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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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S6 Mutations in close proximity have opposite effects on channel gating. (A) The sequence alignment shows the similarity between Kv2.1, Shaker B (ShB) and EXP-2. The cysteine at position 393 in Kv2.1 (in bold) influences open-state stability and K+/Rb+ permeation (Liu and Joho 1998); the nearest residue in Shaker (Hoshi et al. 1991) and in Kv1.3 (Panyi et al. 1995) determines the kinetics of C-type inactivation (in bold in the ShB sequence). The position of the EXP-2 mutations that prevent the channel from reaching long-lived closed states is three residues COOH-terminal from the Kv2.1 position that influences open-state stability (in bold in EXP-2 sequence). The approximate position of the voltage-activated gate in ShB (Liu et al. 1997) is shown by two downward arrowheads. (B) Top and side views of the C480Y EXP-2 mutant (modeled after KcsA) suggest how S6 residues that are only three positions apart may independently influence the voltage-activated gate and the voltage-independent pore gate. The bulky side chain Y480 prevents S6 rotation that is required for the channel to reach a long-lived closed state. In the open state, A477 (C393 in Kv2.1) is close to the conserved T454 (T370 in Kv2.1), which is located in the narrow ion conduction pathway (part of the K+ channel signature sequence; Heginbotham et al. 1994). In Kv2.1, small hydrophilic side chains at position 393 in S6 (A477 in EXP-2) stabilize the open state and affect ion selectivity (Liu and Joho 1998) presumably by interacting with T370 (T454 in EXP-2). In this model, substitutions for C393 in Kv2.1 (A477 in EXP-2) would only affect the stability of the open state as long as S6 is in the appropriate orientation for the side chain at position 393 to interact with T370. In the wild-type Kv2.1 channel, the interaction between T370 (T454 in EXP-2) and C393 (A477 in EXP-2) is broken when S6 rotates counterclockwise at negative membrane potentials. This rotation cannot occur in EXP-2 mutants with aromatic side chains at position 480 “trapping” the mutant channels in an open state without affecting open state stability (mean open time) or ion selectivity.
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Figure 9: S6 Mutations in close proximity have opposite effects on channel gating. (A) The sequence alignment shows the similarity between Kv2.1, Shaker B (ShB) and EXP-2. The cysteine at position 393 in Kv2.1 (in bold) influences open-state stability and K+/Rb+ permeation (Liu and Joho 1998); the nearest residue in Shaker (Hoshi et al. 1991) and in Kv1.3 (Panyi et al. 1995) determines the kinetics of C-type inactivation (in bold in the ShB sequence). The position of the EXP-2 mutations that prevent the channel from reaching long-lived closed states is three residues COOH-terminal from the Kv2.1 position that influences open-state stability (in bold in EXP-2 sequence). The approximate position of the voltage-activated gate in ShB (Liu et al. 1997) is shown by two downward arrowheads. (B) Top and side views of the C480Y EXP-2 mutant (modeled after KcsA) suggest how S6 residues that are only three positions apart may independently influence the voltage-activated gate and the voltage-independent pore gate. The bulky side chain Y480 prevents S6 rotation that is required for the channel to reach a long-lived closed state. In the open state, A477 (C393 in Kv2.1) is close to the conserved T454 (T370 in Kv2.1), which is located in the narrow ion conduction pathway (part of the K+ channel signature sequence; Heginbotham et al. 1994). In Kv2.1, small hydrophilic side chains at position 393 in S6 (A477 in EXP-2) stabilize the open state and affect ion selectivity (Liu and Joho 1998) presumably by interacting with T370 (T454 in EXP-2). In this model, substitutions for C393 in Kv2.1 (A477 in EXP-2) would only affect the stability of the open state as long as S6 is in the appropriate orientation for the side chain at position 393 to interact with T370. In the wild-type Kv2.1 channel, the interaction between T370 (T454 in EXP-2) and C393 (A477 in EXP-2) is broken when S6 rotates counterclockwise at negative membrane potentials. This rotation cannot occur in EXP-2 mutants with aromatic side chains at position 480 “trapping” the mutant channels in an open state without affecting open state stability (mean open time) or ion selectivity.

Mentions: Below, we will discuss our findings for EXP-2 in light of the “two-gate” model that we proposed for the voltage-gated K+ channel Kv2.1 (Liu and Joho 1998). The two-gate model postulates that a pore gate, located between the external and internal TEA-binding site, is responsible for the brief, voltage-independent openings and closings seen in single-channel records of Kv2.1. We proposed that the pore gate interacts with the conserved cysteine in S6 of Kv channels (C393 in Kv2.1) because subtle, conservative substitutions for C393 dramatically affect both ion permeation and open state stability but have no effect on blockade by external or internal TEA (Liu and Joho 1998; Fig. 9). Small hydrophilic side chain substitutions at position 393 stabilize the open state, whereas larger side chains destabilize the open state; however, these S6 mutations in Kv2.1 have little or no effect on voltage-dependent gating, i.e., on the movement of the voltage-controlled activation gate located near the end of S6 (Liu et al. 1997).


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)

S6 Mutations in close proximity have opposite effects on channel gating. (A) The sequence alignment shows the similarity between Kv2.1, Shaker B (ShB) and EXP-2. The cysteine at position 393 in Kv2.1 (in bold) influences open-state stability and K+/Rb+ permeation (Liu and Joho 1998); the nearest residue in Shaker (Hoshi et al. 1991) and in Kv1.3 (Panyi et al. 1995) determines the kinetics of C-type inactivation (in bold in the ShB sequence). The position of the EXP-2 mutations that prevent the channel from reaching long-lived closed states is three residues COOH-terminal from the Kv2.1 position that influences open-state stability (in bold in EXP-2 sequence). The approximate position of the voltage-activated gate in ShB (Liu et al. 1997) is shown by two downward arrowheads. (B) Top and side views of the C480Y EXP-2 mutant (modeled after KcsA) suggest how S6 residues that are only three positions apart may independently influence the voltage-activated gate and the voltage-independent pore gate. The bulky side chain Y480 prevents S6 rotation that is required for the channel to reach a long-lived closed state. In the open state, A477 (C393 in Kv2.1) is close to the conserved T454 (T370 in Kv2.1), which is located in the narrow ion conduction pathway (part of the K+ channel signature sequence; Heginbotham et al. 1994). In Kv2.1, small hydrophilic side chains at position 393 in S6 (A477 in EXP-2) stabilize the open state and affect ion selectivity (Liu and Joho 1998) presumably by interacting with T370 (T454 in EXP-2). In this model, substitutions for C393 in Kv2.1 (A477 in EXP-2) would only affect the stability of the open state as long as S6 is in the appropriate orientation for the side chain at position 393 to interact with T370. In the wild-type Kv2.1 channel, the interaction between T370 (T454 in EXP-2) and C393 (A477 in EXP-2) is broken when S6 rotates counterclockwise at negative membrane potentials. This rotation cannot occur in EXP-2 mutants with aromatic side chains at position 480 “trapping” the mutant channels in an open state without affecting open state stability (mean open time) or ion selectivity.
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Related In: Results  -  Collection

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Figure 9: S6 Mutations in close proximity have opposite effects on channel gating. (A) The sequence alignment shows the similarity between Kv2.1, Shaker B (ShB) and EXP-2. The cysteine at position 393 in Kv2.1 (in bold) influences open-state stability and K+/Rb+ permeation (Liu and Joho 1998); the nearest residue in Shaker (Hoshi et al. 1991) and in Kv1.3 (Panyi et al. 1995) determines the kinetics of C-type inactivation (in bold in the ShB sequence). The position of the EXP-2 mutations that prevent the channel from reaching long-lived closed states is three residues COOH-terminal from the Kv2.1 position that influences open-state stability (in bold in EXP-2 sequence). The approximate position of the voltage-activated gate in ShB (Liu et al. 1997) is shown by two downward arrowheads. (B) Top and side views of the C480Y EXP-2 mutant (modeled after KcsA) suggest how S6 residues that are only three positions apart may independently influence the voltage-activated gate and the voltage-independent pore gate. The bulky side chain Y480 prevents S6 rotation that is required for the channel to reach a long-lived closed state. In the open state, A477 (C393 in Kv2.1) is close to the conserved T454 (T370 in Kv2.1), which is located in the narrow ion conduction pathway (part of the K+ channel signature sequence; Heginbotham et al. 1994). In Kv2.1, small hydrophilic side chains at position 393 in S6 (A477 in EXP-2) stabilize the open state and affect ion selectivity (Liu and Joho 1998) presumably by interacting with T370 (T454 in EXP-2). In this model, substitutions for C393 in Kv2.1 (A477 in EXP-2) would only affect the stability of the open state as long as S6 is in the appropriate orientation for the side chain at position 393 to interact with T370. In the wild-type Kv2.1 channel, the interaction between T370 (T454 in EXP-2) and C393 (A477 in EXP-2) is broken when S6 rotates counterclockwise at negative membrane potentials. This rotation cannot occur in EXP-2 mutants with aromatic side chains at position 480 “trapping” the mutant channels in an open state without affecting open state stability (mean open time) or ion selectivity.
Mentions: Below, we will discuss our findings for EXP-2 in light of the “two-gate” model that we proposed for the voltage-gated K+ channel Kv2.1 (Liu and Joho 1998). The two-gate model postulates that a pore gate, located between the external and internal TEA-binding site, is responsible for the brief, voltage-independent openings and closings seen in single-channel records of Kv2.1. We proposed that the pore gate interacts with the conserved cysteine in S6 of Kv channels (C393 in Kv2.1) because subtle, conservative substitutions for C393 dramatically affect both ion permeation and open state stability but have no effect on blockade by external or internal TEA (Liu and Joho 1998; Fig. 9). Small hydrophilic side chain substitutions at position 393 stabilize the open state, whereas larger side chains destabilize the open state; however, these S6 mutations in Kv2.1 have little or no effect on voltage-dependent gating, i.e., on the movement of the voltage-controlled activation gate located near the end of S6 (Liu et al. 1997).

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