The alpha-subunits of Kv1 channels display characteristic distributions and restricted co-assembly in mammalian brain. The heterogeneous composition of Kv1 channels has made it difficult to use specific toxins to label brain structures. We used autoradiography to analyse the competitive behaviour of three Kv1 channel toxins–alpha-dendrotoxin, kaliotoxin, and mast cell degranulating peptide–for binding to kaliotoxin binding sites in various brain structures. IC(50) varied considerably between brain regions (by up to three orders of magnitude) for each ligand. alpha-dendrotoxin and kaliotoxin competed equally in some regions and to different extents in others, identifying two types of structure. Mast cell degranulating peptide competed with (125)I-kaliotoxin less efficiently than alpha-dendrotoxin and kaliotoxin, in all regions. Thus, differences in the capacity of these three toxins to bind to kaliotoxin binding sites provide evidence of major differences in the composition of the Kv1 channels constituting the kaliotoxin binding sites.Bessone R., et al. (2004) Heterogeneous competition of Kv1 channel toxins with kaliotoxin for binding in rat brain: autoradiographic analysis. Neurochem Int. PMID: 15337303

Olfactory associative learning was used to investigate the involvement of Kv channels containing Kv1.1 and Kv1.3 alpha-subunits in learning and memory. Kaliotoxin (KTX), a specific inhibitor of these Kv channels, was injected intracerebroventricularly in the rat brain, at a dose of 10 ng that did not disturb the rats’ locomotor activity or drinking behaviour. In the first paradigm (odour-reward training), KTX improved learning but not information consolidation. Moreover, KTX increased the long-term retrieval of an odour-reward association tested by a reversal test 1 month after the odour-reward training. The second paradigm (successive odour-pair training) tested reference memory. The first session was an acquisition session where the rats learned a new odour-discrimination problem with the same procedure. The second was a retention session held 24 h later to test retrieval of the learned information. KTX injected before the acquisition or retention session improved performance, but no effect was found when KTX was injected immediately after acquisition. We showed that these effects were not due to the action of KTX on attention processes. Thus, these results suggest that the blockage of Kv1.1 or Kv1.3 channels by KTX facilitates cognitive processes as learning, in particular in a reference representation.Kourrich S., et al.  (2001) Kaliotoxin, a Kv1.1 and Kv1.3 channel blocker, improves associative learning in rats. Behav Brain Res. PMID: 11173083

Kaliotoxin (KTX), a blocker of voltage-gated potassium channels (Kv), is highly selective for Kv1.1 and Kv1.3. First, Kv1.3 is expressed by T lymphocytes. Blockers of Kv1.3 inhibit T lymphocyte activation. Second, Kv1.1 is found in paranodal regions of axons in the central nervous system. Kv blockers improve the impaired neuronal conduction of demyelinated axons in vitro and potentiate the synaptic transmission. Therefore, we investigated the therapeutic properties of KTX via its immunosuppressive and symptomatic neurological effects, using experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. The T line cells used to induce adoptive EAE were myelin basic protein (MBP)-specific, constitutively contained mRNA for Kv1.3. and expressed Kv1.3. These channels were shown to be blocked by KTX. Activation is a crucial step for MBP T cells to become encephalitogenic. The addition of KTX during Ag-T cell activation led to a great reduction in the MBP T cell proliferative response, in the production of IL-2 and TNF, and in Ca(2+) influx. Furthermore, the addition of KTX during T cell activation in vitro led a decreased encephalitogenicity of MBP T cells. Moreover, KTX injected into Lewis rats impaired T cell function such as the delayed-type hypersensitivity. Lastly, the administration of this blocker of neuronal and lymphocyte channels to Lewis rats improved the symptoms of EAE. We conclude that KTX is a potent immunosuppressive agent with beneficial effects on the neurological symptoms of EAE.Beeton C., et al. (2001) Selective blocking of voltage-gated K+ channels improves experimental autoimmune encephalomyelitis and inhibits T cell activation. J Immunol. PMID: 11145670

The distribution of the binding sites for kaliotoxin (KTX), a blocker of voltage-dependent K(+) channels, was studied with quantitative autoradiography in adult rat brain and during postnatal brain maturation. Iodinated KTX bound specifically to tissue sections with a high affinity (K(d) = 82 pM) and a maximal binding capacity of 13.4 fmol/mg protein. The distribution of KTX binding sites within the central nervous system was heterogeneous. The highest densities were found in the neocortex, hypothalamus, dentate gyrus, bed nucleus of the stria terminalis, and parabrachial nuclei. The lowest level was observed in the white matter. From postnatal day 5 onward, KTX binding sites were detectable only in the hindbrain. The density of KTX binding sites in whole brain drastically increased after postnatal day 15 to achieve adult levels at postnatal day 60 in the whole brain. Bath application of KTX to Xenopus laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels potently and Kv1.2 channels less potently, with respective K(d) values of 0.1, 1.5, and 25 nM. KTX affinities for each of these channels expressed in mammalian cells were about 10-fold lower. A comparison of the distribution of KTX binding sites with that of Kv1 channel polypeptides, together with the pharmacology of KTX block, suggests that the principal targets for KTX in rat brain are K(+) channels containing Kv1.1 and Kv1.3 alpha-subunits.Mourre C, et al. (1999) Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits. J Pharmacol Exp Ther. PMID: 10565809

Kaliotoxin (KTX) is a natural peptide blocker of voltage-dependent K+ channels. The 3D structure of a truncated analogue of KTX (Fernandez et al. (1994) Biochemistry 33, 14256-14263) was determined by NMR spectroscopy and showed significant differences from structures established for other related scorpion toxins. A recent publication with the structure of the complete toxin (Aiyar et al. (1995) Neuron 15, 1169-1181) did not confirm these differences. In this communication we report NMR data for KTX at pH 3.0, 5.5 and 7.2 and the 3D structure obtained from data at pH = 5.5. Complete KTX displays a folding similar to that of other toxins with an alpha-helix and a beta-sheet linked by two disulphide bonds. The pKa of His 34 is anomalously low (4.7-5.2 depending on the buffer) owing to its interaction with two Lys residues (including the essential Lys 27), the charged N-terminus and the side chain of Met 29. Charged residues are placed symmetrically with respect to an axis that approximately coincides with one of the principal components of the moment of inertia of the toxin. His 34, which occupies a well-defined position between two conserved Cys, is located on the centre of a layer of charged groups. Positively and negatively charged residues are found at the same position in related toxins. It is suggested that electrostatic effects modulate the distances between positive charges in flexible side chains, contributing to the fine tuning of the selectivity toward different channel subclasses and that the approximate coincidence between the moment of inertia and the charge axis facilitate the approach of the toxin to the channel. The very low pKa of His 34 implies that it will be completely unprotonated at physiological pH.Gairí M, et al. 3D structure of kaliotoxin: is residue 34 a key for channel selectivity? J Pept Sci.  PMID: 9262650

Membrane potential (Vm) is tightly controlled in T cells through the regulated flux of ions across the plasma membrane. To investigate the functional role of voltage-dependent (Kv) and calcium-activated (KCa) potassium channels in T cell activation, we compared the effects of two K+ channel blockers, namely kaliotoxin (KTX) and charybdotoxin (CHTX), on Vm, calcium influx, and cell proliferation. KTX potently inhibited Kv (ID50 = 3 nM) but not KCa (ID50 = 5 microM) currents in T cells. Resting T cells exposed to KTX (300 nM) depolarized from -56 mV to -50 mV. KTX had no effect on the transient membrane hyperpolarization that characteristically follows receptor-mediated T cell stimulation. However, T cells stimulated in the presence of KTX subsequently depolarized to -40 mV. KTX also reduced the steady state intracellular free calcium concentration ([Ca2+]i) in stimulated cells by 19% and inhibited T cell proliferation by 35%. CHTX potently inhibited both Kv and KCa currents (ID50 = approximately 1 nM). CHTX (300 nM) depolarized resting T cells to -48 mV, equivalent to the effect observed for KTX. In stimulated T cells, 300 nM CHTX completely blocked the induced hyperpolarization and subsequently depolarized the cells to -21 mV. These effects were associated with a 45% reduction in peak [Ca2+]i, a 60% decrease in steady state [Ca2+]i, and 63% inhibition of T cell proliferation. These results suggest that both Kv and KCa conductances contribute to the underlying mechanisms of T cell activation.

Rader RK, et al. (1997) T cell activation is regulated by voltage-dependent and calcium-activated potassium channels. J Immunol. PMID: 8568243

Through expression of the cloned mouse (mSlo) or human (hSlo) large-conductance (BK) Ca(2+)-activated K+ channel in Xenopus laevis oocytes and HEK 293 cells, we characterized the effects of reported blockers and openers of BK channels to initiate the study of the molecular determinants of BK channel modulation. In oocytes, iberiotoxin and charybdotoxin, peptidyl scorpion toxins, were both equally effective blockers of BK current, although iberiotoxin was significantly more potent than charybdotoxin. The structurally related peptide kaliotoxin was not a potent blocker of BK current. Paxilline, a fungal tremorgenic alkaloid, was an effective but complex blocker of BK current. Tetrandrine, a putative blocker of type II BK channels, and ketamine were relatively ineffective. The putative BK openers NS004 and NS1619, phloretin, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) increased BK current in oocytes at microM concentrations; many of these produced biphasic concentration-response relationships. Coapplication of representative blockers and openers revealed several patterns of interaction, including competitive and noncompetitive antagonism. NS1619, niflumic acid, and phloretin were tested by using excised inside-out membrane patches from HEK 293 cells and were found to increase the activity of hSlo BK channels and produce a leftward shift in the G/Gmax-versus-voltage relationship of these channels. These results represent the first comprehensive examination of the molecular pharmacology of BK channels.

Gribkoff VK. 1996) Effects of channel modulators on cloned large-conductance calcium-activated potassium channels. Mol Pharmacol. PMID: 8700114

Using the whole-cell configuration of the patch clamp technique, calcium-activated potassium currents (I(K,Ca)) were investigated in ramified murine brain macrophages. In order to induce I(K,Ca) the intracellular concentration of nominal free Ca2+ was adjusted to 1 microM. The Ca2+-activated K+ current of brain macrophages did not show any voltage dependence at test potentials between -120 and +30 mV. A tenfold change in extracellular K+ concentration shifted the reversal potential of I(K,Ca) by 51 mV. The bee venom toxin apamin applied at concentrations of up to 1 microM did not affect I(K,Ca). Ca2+-activated K+ currents of ramified brain macrophages were highly sensitive to extracellularly applied charybdotoxin (CTX). The half-maximal effective concentration of CTX was calculated to be 4.3 nM. In contrast to CTX, the scorpion toxin kaliotoxin did not inhibit I(K,Ca) at concentrations between 1 and 50 nM. Tetraethylammonium (TEA) blocked 8.0% of I(K,Ca) at a concentration of 1 mM, whereas 31.4% of current was blocked by 10 mM TEA. Several inorganic polyvalent cations were tested at a concentration of 2 mM for their ability to block I(K,Ca). La3+ reduced I(K,Ca) by 72.8%, whereas Cd2+ decreased I(K,Ca) by 17.4%; in contrast, Ni2+ did not have any effect on I(K,Ca). Ba2+ applied at a concentration of 1 mM reduced I(K,Ca) voltage-dependently at hyperpolarizing potentials.

Eder C. (1997) Pharmacological properties of Ca2+activated K+ currents of ramified murine brain macrophages. Naunyn Schmiedebergs Arch Pharmacol. PMID: 9272730

A peptidyl inhibitor of the high conductance Ca(2+)-activated K+ channels (KCa) has been purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus. The peptide has been named kaliotoxin (KTX). It is a single 4-kDa polypeptide chain. Its complete amino acid sequence has been determined. KTX displays sequence homology with other scorpion-derived inhibitors of Ca(2+)-activated or voltage-gated K+ channels: 44% homology with charybdotoxin (CTX), 52% with noxiustoxin (NTX), and 44% with iberiotoxin (IbTX). Electrophysiological experiments performed in identified nerve cells from the mollusc Helix pomatia showed that KTX specifically suppressed the whole cell Ca(2+)-activated K+ current. KTX had no detectable effects on voltage-gated K+ current (delayed rectifier and fast transient A current) or on L-type Ca2+ currents. KTX interacts in a one-to-one way with KCa channels with a Kd of 20 nM. Single channel experiments were performed on high conductance KCa channels excised from the above Helix neurons and from rabbit coeliac ganglia sympathetic neurons. KTX acted exclusively at the outer face of the channel. KTX applied on excised outside-out KCa channels induced a transient period of fast-flicker block followed by a persistent channel blockade. The KTX-induced block was not voltage-dependent which suggests differences in the blockade of KCa channels by KTX and by CTX. Comparison of KTX and CTX sequences leads to the identification of a short amino acid sequence (26-33) which may be implicated in the toxin-channel interaction. KTX therefore appears to be a useful tool for elucidating the molecular pharmacology of the high conductance Ca(2+)-activated K+ channel.

Crest M., et al. (1992) Kaliotoxin, a novel peptidyl inhibitor of neuronal BK-type Ca(2+)-activated K+ channels characterized from Androctonus mauretanicus mauretanicus venom. JBC. PMID: 1730708