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Tail end of the s6 segment: role in permeation in shaker potassium channels.

Ding S, Horn R - J. Gen. Physiol. (2002)

Bottom Line: One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma).Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter.Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P(o,max)) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma). Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb(+) ions for K(+) ions reduces the amplitude of single channel currents and makes gamma insensitive to mutations of Y485. These results suggest that Rb(+) ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6(T) residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

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Voltage dependence of TBA block for wild-type (A; n = 3–5) and Y485A (B; n = 3–5) channels. Currents from depolarizations between −80 and 40 mV in the presence of 50 mM intracellular TBA. Estimates of kon and koff are plotted against membrane potential in the bottom panels. The exponential increase in kon was fit by Eq. 2, with parameters kon(0) = 2.22/0.54 s−1mM−1 and d = 0.30/0.31 for WT/Y485A channels, respectively. koff was 0.03/0.014 ms−1, respectively, for WT/Y485A channels.
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fig5: Voltage dependence of TBA block for wild-type (A; n = 3–5) and Y485A (B; n = 3–5) channels. Currents from depolarizations between −80 and 40 mV in the presence of 50 mM intracellular TBA. Estimates of kon and koff are plotted against membrane potential in the bottom panels. The exponential increase in kon was fit by Eq. 2, with parameters kon(0) = 2.22/0.54 s−1mM−1 and d = 0.30/0.31 for WT/Y485A channels, respectively. koff was 0.03/0.014 ms−1, respectively, for WT/Y485A channels.

Mentions: We further examined the possibility that the position of the TBA binding site within the membrane electric field is affected by the Y485A mutation. Fig. 5 shows the effect of membrane potential on TBA block for both wild-type and Y485A channels. The currents shown were elicited by a series of voltage steps from −80 to 40 mV in the constant presence of 50 mM internal TBA. The values of kon and koff were estimated from the kinetics and magnitude of steady-state block at each voltage, and their estimates are plotted on semilogarithmic axes against membrane potential in the bottom panels of Fig. 5. Although the kinetics of the block are quite different for wild-type and mutant channels, the estimates of kon and koff were independent of [TBA] (unpublished data), a manipulation that also affects kinetics; this result supports the reliability of this method of estimation.


Tail end of the s6 segment: role in permeation in shaker potassium channels.

Ding S, Horn R - J. Gen. Physiol. (2002)

Voltage dependence of TBA block for wild-type (A; n = 3–5) and Y485A (B; n = 3–5) channels. Currents from depolarizations between −80 and 40 mV in the presence of 50 mM intracellular TBA. Estimates of kon and koff are plotted against membrane potential in the bottom panels. The exponential increase in kon was fit by Eq. 2, with parameters kon(0) = 2.22/0.54 s−1mM−1 and d = 0.30/0.31 for WT/Y485A channels, respectively. koff was 0.03/0.014 ms−1, respectively, for WT/Y485A channels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2311402&req=5

fig5: Voltage dependence of TBA block for wild-type (A; n = 3–5) and Y485A (B; n = 3–5) channels. Currents from depolarizations between −80 and 40 mV in the presence of 50 mM intracellular TBA. Estimates of kon and koff are plotted against membrane potential in the bottom panels. The exponential increase in kon was fit by Eq. 2, with parameters kon(0) = 2.22/0.54 s−1mM−1 and d = 0.30/0.31 for WT/Y485A channels, respectively. koff was 0.03/0.014 ms−1, respectively, for WT/Y485A channels.
Mentions: We further examined the possibility that the position of the TBA binding site within the membrane electric field is affected by the Y485A mutation. Fig. 5 shows the effect of membrane potential on TBA block for both wild-type and Y485A channels. The currents shown were elicited by a series of voltage steps from −80 to 40 mV in the constant presence of 50 mM internal TBA. The values of kon and koff were estimated from the kinetics and magnitude of steady-state block at each voltage, and their estimates are plotted on semilogarithmic axes against membrane potential in the bottom panels of Fig. 5. Although the kinetics of the block are quite different for wild-type and mutant channels, the estimates of kon and koff were independent of [TBA] (unpublished data), a manipulation that also affects kinetics; this result supports the reliability of this method of estimation.

Bottom Line: One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma).Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter.Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Jefferson Medical College, Philadelphia, PA 19107, USA.

ABSTRACT
The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (P(o,max)) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces P(o,max), whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (gamma). Mutations of residue Y485 have no effect on the Rb(+)/K(+) selectivity, suggesting a local effect on gamma rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb(+) ions for K(+) ions reduces the amplitude of single channel currents and makes gamma insensitive to mutations of Y485. These results suggest that Rb(+) ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6(T) residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

Show MeSH
Related in: MedlinePlus