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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 (A) true single-channel conductivity gtrue from Fig. 5 B, (B) characteristic voltage VG (Fig. 7, Eq. 5), and (C) amplitude factor Rk,0 (Fig. 7, Eq. 5) on [Tl+] with [Tl+] + [ K+] = 150 mM. Data from Table I, free fit.
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fig8: Dependence of (A) true single-channel conductivity gtrue from Fig. 5 B, (B) characteristic voltage VG (Fig. 7, Eq. 5), and (C) amplitude factor Rk,0 (Fig. 7, Eq. 5) on [Tl+] with [Tl+] + [ K+] = 150 mM. Data from Table I, free fit.

Mentions: To test whether all curves in Fig. 7 share the same characteristic voltage (as in the case of the different potassium concentrations in Schroeder and Hansen, 2007), a joint fit with shared VG and free Rk,0 was attempted. This approach failed completely (unpublished data). However, it turned out that a reasonable joint fit with common VG is possible for the pairs 0/25 mM Tl+ and 50/150 mM Tl+ (dashed lines in Fig. 7, A,B and C,D, respectively, and last columns in Table I), even though the bending was somewhat too sharp for the 0 and 50 mM data. The more likely free fit resulted in quite similar VG for 50 and 150 mM Tl+, but in a definitely different VG for 0 and 25 mM Tl+ (Table I, third column), explaining the failure of the joint fit of all concentrations. The amplitude factors Rk,0 obtained at 50 and 150 mM (Table I, second column) are proportional to the concentrations (within the scatter). This does not hold for Rk,0 obtained at 0 and 25 mM Tl+. These numbers seem to indicate that different mechanisms are involved at 0 and 25 mM on one side and at 50 or 150 mM Tl+ on the other side. In Fig. 8, the dependence of gtrue of Tl+ concentration (Fig. 8 A) is compared with the dependence of VG (Fig. 8 B). The similar saturation behavior may lead to the suggestion that both effects result from the same mechanism. Especially, the role of 50 mM Tl+ becomes obvious from these curves. Also the amplitude factor Rk,0 seems to change its behavior at 50 mM (Fig. 8 C) even though the scatter prevents a final statement.


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 (A) true single-channel conductivity gtrue from Fig. 5 B, (B) characteristic voltage VG (Fig. 7, Eq. 5), and (C) amplitude factor Rk,0 (Fig. 7, Eq. 5) on [Tl+] with [Tl+] + [ K+] = 150 mM. Data from Table I, free fit.
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fig8: Dependence of (A) true single-channel conductivity gtrue from Fig. 5 B, (B) characteristic voltage VG (Fig. 7, Eq. 5), and (C) amplitude factor Rk,0 (Fig. 7, Eq. 5) on [Tl+] with [Tl+] + [ K+] = 150 mM. Data from Table I, free fit.
Mentions: To test whether all curves in Fig. 7 share the same characteristic voltage (as in the case of the different potassium concentrations in Schroeder and Hansen, 2007), a joint fit with shared VG and free Rk,0 was attempted. This approach failed completely (unpublished data). However, it turned out that a reasonable joint fit with common VG is possible for the pairs 0/25 mM Tl+ and 50/150 mM Tl+ (dashed lines in Fig. 7, A,B and C,D, respectively, and last columns in Table I), even though the bending was somewhat too sharp for the 0 and 50 mM data. The more likely free fit resulted in quite similar VG for 50 and 150 mM Tl+, but in a definitely different VG for 0 and 25 mM Tl+ (Table I, third column), explaining the failure of the joint fit of all concentrations. The amplitude factors Rk,0 obtained at 50 and 150 mM (Table I, second column) are proportional to the concentrations (within the scatter). This does not hold for Rk,0 obtained at 0 and 25 mM Tl+. These numbers seem to indicate that different mechanisms are involved at 0 and 25 mM on one side and at 50 or 150 mM Tl+ on the other side. In Fig. 8, the dependence of gtrue of Tl+ concentration (Fig. 8 A) is compared with the dependence of VG (Fig. 8 B). The similar saturation behavior may lead to the suggestion that both effects result from the same mechanism. Especially, the role of 50 mM Tl+ becomes obvious from these curves. Also the amplitude factor Rk,0 seems to change its behavior at 50 mM (Fig. 8 C) even though the scatter prevents a final statement.

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