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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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(A) Apparent (Iapp,100) and (B) true single-channel IV curves of MaxiK with different luminal K+/Tl+ mixtures (pipette). The Tl+ concentrations are attached to the curves with [Tl+] + [K+] = 150 mM. (C). Apparent IV curves obtained with cytosolic K+/Tl+ mixtures (bath). Besides in the region of the negative slope at positive potentials, Iapp and Itrue are identical.
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fig5: (A) Apparent (Iapp,100) and (B) true single-channel IV curves of MaxiK with different luminal K+/Tl+ mixtures (pipette). The Tl+ concentrations are attached to the curves with [Tl+] + [K+] = 150 mM. (C). Apparent IV curves obtained with cytosolic K+/Tl+ mixtures (bath). Besides in the region of the negative slope at positive potentials, Iapp and Itrue are identical.

Mentions: The resulting IV curves are shown in Fig. 5 A. At positive membrane potentials where current is solely carried by K+, all IV curves show the same negative slope as the K+ data do (Schroeder and Hansen, 2007); Tl+ does not seem to have any significant effect. However, at negative voltages, when Tl+ is drawn from the lumen into the pore, a second negative slope is induced. The effect increases monotonously with increasing Tl+ concentration in the mixed solution. Similar to the negative slope on the positive side, the involvement of unresolved gating becomes obvious from the appearance of flickery current traces (Fig. 4).


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)

(A) Apparent (Iapp,100) and (B) true single-channel IV curves of MaxiK with different luminal K+/Tl+ mixtures (pipette). The Tl+ concentrations are attached to the curves with [Tl+] + [K+] = 150 mM. (C). Apparent IV curves obtained with cytosolic K+/Tl+ mixtures (bath). Besides in the region of the negative slope at positive potentials, Iapp and Itrue are identical.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: (A) Apparent (Iapp,100) and (B) true single-channel IV curves of MaxiK with different luminal K+/Tl+ mixtures (pipette). The Tl+ concentrations are attached to the curves with [Tl+] + [K+] = 150 mM. (C). Apparent IV curves obtained with cytosolic K+/Tl+ mixtures (bath). Besides in the region of the negative slope at positive potentials, Iapp and Itrue are identical.
Mentions: The resulting IV curves are shown in Fig. 5 A. At positive membrane potentials where current is solely carried by K+, all IV curves show the same negative slope as the K+ data do (Schroeder and Hansen, 2007); Tl+ does not seem to have any significant effect. However, at negative voltages, when Tl+ is drawn from the lumen into the pore, a second negative slope is induced. The effect increases monotonously with increasing Tl+ concentration in the mixed solution. Similar to the negative slope on the positive side, the involvement of unresolved gating becomes obvious from the appearance of flickery current traces (Fig. 4).

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