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Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels.

Lu T, Wu L, Xiao J, Yang J - J. Gen. Physiol. (2001)

Bottom Line: In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold.However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current.This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
We studied the effect of monovalent thallium ion (Tl(+)) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (tau = 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl(+) must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current. These results suggest that Tl(+) ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K(+) with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl(+) greatly diminishes Ba(2+) block. The unblocking rate of Ba(2+) is increased by >22-fold when the external permeant ion is switched from K(+) to Tl(+) regardless of the direction of Ba(2+) exit. This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

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Alteration of single-channel gating by Tl+ ions. (A and B) Consecutive records of single-channel K+ (A) or Tl+ (B) currents recorded at −100 mV in a cell-attached membrane patch. The dashed lines indicate the closed level in this and all subsequent figures. Arrows in B indicate the dominant subconductance level and arrowheads mark a relatively rare subconductance level. (C and D) Open- and closed-time distributions of channels recorded with K+ (C) or Tl+ (D) as the external permeant ion. The open-time distributions were best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are indicated. (E) Voltage dependence of the open-time with either K+ or Tl+ as the permeant ion (n = 4–6). (F and G) Voltage dependence of the closed times with either K+ (F) or Tl+ (G) as the permeant ion (n = 4–12). Error bars are SD and are smaller than the symbols in some cases.
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Figure 2: Alteration of single-channel gating by Tl+ ions. (A and B) Consecutive records of single-channel K+ (A) or Tl+ (B) currents recorded at −100 mV in a cell-attached membrane patch. The dashed lines indicate the closed level in this and all subsequent figures. Arrows in B indicate the dominant subconductance level and arrowheads mark a relatively rare subconductance level. (C and D) Open- and closed-time distributions of channels recorded with K+ (C) or Tl+ (D) as the external permeant ion. The open-time distributions were best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are indicated. (E) Voltage dependence of the open-time with either K+ or Tl+ as the permeant ion (n = 4–6). (F and G) Voltage dependence of the closed times with either K+ (F) or Tl+ (G) as the permeant ion (n = 4–12). Error bars are SD and are smaller than the symbols in some cases.

Mentions: As we reported previously (Lu et al. 2001), Kir2.1 channels expressed in Xenopus oocytes undergo spontaneous open and close transitions when recorded in cell-attached patches at constant membrane potentials more negative than the potassium equilibrium potential. Fig. 2 A shows representative inward single-channel currents recorded at −100 mV with K+ as the external permeant ion. The open- and closed-time distributions show that there are one open and three closed states (Fig. 2 C). The open-time was voltage-dependent, decreasing e-fold with 67-mV hyperpolarization, from 293 ms at −80 mV to 45 ms at −200 mV (Fig. 2 E). The two longer closed-times were also voltage-dependent, decreasing e-fold with 71- and 62-mV hyperpolarization, respectively (Fig. 2 F). The shortest closed-time was relatively independent of membrane voltage. Previous studies have shown that these closed states are not produced by open-pore blockage by external or internal blocking ions (Choe et al. 1999; Lu et al. 2001).


Permeant ion-dependent changes in gating of Kir2.1 inward rectifier potassium channels.

Lu T, Wu L, Xiao J, Yang J - J. Gen. Physiol. (2001)

Alteration of single-channel gating by Tl+ ions. (A and B) Consecutive records of single-channel K+ (A) or Tl+ (B) currents recorded at −100 mV in a cell-attached membrane patch. The dashed lines indicate the closed level in this and all subsequent figures. Arrows in B indicate the dominant subconductance level and arrowheads mark a relatively rare subconductance level. (C and D) Open- and closed-time distributions of channels recorded with K+ (C) or Tl+ (D) as the external permeant ion. The open-time distributions were best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are indicated. (E) Voltage dependence of the open-time with either K+ or Tl+ as the permeant ion (n = 4–6). (F and G) Voltage dependence of the closed times with either K+ (F) or Tl+ (G) as the permeant ion (n = 4–12). Error bars are SD and are smaller than the symbols in some cases.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233840&req=5

Figure 2: Alteration of single-channel gating by Tl+ ions. (A and B) Consecutive records of single-channel K+ (A) or Tl+ (B) currents recorded at −100 mV in a cell-attached membrane patch. The dashed lines indicate the closed level in this and all subsequent figures. Arrows in B indicate the dominant subconductance level and arrowheads mark a relatively rare subconductance level. (C and D) Open- and closed-time distributions of channels recorded with K+ (C) or Tl+ (D) as the external permeant ion. The open-time distributions were best fit by a single exponential and the closed-time distributions by three exponentials. The time constants of the fits are indicated. (E) Voltage dependence of the open-time with either K+ or Tl+ as the permeant ion (n = 4–6). (F and G) Voltage dependence of the closed times with either K+ (F) or Tl+ (G) as the permeant ion (n = 4–12). Error bars are SD and are smaller than the symbols in some cases.
Mentions: As we reported previously (Lu et al. 2001), Kir2.1 channels expressed in Xenopus oocytes undergo spontaneous open and close transitions when recorded in cell-attached patches at constant membrane potentials more negative than the potassium equilibrium potential. Fig. 2 A shows representative inward single-channel currents recorded at −100 mV with K+ as the external permeant ion. The open- and closed-time distributions show that there are one open and three closed states (Fig. 2 C). The open-time was voltage-dependent, decreasing e-fold with 67-mV hyperpolarization, from 293 ms at −80 mV to 45 ms at −200 mV (Fig. 2 E). The two longer closed-times were also voltage-dependent, decreasing e-fold with 71- and 62-mV hyperpolarization, respectively (Fig. 2 F). The shortest closed-time was relatively independent of membrane voltage. Previous studies have shown that these closed states are not produced by open-pore blockage by external or internal blocking ions (Choe et al. 1999; Lu et al. 2001).

Bottom Line: In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold.However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current.This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
We studied the effect of monovalent thallium ion (Tl(+)) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K(+) to Tl(+) decreases the mean open-time by approximately 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (tau = 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl(+) must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl(+) current. These results suggest that Tl(+) ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K(+) with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl(+) greatly diminishes Ba(2+) block. The unblocking rate of Ba(2+) is increased by >22-fold when the external permeant ion is switched from K(+) to Tl(+) regardless of the direction of Ba(2+) exit. This effect cannot be explained solely by ion-ion interactions, but is consistent with the notion that Tl(+) induces conformational changes in the selectivity filter.

Show MeSH
Related in: MedlinePlus