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Results for “Kv1.2 Potassium Channel”

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PubMed1999

Differential asparagine-linked glycosylation of voltage-gated K+ channels in mammalian brain and in transfected cells.

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K+ channel alpha subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K+ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K+ channel alpha subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K+ channel function in mammalian brain.

Animals↗

PubMed1999

Distribution in rat brain of binding sites of kaliotoxin, a blocker of Kv1.1 and Kv1.3 alpha-subunits.

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.

Aging↗

PubMed2002

Hypoxic vasorelaxation inhibition by organ culture correlates with loss of Kv channels but not Ca(2+) channels.

We (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001) have recently shown that organ culture for 24 h specifically inhibits relaxation to acute hypoxia (95% N(2)-5% CO(2)) in the porcine coronary artery. Here we show similar results in the porcine carotid artery and the rat and mouse aorta. In the coronary artery, part of the inability to relax to hypoxia after organ culture is associated with a concomitant loss in ability to reduce intracellular Ca(2+) concentration ([Ca(2+)](i)) during hypoxia (Thorne GD, Shimizu S, and Paul RJ. Am J Physiol Cell Physiol 281: C24-C32, 2001). To elucidate the mechanisms responsible for the loss of relaxation to hypoxia, we investigated changes in K(+) and Ca(2+) channel activity and gene expression that play key roles in [Ca(2+)](i) regulation in vascular smooth muscle (VSM). Reduced mRNA expression of O(2)-sensitive K(+) channels (Kv1.5 and Kv2.1) was shown by reverse transcriptase-polymerase chain reaction in the rat aorta. In contrast, no change in other expressed voltage-gated K(+) channels (Kv1.2 and Kv1.3) or Ca(2+) channel subtypes was found. Modified K(+) channel expression is supported by functional evidence indicating a reduced response to general K(+) channel activation, by pinacidil, and to specific voltage-dependent K(+) (Kv) channel blockade by 4-aminopyridine. In conclusion, organ culture decreases expression of specific Kv channels. These changes are consistent with altered mechanisms of VSM contractility that may be involved in Ca(2+)-dependent pathways of hypoxia-induced vasodilation.

Animals↗

PubMed2001

Subunit composition and novel localization of K+ channels in spinal cord.

Axonal K+ channels involved in normal spinal cord function are candidate targets for therapeutics, which improve sensorimotor function in spinal cord injury. To this end, we have investigated the expression, localization, and coassociation of Kv1 alpha and beta subunits in human, rat, and bovine spinal cord. We find that Kv1.1, Kv1.2, and Kvbeta2 form heteromultimeric complexes at juxtaparanodal zones in myelinated fibers. However, these same complexes are also present in paranodal regions of some spinal cord axons, and staining with antibodies against Caspr, a component of the paranodal axoglial junction, overlaps with these paranodal K+ channels. This latter observation suggests a unique role for these channels in normal spinal cord function and may provide an explanation for the sensitivity of spinal cord to K+ channel blockers. Moreover, the conservation of these characteristics between human, rat, and bovine nodes of Ranvier suggests an essential role for this defined channel complex in spinal cord function.

Animals↗

PubMed2005

Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.

The interaural time difference (ITD) is a cue for localizing a sound source along the horizontal plane and is first determined in the nucleus laminaris (NL) in birds. Neurons in NL are tonotopically organized, such that ITDs are processed separately at each characteristic frequency (CF). Here, we investigated the excitability and coincidence detection of neurons along the tonotopic axis in NL, using a chick brainstem slice preparation. Systematic changes with CF were observed in morphological and electrophysiological properties of NL neurons. These properties included the length of dendrites, the input capacitance, the conductance of hyperpolarization-activated current, and the EPSC time course. In contrast to these gradients, the conductance of low-threshold K+ current and the expression of Kv1.2 channel protein were maximal in the central (middle-CF) region of NL. As a result, the middle-CF neuron had the smallest input resistance and membrane time constant, and consequently the fastest EPSP, and exhibited the most accurate coincidence detection. The specialization of middle-CF neurons as coincidence detectors may account for the high resolution of sound-source localization in the middle-frequency range observed in avians.

Action Potentials↗

PubMed2006

Neuromyotonia and limbic encephalitis sera target mature Shaker-type K+ channels: subunit specificity correlates with clinical manifestations.

Autoantibodies to Shaker-type (Kv1) K+ channels are now known to be associated with three syndromes. Peripheral nerve hyperexcitability is the chief manifestation of acquired neuromyotonia; the combination of neuromyotonia with autonomic and CNS involvement is called Morvan's syndrome (MoS); and CNS manifestations without peripheral involvement is called limbic encephalitis (LE). To determine the cellular basis of these clinical manifestations, we immunostained mouse neural tissues with sera from patients with neuromyotonia (n = 10), MoS (n = 2) or LE (n = 5), comparing with specific antibodies to relevant K+ channel subunits. Fourteen of 17 patients' sera were positive for Kv1.1, Kv1.2 or Kv1.6 antibodies by immunoprecipitation of 125I-alpha-dendrotoxin-labelled rabbit brain K+ channels. Most sera (11 out of 17) labelled juxtaparanodes of peripheral myelinated axons, co-localizing with Kv1.1 and Kv1.2. In the CNS, all sera tested (n = 12) co-localized with one or more areas of high Kv1.1, Kv1.2 or Kv1.6 channel expression: 10 out of 12 sera co-localized with Kv1.1 and Kv1.2 at spinal cord juxtaparanodes or cerebellar layers, while 3 out of 12 sera co-localized additionally (n = 2) or exclusively (n = 1) with Kv1.6 subunits in Purkinje cells, motor and hippocampal neurons. However, only sera from LE patients labelled the hippocampal areas that are enriched in excitatory, Kv1.1-positive axon terminals. All sera (17 out of 17) labelled one or more of these Kv1 subunits when expressed at the cell membrane of transfected HeLa cells, but not when they were retained in the endoplasmic reticulum. Again, LE sera labelled Kv1.1 subunits more prominently than did MoS or neuromyotonia sera, suggesting an association between higher Kv1.1 specificity and limbic manifestations. In contrast, neuromyotonia sera bound more strongly to Kv1.2 subunits than to Kv1.1 or Kv1.6. These studies support the hypothesis that antibodies to mature surface membrane-expressed Shaker-type K+ channels cause acquired neuromyotonia, MoS and LE, and suggest that future assays based on immunofluorescence of cells expressing individual Kv1 subunits will prove more sensitive than the immunoprecipitation assay. Although more than one type of antibody is often detectable in individual sera, higher affinity for certain subunits or subunit combinations may determine the range of clinical manifestations.

Adolescent↗

PubMed1998

The small GTP-binding protein RhoA regulates a delayed rectifier potassium channel.

Tyrosine kinases activated by G protein-coupled receptors can phosphorylate and thereby suppress the activity of the delayed rectifier potassium channel Kv1.2. Using a yeast two-hybrid screen, we identified the small GTP-binding protein RhoA as a necessary component in this process. Coimmunoprecipitation experiments confirmed that RhoA associates with Kv1.2. Electrophysiological analyses revealed that overexpression of RhoA markedly reduced the basal current generated by Kv1.2 expressed in Xenopus oocytes. Furthermore, in 293 cells expressing Kv1.2 and ml muscarinic acetylcholine receptors, inactivating RhoA using C3 exoenzyme blocked the ability of ml receptors to suppress Kv1.2 current. Therefore, these results demonstrate that RhoA regulates Kv1.2 activity and is a central component in the mechanism of receptor-mediated tyrosine kinase-dependent suppression of Kv1.2.

ADP Ribose Transferases↗

PubMed2003

Kv3.1b is a novel component of CNS nodes.

We herein demonstrate that Kv3.1b subunits are present at nodes of Ranvier in the CNS of both rats and mice. Kv3.1b colocalizes with voltage-gated Na+ channels in a subset of nodes in the spinal cord, particularly those of large myelinated axons. Kv3.1b is abundantly expressed in the gray matter of the spinal cord, but does not colocalize with Na+ channels in initial segments. In the PNS, few nodes are Kv3.1b-positive. During the development of the CNS, Kv3.1b clustering at nodes occurs later than that of Na+ channels, but precedes the juxtaparanodal clustering of Kv1.2. Moreover, in myelin-deficient rats, which have severe CNS dysmyelination, node-like clusters of Kv3.1b and Na+ channels are observed even in regions devoid of oligodendrocytes. Ankyrin G coimmunoprecipitates Kv3.1b in vivo, indicating that these two proteins may interact in the CNS at nodes. 4-Aminopyridine, a K+ channel blocker, broadened the compound action potential recorded from adult rat optic nerve and spinal cord, but not from the sciatic nerve. These effects were also observed in Kv3.1-deficient mice. In conclusion, Kv3.1b is the first K+ channel subunit to be identified in CNS nodes; but Kv3.1b does not account for the effects of 4-aminopyridine on central myelinated tracts.

4-Aminopyridine↗

PubMed2000

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.

Amino Acid Sequence↗

PubMed2005

Sequential antibodies to potassium channels and glutamic acid decarboxylase in neuromyotonia.

A patient with thymoma-associated neuromyotonia and voltage-gated potassium channel (Kv1.2 and Kv1.6) antibodies by immunoprecipitation and rat brain immunolabeling was treated successfully with immunoadsorption and cyclophosphamide. Curiously, glutamic acid decarboxylase antibodies, absent at onset, appeared later. Stiff-person syndrome was absent, but fast blink reflex recovery suggested enhanced brainstem excitability. The range of antibodies produced in thymoma-associated neuromyotonia is richer, and the timing of antibody appearance more complex, than previously suspected.

Adult↗

PubMed2000

Potential role for kv3.1b channels as oxygen sensors.

Hypoxia inhibits voltage-gated K channels in pulmonary artery smooth muscle (PASM). This is thought to contribute to hypoxic pulmonary vasoconstriction by promoting membrane depolarization, Ca(2+) influx, and contraction. Several of the K-channel subtypes identified in pulmonary artery have been implicated in the response to hypoxia, but contradictory evidence clouds the identity of the oxygen-sensing channels. Using patch-clamp techniques, this study investigated the effect of hypoxia on recombinant Kv1 channels previously identified in pulmonary artery (Kv1.1, Kv1.2, and Kv1.5) and Kv3.1b, which has similar kinetic and pharmacological properties to native oxygen-sensitive currents. Hypoxia failed to inhibit any Kv1 channel, but it inhibited Kv3.1b channels expressed in L929 cells, as shown by a reduction of whole-cell current and single-channel activity, without affecting unitary conductance. Inhibition was retained in excised membrane patches, suggesting a membrane-delimited mechanism. Using reverse transcription-polymerase chain reaction and immunocytochemistry, Kv3.1b expression was demonstrated in PASM cells. Moreover, hypoxia inhibited a K(+) current in rabbit PASM cells in the presence of charybdotoxin and capsaicin, which preserve Kv3.1b while blocking most other Kv channels, but not in the presence of millimolar tetraethylammonium ions, which abolish Kv3.1b current. Kv3.1b channels may therefore contribute to oxygen sensing in pulmonary artery.

Animals↗

PubMedDate not supplied

K+ channel distribution and clustering in developing and hypomyelinated axons of the optic nerve.

The localization of Shaker-type K(+) channels in specialized domains of myelinated central nervous system axons was studied during development of the optic nerve. In adult rats Kv1.1, Kv1.2, Kv1.6, and the cytoplasmic beta-subunit Kvbeta2 were colocalized in juxtaparanodal zones. During development, clustering of K(+) channels lagged behind that for nodal Na(+) channels by about 5 days. In contrast to the PNS, K(+) channels were initially expressed fully segregated from nodes and paranodes, the latter identified by immunofluorescence of Caspr, a component of axoglial junctions. Clusters of K(+) channels were first detected at postnatal day 14 (P14) at a limited number of sites. Expression increased until all juxtaparanodes had immunoreactivity by P40. Developmental studies in hypomyelinating Shiverer mice revealed dramatically disrupted axoglial junctions, aberrant Na(+) channel clusters, and little or no detectable clustering of K(+) channels at all ages. These results suggest that in the optic nerve, compact myelin and normal axoglial junctions are essential for proper K(+) channel clustering and localization.

Action Potentials↗

PubMed2005

Structural and functional alterations of spinal cord axons in adult Long Evans Shaker (LES) dysmyelinated rats.

Abnormal formation or loss of myelin is a distinguishing feature of many neurological disorders and contributes to the pathobiology of neurotrauma. In this study we characterize the functional and molecular changes in CNS white matter in Long Evans Shaker (LES) rats. These rats have a spontaneous mutation of the gene encoding myelin basic protein which results in severe dysmyelination of the central nervous system (CNS), providing a unique model for demyelinating/dysmyelinating disorders. To date, the functional and molecular changes in CNS white matter in this model are not well understood. We have used in vivo somatosensory evoked potential (SSEP), in vitro compound action potential (CAP) recording in isolated dorsal columns, confocal immunohistochemistry, Western blotting and real-time PCR to examine the electrophysiological, molecular and cellular changes in spinal cord white matter in LES rats. We observed that dysmyelination is associated with dispersed labeling of Kv1.1 and Kv1.2 K+ channel subunits, as well as Caspr, a protein normally confined to paranodes, along the LES rat spinal cord axons. Abnormal electrophysiological properties including attenuation of CAP amplitude and conduction velocity, high frequency conduction failure and enhanced sensitivity to K+ channel blockers 4-aminopyridine and dendrotoxin-I were observed in spinal cord axons from LES rats. Our results in LES rats clarify some of the key molecular, cellular and functional consequences of dysmyelination and myelin-axon interactions. Further understanding of these issues in this model could provide critical insights for neurological disorders characterized by demyelination.

Action Potentials↗

PubMed2004

The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system.

The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Animals↗

PubMed2000

PSD-95 and SAP97 exhibit distinct mechanisms for regulating K(+) channel surface expression and clustering.

Mechanisms of ion channel clustering by cytoplasmic membrane-associated guanylate kinases such as postsynaptic density 95 (PSD-95) and synapse-associated protein 97 (SAP97) are poorly understood. Here, we investigated the interaction of PSD-95 and SAP97 with voltage-gated or Kv K(+) channels. Using Kv channels with different surface expression properties, we found that clustering by PSD-95 depended on channel cell surface expression. Moreover, PSD-95-induced clusters of Kv1 K(+) channels were present on the cell surface. This was most dramatically demonstrated for Kv1.2 K(+) channels, where surface expression and clustering by PSD-95 were coincidentally promoted by coexpression with cytoplasmic Kvbeta subunits. Consistent with a mechanism of plasma membrane channel-PSD-95 binding, coexpression with PSD-95 did not affect the intrinsic surface expression characteristics of the different Kv channels. In contrast, the interaction of Kv1 channels with SAP97 was independent of Kv1 surface expression, occurred intracellularly, and prevented further biosynthetic trafficking of Kv1 channels. As such, SAP97 binding caused an intracellular accumulation of each Kv1 channel tested, through the accretion of SAP97 channel clusters in large (3-5 microm) ER-derived intracellular membrane vesicles. Together, these data show that ion channel clustering by PSD-95 and SAP97 occurs by distinct mechanisms, and suggests that these channel-clustering proteins may play diverse roles in regulating the abundance and distribution of channels at synapses and other neuronal membrane specializations.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Altered ion channels in an animal model of Charcot-Marie-Tooth disease type IA.

How demyelination and remyelination affect the function of myelinated axons is a fundamental aspect of demyelinating diseases. We examined this issue in Trembler-J mice, a genetically authentic model of a dominantly inherited demyelinating neuropathy of humans. The K+ channels Kv1.1 and Kv1.2 channels were often improperly located in the paranodal axon membrane, typically associated with improperly formed paranodes, and in unmyelinated segments between internodes. As in wild-type nerves, Trembler-J nodes contained Nav1.6, ankyrin-G, betaIV-spectrin, and KCNQ2, but, unlike wild-type nerves, they also contained Kv3.1b and Nav1.8. In unmyelinated segments bordered by myelin sheaths, these proteins were clustered in heminodes and did not appear to be diffusely localized in the unmyelinated segments themselves. Nodes and heminodes were contacted by Schwann cells processes that did not have the ultrastructural or molecular characteristics of mature microvilli. Despite the presence of Nav1.8, a tetrodotoxin-resistant sodium channel, sciatic nerve conduction was at least as sensitive to tetrodotoxin in Trembler-J nerves as in wild-type nerves. Thus, the profound reorganization of axonal ion channels and the aberrant expression of novel ion channels likely contribute to the altered conduction in Trembler-J nerves.

Amino Acid Substitution↗

PubMed2000

Short-term effects of rapid pacing on mRNA level of voltage-dependent K(+) channels in rat atrium: electrical remodeling in paroxysmal atrial tachycardia.

BACKGROUND: Atrial fibrillation causes electrophysiological changes of the atrium, thereby facilitating its maintenance. Although the expression of ion channels is modulated in chronic atrial fibrillation, it is yet unknown whether paroxysmal atrial fibrillation can also lead to electrical remodeling by affecting gene expression. METHODS AND RESULTS: To examine the short-term effects of rapid pacing on the mRNA level of voltage-dependent K(+) channels, high-rate atrial pacing was performed in Sprague-Dawley rat hearts. Total RNA was prepared from the atrial appendages from 0 to 8 hours after the onset of pacing, and mRNA levels of Kv1.2, Kv1. 4, Kv1.5, Kv2.1, Kv4.2, Kv4.3, erg, KvLQT1, and minK were determined by RNase protection assay. Among these 9 genes, the mRNA level of the Kv1.5 channel immediately and transiently increased, with bimodal peaks at 0.5 and 2 hours after the onset of pacing. Conversely, the pacing gradually and progressively decreased the mRNA levels of the Kv4.2 and Kv4.3 channels. The increase of Kv1.5 and the decrease of Kv4.2 and Kv4.3 mRNA levels were both rate dependent. In correspondence with the changes in the mRNA level, Kv1. 5 channel protein transiently increased in the membrane fraction of the atrium during a 2- to 8-hour pacing period. Electrophysiological findings that the shortening of the action potential produced by 4-hour pacing was almost abolished by a low concentration of 4-aminopyridine implied that the increased Kv1.5 protein was functioning. CONCLUSIONS: Even short-term high-rate atrial excitation could differentially alter the mRNA levels of Kv1.5, Kv4.2, and Kv4.3 in a rate-dependent manner. In particular, increased Kv1.5 gene expression, having a transient nature, implied the possible biochemical electrical remodeling unique to paroxysmal tachycardia.

Action Potentials↗

PubMed1999

Identification of residues in dendrotoxin K responsible for its discrimination between neuronal K+ channels containing Kv1.1 and 1.2 alpha subunits.

Dendrotoxin (DTX) homologues are powerful blockers of K+ channels that contain certain subfamily Kv1 (1.1-1.6) alpha- and beta-subunits, in (alpha)4(beta)4 stoichiometry. DTXk inhibits potently Kv1.1-containing channels only, whereas alphaDTX is less discriminating, but exhibits highest affinity for Kv1.2. Herein, the nature of interactions of DTXk with native K+ channels composed of Kv1.1 and 1.2 (plus other) subunits were examined, using 15 site-directed mutants in which amino acids were altered in the 310-helix, beta-turn, alpha-helix and random-coil regions. The mutants' antagonism of high-affinity [125I]DTXk binding to Kv1. 1-possessing channels in rat brain membranes and blockade of the Kv1. 1 current expressed in oocytes were quantified. Also, the levels of inhibition of [125I]alphaDTX binding to brain membranes by the DTXk mutants were used to measure their high- and low-affinity interactions, respectively, with neuronal Kv1.2-containing channels that possess Kv1.1 as a major or minor constituent. Displacement of toxin binding to either of these subtypes was not altered by single substitution with alanine of three basic residues in the random-coil region, or R52 or R53 in the alpha-helix; accordingly, representative mutants (K17A, R53A) blocked the Kv1.1 current with the same potency as the natural toxin. In contrast, competition of the binding of the radiolabelled alphaDTX or DTXk was dramatically reduced by alanine substitution of K26 or W25 in the beta-turn whereas changing nearby residues caused negligible alterations. Consistently, W25A and K26A exhibited diminished functional blockade of the Kv1.1 homo-oligomer. The 310-helical N-terminal region of DTXk was found to be responsible for recognition of Kv1.1 channels because mutation of K3A led to approximately 1246-fold reduction in the inhibitory potency for [125I]DTXk binding and a large decrease in its ability to block the Kv1.1 current; the effect of this substitution on the affinity of DTXk for Kv1.2-possessing oligomers was much less dramatic (approximately 16-fold).

Amino Acid Sequence↗