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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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Sections of four current records with different K+/Tl+ mixtures on the luminal side (pipette). [Tl+] is given at the traces with [K+] = 150 mM − [Tl+]. Cytosolic solution contained 150 mM K+. Membrane potential was −100 mV for all records. Channel openings result in downward deflections. With increasing Tl+ concentration, the open-channel noise increases and the apparent current decreases, indicating the increase of unresolved gating events. The upper horizontal lines give the baseline (closed channel). The dotted lines show the apparent current Iapp,∞ taken from the plateau in Fig. 3. Iapp,1 obtained from fit-by-eye (Riessner et al., 2002) is presented by solid horizontal lines.
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fig4: Sections of four current records with different K+/Tl+ mixtures on the luminal side (pipette). [Tl+] is given at the traces with [K+] = 150 mM − [Tl+]. Cytosolic solution contained 150 mM K+. Membrane potential was −100 mV for all records. Channel openings result in downward deflections. With increasing Tl+ concentration, the open-channel noise increases and the apparent current decreases, indicating the increase of unresolved gating events. The upper horizontal lines give the baseline (closed channel). The dotted lines show the apparent current Iapp,∞ taken from the plateau in Fig. 3. Iapp,1 obtained from fit-by-eye (Riessner et al., 2002) is presented by solid horizontal lines.

Mentions: Sample recordings at −100 mV for four different concentrations of luminal Tl+ are shown in Fig. 4. At constant membrane potential, an increase in Tl+ concentration causes an increase in open-channel noise and a decrease in apparent single-channel current. The analysis here deals only with 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)

Sections of four current records with different K+/Tl+ mixtures on the luminal side (pipette). [Tl+] is given at the traces with [K+] = 150 mM − [Tl+]. Cytosolic solution contained 150 mM K+. Membrane potential was −100 mV for all records. Channel openings result in downward deflections. With increasing Tl+ concentration, the open-channel noise increases and the apparent current decreases, indicating the increase of unresolved gating events. The upper horizontal lines give the baseline (closed channel). The dotted lines show the apparent current Iapp,∞ taken from the plateau in Fig. 3. Iapp,1 obtained from fit-by-eye (Riessner et al., 2002) is presented by solid horizontal lines.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Sections of four current records with different K+/Tl+ mixtures on the luminal side (pipette). [Tl+] is given at the traces with [K+] = 150 mM − [Tl+]. Cytosolic solution contained 150 mM K+. Membrane potential was −100 mV for all records. Channel openings result in downward deflections. With increasing Tl+ concentration, the open-channel noise increases and the apparent current decreases, indicating the increase of unresolved gating events. The upper horizontal lines give the baseline (closed channel). The dotted lines show the apparent current Iapp,∞ taken from the plateau in Fig. 3. Iapp,1 obtained from fit-by-eye (Riessner et al., 2002) is presented by solid horizontal lines.
Mentions: Sample recordings at −100 mV for four different concentrations of luminal Tl+ are shown in Fig. 4. At constant membrane potential, an increase in Tl+ concentration causes an increase in open-channel noise and a decrease in apparent single-channel current. The analysis here deals only with 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