Perez-Cornejo P et al. (1998), Proton probing of the charybdotoxin binding sit...

XB-ART-15173

J Gen Physiol 1998 Mar 01;1113:441-50.

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Proton probing of the charybdotoxin binding site of Shaker K+ channels.

Perez-Cornejo P , Stampe P , Begenisich T .

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We have investigated the interaction of charybdotoxin (CTX) with Shaker K channels. We substituted a histidine residue for the wild-type phenylalanine (at position 425) in an inactivation-removed channel. The nature of the imidazole ring of the histidine provides the ability to change the charge on this amino acid side chain with solution hydrogen ion concentration. Wild-type, recombinant CTX blocked wild-type Shaker channels in a bimolecular fashion with a half-blocking concentration (Kd) of 650 nM (at a membrane potential of 0 mV). The F425H mutant channels were much more sensitive to CTX block with an apparent Kd (at pH 7.0) of 75 nM. Block of F425H but not wild-type channels was strongly pH sensitive. A pH change from 7 to 5.5 rendered the F425H channels >200-fold less sensitive to CTX. The pH dependence of CTX block was steeper than expected for inhibition produced by H+ ions binding to identical, independent sites. The data were consistent with H+ ions interacting with subunits of the channel homotetrameric structure. The in situ pK for the imidazole group on the histidine at channel position 425 was determined to be near 6.4 and the dissociation constant for binding of toxin to the unprotonated channel was near 50 nM. We estimate that the binding of a H+ ion to each subunit adds 0.8 kcal/mol or more of interaction energy with CTX. We used mutant toxins to test electrostatic and steric interactions between specific CTX residues and channel position 425. Our results are consistent with a model in which protons on F425H channel subunits interact with three positive charges on CTX at an effective distance 6-7 A from this channel position.

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Species referenced: Xenopus laevis
Genes referenced: pkm vsig1

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	Figure 6. pH dependence of mutant CTX block of wild-type and F425H channels. K31Q block of wild-type (○) and F425H (•) channels and K11Q block of F425H channels (▪). Toxin concentrations of 0.5 μM; currents measured at +40 mV. Dotted line: fit of Eq. 1 to data with K31Q CTX and wild-type channels with Kd and pKH values of 0.78 μM and 5.4, respectively. Solid line: see discussion. Data values are averages of 3–4 (○), 4–5 (▪), or 5–6 (•); measurements shown with SEM limits.
	Figure 7. pH dependence of T8ST9G CTX block of F425H channels. Currents before, during, and after application of 0.25 μM T8ST9G CTX recorded at 0, 20, and 40 mV are shown at pH 5 (top) and 7 (bottom).
	Figure 2. Block of wild-type and mutant Shaker K channels by CTX at pH 7.0. Ordinate: amount of current inhibited by CTX expressed as a fraction of the average of current before and after toxin application. Currents measured at the end of 20-ms pulses to a potential of 0 mV. Data from wild-type (•) and F425H (○) channels are illustrated with standard error limits. The solid curve is the fit to the experimental data described by the relation:\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation}Ordinate=\frac{[CTX]}{[CTX]+K_{d}}.\end{equation}\end{document} Kd values obtained from these fits are indicated. The number of determinations (if greater than three) are given next to the data points.
	Figure 3. CTX block of F425H channels at pH 7 and 5.5. Ordinate values as described in Fig. 2. Data at pH 7 (○) and 5.5 (•) are illustrated. Currents were measured at the end of 20-ms pulses to +40 mV. Solid lines are as in Fig. 2, with indicated Kd values. The number of determinations (if greater than three) are given next to the data points.
	Figure 4. pH dependence of CTX block of wild-type channels. (A) Block of current at the indicated CTX concentration at pH 7 (•) and 5.5 (○). Data at pH 7 are the averages of 3–10 observations, except for a single determination at 5 μM. Solid line as in Fig. 2 with indicated Kd value. (B) pH dependence of block by 0.5 μM CTX. Data are the averages of 5–10 measurements; standard error limits omitted if smaller than symbol. Line was fit to data of Eq. 1 with indicated parameters.
	Figure 5. pH dependence of CTX block of F425H channels. Currents measured at +40 mV. The CTX concentration used was 100 nM. Data are the averages of 4–7 observations and indicated next to the mean values. SEM limits as shown. (A) Solid line: fit of Eq. 1 to data with Kd and pKH values of 42 nM and 7.2, respectively. Dotted line: see text. Dashed line: fit of Eq. 2 to data with Kd and pKH values of 48 nM and 5.9, respectively. (B) Data as in A. Lines: fits of Eq. 3 to data. See text for details.

References [+] :

Aiyar, Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins. 1995, Pubmed