Characterization of High Affinity Binding Sites for Charybdotoxin in Sarcolemmal Membranes from Bovine Aortic Smooth Muscle

Vázquez, Jesús; Feigenbaum, P; Katz, George M.; King, V F; Reuben, John P.; Roy-Contancin, L; Slaughter, Robert S.; Kaczorowski, Gregory J.; García, María L.

doi:10.1016/s0021-9258(19)30021-3

Cited by 87 publications

(42 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Elution of bound material was achieved in the presence of a linear gradient of NaCl (0-750 mM NaCl over 60 min) at a flow rate of 0.5 mL/min. The fraction which contains IbTX-D19Y/Y36F was identified in radioligand binding studies (Va ´zquez et al, 1989) and subsequently incubated with 5% acetic acid at 45 °C for 72 h to achieve complete cyclization of the amino-terminal glutamic acid residue. The sample was then applied to a C 18 reversedphase HPLC column (Vydac, 1.0 × 25 cm) and elution was carried out using conditions identical to those previously reported for wild-type IbTX (Galvez et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

“…Identical binding parameters have been measured for purified bovine aortic smooth muscle sarcolemmal membrane vesicles (data not shown). Under similar conditions, [ 125 labeled bovine tracheal smooth muscle maxi-K channels with lower affinity (K d ≈ 20 pM) whereas the respective B max value was very similar (Va ´zquez et al, 1989).…”

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

confidence: 99%

“…Several ions such as K + , TEA, and Ca 2+ which are known to interact with smooth muscle maxi-K channels by binding to sites along the ion conduction pathway, and which have been shown to modulate [ 125 I]ChTX binding to maxi-K channels (Va ´zquez et al, 1989;McManus et al, 1995) were also investigated. When membrane vesicles were incubated with [ 125 I]IbTX-D19Y/Y36F and increasing concentrations of either Ca 2+ or TEA, a concentration-dependent inhibition of toxin binding is observed (Figure 6B).…”

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

confidence: 99%

“…High-conductance Ca 2+ -activated K + (maxi-K) 1 channels comprise a group of ion channels that are expressed in skeletal and smooth muscle, neurons, kidney, adrenal chromafin cells, insulin-producing cells, and pituitary cells, as well as fibroblasts (Miller et al, 1985;Reinhart et al, 1989;Va ´zquez et al, 1989;Knaus et al, 1996;Zweifach et al, 1991;Solaro & Lingle, 1992;Oosawa et al, 1992;Rane, 1991;Kaczorowski et al, 1996). Gating of these channels is modulated by elevation of intracellular Ca 2+ and membrane depolarization, and they display a large single-channel conductance (200-300 pS), as well as high selectivity for K + (Latorre et al, 1989).…”

mentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 1997

View full text Add to dashboard Cite

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.

show abstract

Section: Methodsmentioning

confidence: 99%

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

confidence: 99%

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

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 1997

View full text Add to dashboard Cite

show abstract

“…This peptidyl toxin from the scorpion, Leiurus quinquestriatus, blocks several Ca 2ϩ -activated K ϩ channels and also voltage-gated potassium channels (Miller et al, 1985;Garcia et al, 1995). It was tested on three nonpregnant and seven late-pregnant myocytes at 100 nM (IC 50 , 100 pM, Vasquez et al, 1989). On nonpregnant Traces are difference currents, I control Ϫ I TEA , at all voltages.…”

Section: Charybdotoxinmentioning

confidence: 99%

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

Wang

Yamada

Sui

et al. 1998

The Journal of General Physiology

View full text Add to dashboard Cite

In freshly dissociated uterine myocytes, the outward current is carried by K+ through channels highly selective for K+. Typically, nonpregnant myocytes have rather noisy K+ currents; half of them also have a fast-inactivating transient outward current (ITO). In contrast, the current records are not noisy in late pregnant myocytes, and ITO densities are low. The whole-cell IK of nonpregnant myocytes respond strongly to changes in [Ca2+]o or changes in [Ca2+]i caused by photolysis of caged Ca2+ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca2+ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell IK of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca2+-activated K+ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca2+-sensitive K+ channels also have a half-open probability at 68 mV, and are less sensitive to Ca2+ than similar channels in taenia coli myocytes. Ca2+-activated K+ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total IK in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total IK in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K+ currents that include a suppression of the ITO, a redistribution of IK expression from large-conductance Ca2+-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca2+ sensitivity, and a positive shift of the activation of some large-conductance Ca2+-activated channels.

show abstract

Pharmacology of High-Conductance, Ca2+-Activated Potassium Channels

García

Kaczorowski

2001

Potassium Channels in Cardiovascular Biology

View full text Add to dashboard Cite

Characterization of High Affinity Binding Sites for Charybdotoxin in Sarcolemmal Membranes from Bovine Aortic Smooth Muscle

Cited by 87 publications

References 33 publications

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

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

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

Pharmacology of High-Conductance, Ca2+-Activated Potassium Channels

Contact Info

Product

Resources

About

Characterization of High Affinity Binding Sites for Charybdotoxin in Sarcolemmal Membranes from Bovine Aortic Smooth Muscle

Cited by 87 publications

References 33 publications

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

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

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

Pharmacology of High-Conductance, Ca2+-Activated Potassium Channels

Contact Info

Product

Resources

About

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

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