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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 rate constants of a two-state model on luminal Tl+ concentration at different membrane potentials as obtained from the β distributions of the current amplitudes in the bursts for (A) kOC and (B) kCO. C and D present tests of whether the absolute values in A and B are determined correctly. The black histograms are generated from the measured time series. The coinciding red curves result from simulated time series using the rate constants in A and B with (C) 50 mM Tl+ at −100 mV and (D) 150 mM Tl+ at −80 mV (a = 1 in Eq. 2). The deviating histograms (green) are obtained by multiplying the rate constants kCO and kOC in Eq. 2 with a common factor of (C) a = 0.33 and (D) a = 2.
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fig6: Dependence of the rate constants of a two-state model on luminal Tl+ concentration at different membrane potentials as obtained from the β distributions of the current amplitudes in the bursts for (A) kOC and (B) kCO. C and D present tests of whether the absolute values in A and B are determined correctly. The black histograms are generated from the measured time series. The coinciding red curves result from simulated time series using the rate constants in A and B with (C) 50 mM Tl+ at −100 mV and (D) 150 mM Tl+ at −80 mV (a = 1 in Eq. 2). The deviating histograms (green) are obtained by multiplying the rate constants kCO and kOC in Eq. 2 with a common factor of (C) a = 0.33 and (D) a = 2.

Mentions: The β fit of the time series in Figs. 4 and 5 also delivers the rate constants (Fig. 6) of the two-state O-C model causing the gating in the bursts.


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 rate constants of a two-state model on luminal Tl+ concentration at different membrane potentials as obtained from the β distributions of the current amplitudes in the bursts for (A) kOC and (B) kCO. C and D present tests of whether the absolute values in A and B are determined correctly. The black histograms are generated from the measured time series. The coinciding red curves result from simulated time series using the rate constants in A and B with (C) 50 mM Tl+ at −100 mV and (D) 150 mM Tl+ at −80 mV (a = 1 in Eq. 2). The deviating histograms (green) are obtained by multiplying the rate constants kCO and kOC in Eq. 2 with a common factor of (C) a = 0.33 and (D) a = 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2279167&req=5

fig6: Dependence of the rate constants of a two-state model on luminal Tl+ concentration at different membrane potentials as obtained from the β distributions of the current amplitudes in the bursts for (A) kOC and (B) kCO. C and D present tests of whether the absolute values in A and B are determined correctly. The black histograms are generated from the measured time series. The coinciding red curves result from simulated time series using the rate constants in A and B with (C) 50 mM Tl+ at −100 mV and (D) 150 mM Tl+ at −80 mV (a = 1 in Eq. 2). The deviating histograms (green) are obtained by multiplying the rate constants kCO and kOC in Eq. 2 with a common factor of (C) a = 0.33 and (D) a = 2.
Mentions: The β fit of the time series in Figs. 4 and 5 also delivers the rate constants (Fig. 6) of the two-state O-C model causing the gating in the bursts.

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