Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit

Scott, Victoria E.; Parcej, David; Keen, Jeff N.; Findlay, John B. C.; Dolly, J. Oliver

doi:10.1016/s0021-9258(17)30474-x

Cited by 100 publications

(28 citation statements)

References 28 publications

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“…Labeling of the MgTX receptor in rat brain synaptic plasma membrane vesicles using a bifunctional cross-linking reagent resulted in specific incorporation of the radioligand into a diffuse glycosylated polypeptide with an of 74 000 that after enzymatic deglycosylation yielded a core protein of 63 000. This observation very much parallels findings that have been reported for the bovine and rat a-DTX receptor, where A'-glycosidase F treatment resulted in a shift in apparent molecular mass from 78 to 65 kDa (Rehm & Lazdunski, 1988;Scott et al, 1990). Even after deglycosylation, the diffuse appearance of the MgTX receptor remained unchanged.…”

Section: Molecular Components Of the Mgtx Receptor In Ratsupporting

confidence: 89%

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

Knaus¹,

Koch²,

Eberhart³

et al. 1995

Biochemistry

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Monoiodotyrosine margatoxin ([125I]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM). Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [125I]MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [125I]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [125I]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Molecular Components Of the Mgtx Receptor In Ratsupporting

confidence: 89%

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

Knaus¹,

Koch²,

Eberhart³

et al. 1995

Biochemistry

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“…In order to extend the pharmacology of [ 125 I]-IbTX-D19Y/Y36F and to obtain additional evidence for specificity, we investigated the binding profile of this ligand with rat brain synaptic plasma membrane vesicles (Table 1). In this tissue, the dissociation constant, as well as the maximal density of sites labeled by For these radioligands it has been shown that the voltage-gated K + channel K v 1.2 (and perhaps K v 1.1 as well as K v 1.3) constitute their high-affinity receptor in mammalian brain (Scott et al, 1990;Grissmer et al, 1994;Chandy & Gutman, 1995). In summary, introducing two amino acid substitutions into IbTX at positions 19 and 36 does not result in any detectable alteration of its pharmacological profile of this ligand but provides a tool with which to obtain a selective, high specific activity radioligand for maxi-K channels.…”

Section: B Amentioning

confidence: 96%

[¹²⁵I]Iberiotoxin-D19Y/Y36F, the First Selective, High Specific Activity Radioligand for High-Conductance Calcium-Activated Potassium Channels

et al. 1997

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Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore. Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I. IbTX-D19Y/Y36F and [127I]IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM). Under low ionic strength conditions, [125I]IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein. Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding. Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate [125I]IbTX-D19Y/Y36F binding in a complex manner. This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent. In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of [125I]MgTX or [125I]-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit [125I]IbTX-D19Y/Y36F binding. Taken together, these results demonstrate that [125I]IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity.

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“…have also been shown to contain the Kv1.2 ␣ subunit (Scott et al, 1990(Scott et al, , 1994b. To confirm that Kv␤2 and Auxiliary subunits in tight association with the ␣ subunits of Na ϩ and Ca 2ϩ channels have been commonly…”

Section: Introductionmentioning

confidence: 95%

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

Shi

Nakahira

Hammond

et al. 1996

Neuron

367

321

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Voltage-gated K+ channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic beta subunits. Here, we show that, in transfected mammalian cells, the predominant beta subunit isoform in brain, Kv beta 2, associates with the Kv1.2 alpha subunit early in channel biosynthesis and that Kv beta 2 exerts multiple chaperone-like effects on associated Kv1.2 including promotion of cotranslational N-linked glycosylation of the nascent Kv1.2 polypeptide, increased stability of Kv beta 2/Kv1.2 complexes, and increased efficiency of cell surface expression of Kv1.2. Taken together, these results indicate that while some cytoplasmic K+ channel beta subunits affect the inactivation kinetics of alpha subunits, a more general, and perhaps more fundamental, role is to mediate the biosynthetic maturation and surface expression of voltage-gated K+ channel complexes. These findings provide a molecular basis for recent genetic studies indicating that beta subunits are key determinants of neuronal excitability.

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Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit

Cited by 100 publications

References 28 publications

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

[¹²⁵I]Iberiotoxin-D19Y/Y36F, the First Selective, High Specific Activity Radioligand for High-Conductance Calcium-Activated Potassium Channels

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

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Alpha-dendrotoxin acceptor from bovine brain is a K+ channel protein. Evidence from the N-terminal sequence of its larger subunit

Cited by 100 publications

References 28 publications

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

[125I]Margatoxin, an Extraordinarily High Affinity Ligand for Voltage-Gated Potassium Channels in Mammalian Brain

[125I]Iberiotoxin-D19Y/Y36F, the First Selective, High Specific Activity Radioligand for High-Conductance Calcium-Activated Potassium Channels

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

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Product

Resources

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[¹²⁵I]Iberiotoxin-D19Y/Y36F, the First Selective, High Specific Activity Radioligand for High-Conductance Calcium-Activated Potassium Channels