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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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The effect of filtering on the apparent single-channel current. (A and C) No moving average filter (=raw data). (B and D) The same data with moving average filter over 100 data points separated by 5 μs. (A and B) The horizontal lines present Iapp as obtained by fit-by-eye (Riessner et al., 2002). (C and D) The vertical lines present Iapp as obtained from the maxima of the amplitude histograms of the time series above them. The time series was recorded at −120 mV with 50 mM Tl+ + 100 mM K+ in the pipette (luminal) and 150 mM K+ in the bathing medium (cytosolic).
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fig2: The effect of filtering on the apparent single-channel current. (A and C) No moving average filter (=raw data). (B and D) The same data with moving average filter over 100 data points separated by 5 μs. (A and B) The horizontal lines present Iapp as obtained by fit-by-eye (Riessner et al., 2002). (C and D) The vertical lines present Iapp as obtained from the maxima of the amplitude histograms of the time series above them. The time series was recorded at −120 mV with 50 mM Tl+ + 100 mM K+ in the pipette (luminal) and 150 mM K+ in the bathing medium (cytosolic).

Mentions: In the μs range, the gating during the often nonresolved bursts is investigated. A major issue is the distinction between Itrue and Iapp and the question of whether a putative molecular mechanism can provide a prediction of the measured current reduction RI.(1)\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_{I}=\frac{I_{true}}{I_{app}},\end{equation*}\end{document}as defined in Materials and methods. In flickery time series like that one in Fig. 2 A, there are two problems, i.e., determining Itrue and an appropriate value for Iapp.Fig. 2 illustrates the problem of estimating Iapp. The apparent single-channel current Iapp as obtained by fit-by-eye (horizontal lines in Fig. 2 [A and B]; Riessner et al., 2002) or from the evaluation of the amplitude histograms (vertical lines in Fig. 2, C and D) depends strongly on the integration time of the anti-aliasing filter of the recording set-up. In Fig. 2, different integration times are mimicked by the length of an offline moving average filter.


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)

The effect of filtering on the apparent single-channel current. (A and C) No moving average filter (=raw data). (B and D) The same data with moving average filter over 100 data points separated by 5 μs. (A and B) The horizontal lines present Iapp as obtained by fit-by-eye (Riessner et al., 2002). (C and D) The vertical lines present Iapp as obtained from the maxima of the amplitude histograms of the time series above them. The time series was recorded at −120 mV with 50 mM Tl+ + 100 mM K+ in the pipette (luminal) and 150 mM K+ in the bathing medium (cytosolic).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2279167&req=5

fig2: The effect of filtering on the apparent single-channel current. (A and C) No moving average filter (=raw data). (B and D) The same data with moving average filter over 100 data points separated by 5 μs. (A and B) The horizontal lines present Iapp as obtained by fit-by-eye (Riessner et al., 2002). (C and D) The vertical lines present Iapp as obtained from the maxima of the amplitude histograms of the time series above them. The time series was recorded at −120 mV with 50 mM Tl+ + 100 mM K+ in the pipette (luminal) and 150 mM K+ in the bathing medium (cytosolic).
Mentions: In the μs range, the gating during the often nonresolved bursts is investigated. A major issue is the distinction between Itrue and Iapp and the question of whether a putative molecular mechanism can provide a prediction of the measured current reduction RI.(1)\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_{I}=\frac{I_{true}}{I_{app}},\end{equation*}\end{document}as defined in Materials and methods. In flickery time series like that one in Fig. 2 A, there are two problems, i.e., determining Itrue and an appropriate value for Iapp.Fig. 2 illustrates the problem of estimating Iapp. The apparent single-channel current Iapp as obtained by fit-by-eye (horizontal lines in Fig. 2 [A and B]; Riessner et al., 2002) or from the evaluation of the amplitude histograms (vertical lines in Fig. 2, C and D) depends strongly on the integration time of the anti-aliasing filter of the recording set-up. In Fig. 2, different integration times are mimicked by the length of an offline moving average filter.

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