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Tl+-induced micros gating of current indicates instability of the MaxiK selectivity filter as caused by ion/pore interaction.

Schroeder I, Hansen UP - J. Gen. Physiol. (2008)

Bottom Line: With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app).The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter.Different models were considered.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, University of Kiel, 24098 Kiel, Germany.

ABSTRACT
Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.

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Dependence of the gating factor Rk (closed squares, Eq. 4a) and RI (open circles, Eq. 4b) on membrane potential as calculated from the data in Fig. 6, A and B, and Fig. 5, A and B, respectively. The flickering bursts occurring in records at 50 mM Tl+ were very short due to sojourns in very short closed events. There, Iapp,∞ was obtained from the expectance of current from the distributions per level (Schroeder et al., 2004). They coincided with Iapp,k, indicating the high reliability of the β fit. The smooth lines present the free fits of Rk (Eq. 5, with the parameters in Table I). Dashed lines are joint fits for 0/25 mM Tl+ and 50/150 mM Tl+, respectively. Errors bars represent SEM. Number of data points are given at the curves.
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fig7: Dependence of the gating factor Rk (closed squares, Eq. 4a) and RI (open circles, Eq. 4b) on membrane potential as calculated from the data in Fig. 6, A and B, and Fig. 5, A and B, respectively. The flickering bursts occurring in records at 50 mM Tl+ were very short due to sojourns in very short closed events. There, Iapp,∞ was obtained from the expectance of current from the distributions per level (Schroeder et al., 2004). They coincided with Iapp,k, indicating the high reliability of the β fit. The smooth lines present the free fits of Rk (Eq. 5, with the parameters in Table I). Dashed lines are joint fits for 0/25 mM Tl+ and 50/150 mM Tl+, respectively. Errors bars represent SEM. Number of data points are given at the curves.

Mentions: This can be used for checking whether the relationship Iapp,∞ = Iapp,k holds and for the calculation of the gating factors Rk and RI.(4a,b)\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*}R_{k}=\frac{k_{CO}+k_{OC}}{k_{CO}}=1+\frac{k_{OC}}{k_{CO}}\hspace{.167em}{\mathrm{and}}\hspace{.167em}R_{I}=\frac{I_{true}}{I_{app,{\infty}}}\end{equation*}\end{document}In Fig. 7, Rk as calculated by Eq. 4a is compared with RI as obtained from Eq. 4b. Both gating factors are plotted vs. membrane potential for different Tl+ concentrations. The coincidence of RI and Rk indicates that the relationship Iapp,∞ = Iapp,k holds.


Tl+-induced micros gating of current indicates instability of the MaxiK selectivity filter as caused by ion/pore interaction.

Schroeder I, Hansen UP - J. Gen. Physiol. (2008)

Dependence of the gating factor Rk (closed squares, Eq. 4a) and RI (open circles, Eq. 4b) on membrane potential as calculated from the data in Fig. 6, A and B, and Fig. 5, A and B, respectively. The flickering bursts occurring in records at 50 mM Tl+ were very short due to sojourns in very short closed events. There, Iapp,∞ was obtained from the expectance of current from the distributions per level (Schroeder et al., 2004). They coincided with Iapp,k, indicating the high reliability of the β fit. The smooth lines present the free fits of Rk (Eq. 5, with the parameters in Table I). Dashed lines are joint fits for 0/25 mM Tl+ and 50/150 mM Tl+, respectively. Errors bars represent SEM. Number of data points are given at the curves.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279167&req=5

fig7: Dependence of the gating factor Rk (closed squares, Eq. 4a) and RI (open circles, Eq. 4b) on membrane potential as calculated from the data in Fig. 6, A and B, and Fig. 5, A and B, respectively. The flickering bursts occurring in records at 50 mM Tl+ were very short due to sojourns in very short closed events. There, Iapp,∞ was obtained from the expectance of current from the distributions per level (Schroeder et al., 2004). They coincided with Iapp,k, indicating the high reliability of the β fit. The smooth lines present the free fits of Rk (Eq. 5, with the parameters in Table I). Dashed lines are joint fits for 0/25 mM Tl+ and 50/150 mM Tl+, respectively. Errors bars represent SEM. Number of data points are given at the curves.
Mentions: This can be used for checking whether the relationship Iapp,∞ = Iapp,k holds and for the calculation of the gating factors Rk and RI.(4a,b)\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*}R_{k}=\frac{k_{CO}+k_{OC}}{k_{CO}}=1+\frac{k_{OC}}{k_{CO}}\hspace{.167em}{\mathrm{and}}\hspace{.167em}R_{I}=\frac{I_{true}}{I_{app,{\infty}}}\end{equation*}\end{document}In Fig. 7, Rk as calculated by Eq. 4a is compared with RI as obtained from Eq. 4b. Both gating factors are plotted vs. membrane potential for different Tl+ concentrations. The coincidence of RI and Rk indicates that the relationship Iapp,∞ = Iapp,k holds.

Bottom Line: With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app).The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter.Different models were considered.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Biology, University of Kiel, 24098 Kiel, Germany.

ABSTRACT
Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.

Show MeSH
Related in: MedlinePlus