Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Vázquez, Jesús; Feigenbaum, P; King, V F; Kaczorowski, Gregory J.; García, María L.

doi:10.1016/s0021-9258(18)55434-x

Cited by 86 publications

(21 citation statements)

References 32 publications

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“…, while IbTX is without effect up to 4 orders of magnitude higher in concentration (Vazquez et al, 1990). Similar findings are presented here with synthetic ChTX and IbTX.…”

Section: Resultssupporting

confidence: 89%

“…Binding Studies. The interaction of [125I]ChTX with either bovine aortic sarcolemmal membrane vesicles or rat brain synaptic membrane vesicles was monitored as previously described (Vazquez et al, 1989(Vazquez et al, ,1990. Binding assays were performed in triplicate, and the standard error of the mean was less than 3%.…”

Section: Methodsmentioning

confidence: 99%

“…Despite sequence homology with ChTX, IbTX does not block Kvu in human T lymphocytes (Deutsch et al, 1991;Leonard et al, 1992). Binding studies with [125I]ChTX have revealed a single class of sites in rat brain (Vazquez et al, 1990) and human T lymphocytes (Deutsch et al, 1991) that is completely inhibitable by other peptidyl inhibitors of voltage-dependent K+ channels such as noxiustoxin and -dendrotoxin but is insensitive to IbTX. This pharmacological profile is consistent with that of the Kv13 currents found in these tissues.…”

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confidence: 99%

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Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

Giangiacomo¹,

Sugg²,

García‐Calvo³

et al. 1993

Biochemistry

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Charybdotoxin (ChTX) and iberiotoxin (IbTX) are highly charged peptidyl toxins which exhibit 68% sequence identity and share a similar three-dimensional structure. Despite these structural similarities, IbTX and ChTX differ in their selectivity for two types of potassium channels; large conductance calcium-activated potassium (maxi-K) channels and slowly inactivating voltage-gated (Kv1.3) potassium channels. ChTX blocks with high affinity both maxi-K and Kv1.3 channels, while IbTX blocks the maxi-K but not the voltage-gated channel. To identify regions of the toxins which impart this this selectivity, we have constructed by solid-phase synthesis two chimeric toxins, ChTX1-19IbTX20-37 (Ch-IbTX) and IbTX1-19ChTX20-37 (Ib-ChTX), as well as a truncated peptide, ChTX7-37. These peptides were assayed for their ability to inhibit [125I]ChTX binding in sarcolemmal vesicles from smooth muscle (maxi-K binding) and [125I]ChTX binding to plasma membranes from brain (Kv1.3 binding). The ability of the peptides to block the maxi-K channel was determined from recordings of single maxi-K channels incorporated into planar lipid bilayers. Block of Kv1.3 was determined from recordings of whole cell currents in Xenopus oocytes injected with mRNA encoding the cloned Kv1.3 channel. Both chimeric toxins inhibited [125I]ChTX binding to sarcolemmal membranes from smooth muscle, and they both blocked the maxi-K channel in planar lipid bilayers. In contrast, [125I]ChTX binding in brain and Kv1.3 currents expressed in oocytes were inhibited only by the chimera Ib-ChTX.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…, while IbTX is without effect up to 4 orders of magnitude higher in concentration (Vazquez et al, 1990). Similar findings are presented here with synthetic ChTX and IbTX.…”

Section: Resultssupporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

Giangiacomo¹,

Sugg²,

García‐Calvo³

et al. 1993

Biochemistry

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“…As K + was further increased in the low mM range, partial reversal of the inhibition of [ 125 I]IbTX-D19Y/Y36F binding was observed. This is a phenomena which had previously been described for [ 125 I]ChTX and [ 125 I]MgTX binding to voltage-gated K + channels in rat brain synaptic membranes (Va ´zquez et al, 1990;Knaus et al, 1995). Further increase in K + concentration was followed by a complete inhibition of [ 125 I]IbTX-D19Y/Y36F binding to smooth muscle membranes.…”

Section: Binding Properties Of [ 125 I]ibtx-d19y/y36f In Purified Bov...supporting

confidence: 70%

“…Amino acid sequence obtained from proteolytic fragments of the R-and β-subunits, as well as by low-stringency hybridization protocols enabled the cloning of the maxi-K channel R-and β-subunits (Butler et al, 1993;Knaus et al, 1994a,b;Tseng-Crank et al, 1994). In addition to its interaction with maxi-K channels, ChTX also inhibits representatives of the Shaker family of voltagegated K + channels (e.g., K v 1.2 and K v 1.3) which are expressed in neurons, T-lymphocytes, and osteoclasts (Va ´zquez et al, 1990;Deutsch et al, 1991;Arkett et al, 1994), and [ 125 I]ChTX binds with high affinity to these channels in brain and T-lymphocytes (Va ´zquez et al, 1990;Deutsch et al, 1991). Moreover, high-affinity interaction of ChTX has also been reported for low-and intermediate-conductance Ca 2+ -activated K + channels in smooth muscle cell lines, Aplysia neurons, human T-lymphocytes, and erythrocytes (Hermann & Erxleben, 1987;Van Renterghem & Lazdunski, 1992;Leonard et al, 1992;Brugnara et al, 1993a,b).…”

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confidence: 99%

[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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The scorpion toxin BeKm‐1 blocks hERG cardiac potassium channels using an indispensable arginine residue

Zavarzina,

Kuzmenkov,

Dobrokhotov

et al. 2024

FEBS Letters

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BeKm‐1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm‐1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double‐mutant cycle analysis data, to obtain an in silico model of the hERG–BeKm‐1 complex. Adding to the previous mutagenesis study of BeKm‐1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm‐1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

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Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Cited by 86 publications

References 32 publications

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

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

The scorpion toxin BeKm‐1 blocks hERG cardiac potassium channels using an indispensable arginine residue

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Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Cited by 86 publications

References 32 publications

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

Synthetic charybdotoxin-iberiotoxin chimeric peptides define toxin binding sites on calcium-activated and voltage-dependent potassium channels

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

The scorpion toxin BeKm‐1 blocks hERG cardiac potassium channels using an indispensable arginine residue

Contact Info

Product

Resources

About

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