hKv1.3 (H399T) current block. These results might indicate that the alteration of Ala413 modulates the access of phenylalkylamines to their binding site depending on the occupancy of the phenyl rings with methoxy groups. A computer-based docking model shows a subset of docked PAA conformations, with a spatial proximity between the (4-methoxyphenyl)ethyl-methyl-amino group and Ala413. The PAA binding site might therefore include a binding pocket for the aromatic ring of the ethyl-methyl-amino part in an S6-S6 interface gap.
Molecular Pharmacology 11/2005; 68(4):966-73. · 4.88 Impact Factor
-
Sarrah M'Barek,
Benjamin Chagot,
Nicolas Andreotti,
Violeta Visan,
Pascal Mansuelle, Stephan Grissmer,
Mohamed Marrakchi,
Mohamed El Ayeb,
François Sampieri,
Hervé Darbon,
Ziad Fajloun,
Michel De Waard,
Jean-Marc Sabatier
[show abstract]
[hide abstract]
ABSTRACT: 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.
Proteins Structure Function and Bioinformatics 09/2005; 60(3):401-11. · 3.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Biochemical Journal 02/2005; 385(Pt 1):95-104. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Molecular Pharmacology 12/2004; 66(5):1103-12. · 4.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 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.
Toxicon 07/2004; 43(8):973-80. · 2.51 Impact Factor
