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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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Dwell-time histograms of the open (A, C, E, and G) and the closed (B, D, F, and H) states obtained from MaxiK channels in the presence and absence of Tl+ with the following solutions: 150 mM KNO3 on both sides (‘K+’, black), 50 mM Tl+ and 100 mM K+ on the luminal side (Tl+ lum, green) or on the cytosolic side (Tl+ cyt, red). On the opposite side there was always 150 mM KNO3. 2.5 mM Mg(NO3)2 and 2.5 mM Ca(NO3)2 were included in all solutions. For the sake of clarity, only representative error bars are given, which were obtained from averaging over the error bars of bins of 25% of the τ value.
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fig1: Dwell-time histograms of the open (A, C, E, and G) and the closed (B, D, F, and H) states obtained from MaxiK channels in the presence and absence of Tl+ with the following solutions: 150 mM KNO3 on both sides (‘K+’, black), 50 mM Tl+ and 100 mM K+ on the luminal side (Tl+ lum, green) or on the cytosolic side (Tl+ cyt, red). On the opposite side there was always 150 mM KNO3. 2.5 mM Mg(NO3)2 and 2.5 mM Ca(NO3)2 were included in all solutions. For the sake of clarity, only representative error bars are given, which were obtained from averaging over the error bars of bins of 25% of the τ value.

Mentions: Fig. 1 presents dwell-time histograms obtained at different membrane potentials and with 150 mM K+ or 50 mM Tl+ + 100 mM K+ on the luminal or on the cytosolic side. Histograms were obtained from −120 to +120 mV in steps of 20 mV, but here only the characteristic phenomena are shown. In the range of −40 to +40 mV, there is no significant influence of luminal or cytosolic Tl+ on the dwell-time histograms of the open state (Fig. 1, C and E). A strong influence is found in the open-state histogram for luminal Tl+ at potentials more negative than −80 mV (Fig. 1 A) and for cytosolic Tl+ at potentials more positive than +80 mV (Fig. 1 G). This sidedness of the Tl+ effect holds also for the closed-state histograms (Fig. 1, B, D, F, and H) with the difference that a strong effect is only observed at potentials more positive than +80 mV.


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)

Dwell-time histograms of the open (A, C, E, and G) and the closed (B, D, F, and H) states obtained from MaxiK channels in the presence and absence of Tl+ with the following solutions: 150 mM KNO3 on both sides (‘K+’, black), 50 mM Tl+ and 100 mM K+ on the luminal side (Tl+ lum, green) or on the cytosolic side (Tl+ cyt, red). On the opposite side there was always 150 mM KNO3. 2.5 mM Mg(NO3)2 and 2.5 mM Ca(NO3)2 were included in all solutions. For the sake of clarity, only representative error bars are given, which were obtained from averaging over the error bars of bins of 25% of the τ value.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Dwell-time histograms of the open (A, C, E, and G) and the closed (B, D, F, and H) states obtained from MaxiK channels in the presence and absence of Tl+ with the following solutions: 150 mM KNO3 on both sides (‘K+’, black), 50 mM Tl+ and 100 mM K+ on the luminal side (Tl+ lum, green) or on the cytosolic side (Tl+ cyt, red). On the opposite side there was always 150 mM KNO3. 2.5 mM Mg(NO3)2 and 2.5 mM Ca(NO3)2 were included in all solutions. For the sake of clarity, only representative error bars are given, which were obtained from averaging over the error bars of bins of 25% of the τ value.
Mentions: Fig. 1 presents dwell-time histograms obtained at different membrane potentials and with 150 mM K+ or 50 mM Tl+ + 100 mM K+ on the luminal or on the cytosolic side. Histograms were obtained from −120 to +120 mV in steps of 20 mV, but here only the characteristic phenomena are shown. In the range of −40 to +40 mV, there is no significant influence of luminal or cytosolic Tl+ on the dwell-time histograms of the open state (Fig. 1, C and E). A strong influence is found in the open-state histogram for luminal Tl+ at potentials more negative than −80 mV (Fig. 1 A) and for cytosolic Tl+ at potentials more positive than +80 mV (Fig. 1 G). This sidedness of the Tl+ effect holds also for the closed-state histograms (Fig. 1, B, D, F, and H) with the difference that a strong effect is only observed at potentials more positive than +80 mV.

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