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Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel.

Espinosa F, Fleischhauer R, McMahon A, Joho RH - J. Gen. Physiol. (2001)

Bottom Line: Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H.Joho. 1998.Pflügers Arch. 435:654-661).

View Article: PubMed Central - PubMed

Affiliation: Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
A gain-of-function mutation in the Caenorhabditis elegans exp-2 K(+)-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at -160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K(+) channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K(+) ions at potentials as negative as -120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654-661).

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Open and closed times are not significantly affected by mutations in S6. Histograms of open and closed times show no differences between C480 wild-type and C480I and C480F mutant channels. Open-time distributions were fit with single exponentials, closed-time distributions with two exponentials. The mean open and closed times in milliseconds (mean ± SEM) and the number of experiments are shown for each channel.
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Figure 8: Open and closed times are not significantly affected by mutations in S6. Histograms of open and closed times show no differences between C480 wild-type and C480I and C480F mutant channels. Open-time distributions were fit with single exponentials, closed-time distributions with two exponentials. The mean open and closed times in milliseconds (mean ± SEM) and the number of experiments are shown for each channel.

Mentions: The expanded current traces for C480, C480I, and C480F suggested that there were no big changes in the open times and brief, intraburst closed times among the studied channels (Fig. 7). The open- and closed-time histograms confirmed the initial impression (Fig. 8). Although there were dramatic differences in Po, corresponding to the large changes in Iss, there were little or no differences in mean open times and mean intraburst closed times. The distribution of open times for C480, C480I, and C480F could be well fit with single exponentials yielding mean open times of 0.24 ± 0.01 ms (n = 7), 0.20 ± 0.02 ms (n = 4), and 0.22 ± 0.01 ms (n = 6) for C480, C480I, and C480F, respectively. The closed time distributions (determined from the 200-ms recordings) could be better fit with two exponentials. The mean closed times were ∼0.1 ms and ∼0.6 ms for wild-type and mutant channels.


Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel.

Espinosa F, Fleischhauer R, McMahon A, Joho RH - J. Gen. Physiol. (2001)

Open and closed times are not significantly affected by mutations in S6. Histograms of open and closed times show no differences between C480 wild-type and C480I and C480F mutant channels. Open-time distributions were fit with single exponentials, closed-time distributions with two exponentials. The mean open and closed times in milliseconds (mean ± SEM) and the number of experiments are shown for each channel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233824&req=5

Figure 8: Open and closed times are not significantly affected by mutations in S6. Histograms of open and closed times show no differences between C480 wild-type and C480I and C480F mutant channels. Open-time distributions were fit with single exponentials, closed-time distributions with two exponentials. The mean open and closed times in milliseconds (mean ± SEM) and the number of experiments are shown for each channel.
Mentions: The expanded current traces for C480, C480I, and C480F suggested that there were no big changes in the open times and brief, intraburst closed times among the studied channels (Fig. 7). The open- and closed-time histograms confirmed the initial impression (Fig. 8). Although there were dramatic differences in Po, corresponding to the large changes in Iss, there were little or no differences in mean open times and mean intraburst closed times. The distribution of open times for C480, C480I, and C480F could be well fit with single exponentials yielding mean open times of 0.24 ± 0.01 ms (n = 7), 0.20 ± 0.02 ms (n = 4), and 0.22 ± 0.01 ms (n = 6) for C480, C480I, and C480F, respectively. The closed time distributions (determined from the 200-ms recordings) could be better fit with two exponentials. The mean closed times were ∼0.1 ms and ∼0.6 ms for wild-type and mutant channels.

Bottom Line: Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H.Joho. 1998.Pflügers Arch. 435:654-661).

View Article: PubMed Central - PubMed

Affiliation: Center for Basic Neuroscience, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
A gain-of-function mutation in the Caenorhabditis elegans exp-2 K(+)-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W., R. Fleischhauer, J.A. Dent, R.H. Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at -160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K(+) channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K(+) ions at potentials as negative as -120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact with a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y., and R.H. Joho. 1998. Pflügers Arch. 435:654-661).

Show MeSH
Related in: MedlinePlus