The Concise Guide to PHARMACOLOGY 2023/24: Ion channels.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

THE CONCISE GUIDE TO PHARMACOLOGY 2021/22: Ion channels.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15539. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Characterization of the σ-Pore in Mutant hKv1.3 Potassium Channels.

BACKGROUND/AIMS: The replacement of the amino acid valine at position 388 (Shaker position 438) in hKv1.3 channels or at the homologue position 370 in hKv1.2 channels resulted in a channel with two different ion conducting pathways: One pathway was the central, potassium-selective α-pore, that was sensitive to block by peptide toxins (CTX or KTX in the hKv1.3_V388C channel and CTX or MTX in the hKv1.2_V370C channel). The other pathway (σ-pore) was behind the central α-pore creating an inward current at potentials more negative than -100 mV, a potential range where the central α-pore was closed. In addition, current through the σ-pore could not be reduced by CTX, KTX or MTX in the hKv1.3_V388C or the hKv1.2_V370C channel, respectively. METHODS: For a more detailed characterization of the σ-pore, we created a trimer consisting of three hKv1.3_V388C α-subunits linked together and characterized current through this trimeric hKv1.3_V388C channel. Additionally, we determined which amino acids line the σ-pore in the tetrameric hKv1.3_V388C channel by replacing single amino acids in the tetrameric hKv1.3_V388C mutant channel that could be involved in σ-pore formation. RESULTS: Overexpression of the trimeric hKv1.3_V388C channel in COS-7 cells yielded typical σ-pore currents at potentials more negative than -100 mV similar to what was observed for the tetrameric hKv1.3_V388C channel. Electrophysiological properties of the trimeric and tetrameric channel were similar: currents could be observed at potentials more negative than -100 mV, were not carried by protons or chloride ions, and could not be reduced by peptide toxins (CTX, MTX) or TEA. The σ-pore was mostly permeable to Na+ and Li+. In addition, in our site-directed mutagenesis experiments, we created a number of new double mutant channels in the tetrameric hKv1.3_V388C background channel. Two of these tetrameric double mutant channels (hKv1.3_V388C_T392Y and hKv1.3_V388C_Y395W) did not show currents through the σ-pore. CONCLUSIONS: From our experiments with the trimeric hKv1.3_V388C channel we conclude that the σ-pore exists in hKv1.3_V388C channels independently of the α-pore. From our site-directed mutagenesis experiments in the tetrameric hKv1.3_V388C channel we conclude that amino acid position 392 and 395 (Shaker position 442 and 445) line the σ-pore.

Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels.

BACKGROUND/AIMS: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated. METHODS: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil. RESULTS: The on-rate of verapamil to block current through hKv1.3_T419C mutant channels is similar to that obtained for hKv1.3_wt channels whereas the on-rate of verapamil to block currents through hKv1.3_L417C and hKv1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3_L346C and the double mutant hKv1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hKv1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold. CONCLUSIONS: We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3_L346C and hKv1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.

Observation of σ-pore currents in mutant hKv1.2_V370C potassium channels.

Current through the σ-pore was first detected in hKv1.3_V388C channels, where the V388C mutation in hKv1.3 channels opened a new pathway (σ-pore) behind the central α-pore. Typical for this mutant channel was inward current at potentials more negative than -100 mV when the central α-pore was closed. The α-pore blockers such as TEA+ and peptide toxins (CTX, MTX) could not reduce current through the σ-pore of hKv1.3_V388C channels. This new pathway would proceed in parallel to the α-pore in the S6-S6 interface gap. To see whether this phenomenon is restricted to hKv1.3 channels we mutated hKv1.2 at the homologue position (hKv1.2_V370C). By overexpression of hKv1.2_V370C mutant channels in COS-7 cells we could show typical σ-currents. The electrophysiological properties of the σ-pore in hKv1.3_V388C and hKv1.2_V370C mutant channels were similar. The σ-pore of hKv1.2_V370C channels was most permeable to Na+ and Li+ whereas Cl- and protons did not influence current through the σ-pore. Tetraethylammonium (TEA+), charybdotoxin (CTX) and maurotoxin (MTX), known α-pore blockers, could not reduce current through the σ-pore of hKv1.2_V370C channels. Taken together we conclude that the observation of σ-pore currents is not restricted to Kv1.3 potassium channels but can also be observed in a closely related potassium channel. This finding could have implications in the treatment of different ion channel diseases linked to mutations of the respective channels in regions close to homologue position investigated by us.

International Union of Basic and Clinical Pharmacology. C. Nomenclature and Properties of Calcium-Activated and Sodium-Activated Potassium Channels.

A subset of potassium channels is regulated primarily by changes in the cytoplasmic concentration of ions, including calcium, sodium, chloride, and protons. The eight members of this subfamily were originally all designated as calcium-activated channels. More recent studies have clarified the gating mechanisms for these channels and have documented that not all members are sensitive to calcium. This article describes the molecular relationships between these channels and provides an introduction to their functional properties. It also introduces a new nomenclature that differentiates between calcium- and sodium-activated potassium channels.

Evidence for the interaction of endophilin a3 with endogenous Kca2.3 channels in PC12 cells.

BACKGROUND/AIMS: Small-conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of K(Ca)2.3 channels and Endophilin A3 in different tissues. Endophilin A3 belongs to a family of BAR- and SH3 domain containing proteins that bind to dynamin and are involved in the process of vesicle scission in clathrin-mediated endocytosis. METHODS: Using the yeast two-hybrid system and the GST pull down assay we demonstrated that Endophilin A3 interacts with the N-terminal part of K(Ca)2.3 channels. In addition, we studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous K(Ca)2.3 channels. K(Ca)2.3 currents were activated by using pipette solutions containing 1 µM free Ca(2+). RESULTS: Whole-cell measurements of PC12 cells transfected with Endophilin A3 showed a reduction of KCa2.3 specific Cs(+) currents indicating that the interaction of Endophilin A3 with K(Ca)2.3 channels also occurs in mammalian cells and that this interaction has functional consequences for current flowing through K(Ca)2.3 channels. Since K(Ca)2.3 specific currents could be increased in PC12 cells transfected with Endophilin A3 with DC-EBIO (30 µM), a known SK-channel activator, these data also implicate that Endophilin A3 did not significantly remove K(Ca)2.3 channels from the membrane but changed the sensitivity of the channels to Ca(2+) which could be overcome by DC-EBIO. CONCLUSION: This interaction seems to be important for the function of K(Ca)2.3 channels and might therefore play a significant role in situations where channel activation is pivotal for cellular function.

Verapamil- and state-dependent effect of 2-aminoethylmethanethiosulphonate (MTSEA) on hK(v)1.3 channels.

BACKGROUND AND PURPOSE: T-cells usually express voltage-gated K(v) 1.3 channels. These channels are distinguished by their typical C-type inactivation. Therefore, to be able to rationally design drugs specific for the C-type inactivation state that may have therapeutic value in autoimmune disease therapy, it is necessary to identify those amino acids that are accessible for drug binding in C-type inactivated channels. EXPERIMENTAL APPROACH: The influence of 2-aminoethylmethanethiosulphonate (MTSEA) on currents through wild-type human K(v)1.3 (hK(v)1.3) and three mutant channels, hK(v)1.3_L418C, hK(v)1.3_T419C and hK(v)1.3_I420C, in the closed, open and inactivated states was investigated by the patch-clamp technique. KEY RESULTS: Currents through hK(v)1.3_L418C and hK(v)1.3_T419C channels were irreversibly reduced after the external application of MTSEA in the open state but not in the inactivated and closed states. Currents through hK(v)1.3_I420C channels were irreversibly reduced in the open and inactivated states but not in the closed state. In the presence of verapamil, the MTSEA modification of hK(v)1.3_T419C and hK(v)1.3_I420C channels was prevented, while the MTSEA modification of hK(v)1.3_L418C channels was unaffected. CONCLUSION AND IMPLICATIONS: From our experiments, we conclude that the activation gate of all mutant channels must be open for modification by MTSEA and must also be open during inactivation. In addition, the relative movement of the S6 segments that occur during C-type inactivation includes a movement of the side chains of the amino acids at positions 418 and 419 away from the pore lining. Furthermore, the overlapping binding site for MTSEA and verapamil does not include position 418 in hK(v) 1.3 channels.

Charybdotoxin and margatoxin acting on the human voltage-gated potassium channel hKv1.3 and its H399N mutant: an experimental and computational comparison.

The effect of the pore-blocking peptides charybdotoxin and margatoxin, both scorpion toxins, on currents through human voltage-gated hK(v)1.3 wild-type and hK(v)1.3_H399N mutant potassium channels was characterized by the whole-cell patch clamp technique. In the mutant channels, both toxins hardly blocked current through the channels, although they did prevent C-type inactivation by slowing down the current decay during depolarization. Molecular dynamics simulations suggested that the fast current decay in the mutant channel was a consequence of amino acid reorientations behind the selectivity filter and indicated that the rigidity-flexibility in that region played a key role in its interactions with scorpion toxins. A channel with a slightly more flexible selectivity filter region exhibits distinct interactions with scorpion toxins. Our studies suggest that the toxin-channel interactions might partially restore rigidity in the selectivity filter and thereby prevent the structural rearrangements associated with C-type inactivation.

Charybdotoxin unbinding from the mKv1.3 potassium channel: a combined computational and experimental study.

Charybdotoxin, belonging to the group of so-called scorpion toxins, is a short peptide able to block many voltage-gated potassium channels, such as mKv1.3, with high affinity. We use a reliable homology model based on the high-resolution crystal structure of the 94% sequence identical homologue Kv1.2 for charybdotoxin docking followed by molecular dynamics simulations to investigate the mechanism and energetics of unbinding, tracing the behavior of the channel protein and charybdotoxin during umbrella-sampling simulations as charybdotoxin is moved away from the binding site. The potential of mean force is constructed from the umbrella sampling simulations and combined with K(d) and free energy values gained experimentally using the patch-clamp technique to study the free energy of binding at different ion concentrations and the mechanism of the charybdotoxin-mKv1.3 binding process. A possible charybdotoxin binding mechanism is deduced that includes an initial hydrophobic contact followed by stepwise electrostatic interactions and finally optimization of hydrogen bonds and salt bridges.

A novel current pathway parallel to the central pore in a mutant voltage-gated potassium channel.

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.

Why does the inner-helix mutation A413C double the stoichiometry of Kv1.3 channel block by emopamil but not by verapamil?

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.

cAMP-dependent chloride conductance evokes ammonia-induced blebbing in the microglial cell line, BV-2.

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.

Inhibitors of potassium channels KV1.3 and IK-1 as immunosuppressants.

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.

AQP4 expression in striatal primary cultures is regulated by dopamine--implications for proliferation of astrocytes.

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.

Identification and characterization of a novel, shorter isoform of the small conductance Ca2+ -activated K+ channel SK2.

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.

Chloride influx provokes lamellipodium formation in microglial cells.

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.

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins.

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.

Formation of cellular projections in neural progenitor cells depends on SK3 channel activity.

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.

Interactions of N-terminal and C-terminal parts of the small conductance Ca2+ activated K+ channel, hSK3.

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.