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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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S5-Pore-S6 alignment and pore model of EXP-2. (A) Sequence alignment for the S5-pore-S6 region of several K+ channels from different Kv subfamilies. The side chains highlighted by asterisks above the EXP-2 sequence are drawn as sticks in B. (“-” indicates identity to the sequence of Kv2.1; “.” indicates a gap required to maintain alignment. Conserved amino acids are shown by letters on the top; residues that are absolutely conserved in Kv channels are in bold.) (B) Top and side views of the S5-pore-S6 region of EXP-2. The four conserved side chains in S5 that are marked by asterisks (M414, L418, G421, and F425) are on the same side of the S5 helix and project toward S6. The side chain at position 480 in S6 points toward the conserved G421 that is flanked by L418 and F425 in S5. The model suggests possible interaction between G421 in S5 and the side chain at position 480 in S6. In case of an aromatic side chain at position 480, there may be aromatic–aromatic interaction with the invariant F425 in S5.
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Figure 1: S5-Pore-S6 alignment and pore model of EXP-2. (A) Sequence alignment for the S5-pore-S6 region of several K+ channels from different Kv subfamilies. The side chains highlighted by asterisks above the EXP-2 sequence are drawn as sticks in B. (“-” indicates identity to the sequence of Kv2.1; “.” indicates a gap required to maintain alignment. Conserved amino acids are shown by letters on the top; residues that are absolutely conserved in Kv channels are in bold.) (B) Top and side views of the S5-pore-S6 region of EXP-2. The four conserved side chains in S5 that are marked by asterisks (M414, L418, G421, and F425) are on the same side of the S5 helix and project toward S6. The side chain at position 480 in S6 points toward the conserved G421 that is flanked by L418 and F425 in S5. The model suggests possible interaction between G421 in S5 and the side chain at position 480 in S6. In case of an aromatic side chain at position 480, there may be aromatic–aromatic interaction with the invariant F425 in S5.

Mentions: To understand the molecular mechanism that may be responsible in keeping mutant EXP-2 channels from closing at negative membrane potentials, we used the crystal structure of the KcsA K+ channel (Doyle et al. 1998) as a scaffold to generate a 3-D model of the EXP-2 pore, including the transmembrane segments S5 and S6 (Davis et al. 1999). In this model, the large tyrosine side chain at position 480 in the C480Y mutant protrudes from S6 and projects toward S5 (see Fig. 1 B). In contrast, the much smaller cysteine side chain in the wild-type is buried close to S6 and is not in contact with S5. It was very interesting in this context that Perozo et al. 1999 had suggested that S6 may rotate counterclockwise (viewed from the outside) on KcsA channel opening. If the EXP-2 channel underwent a similar conformational change, then, in analogy to KcsA, the clockwise rotation of S6 that would be required for channel closing might be impaired due to steric hindrance in the presence of a bulky residue like tyrosine. Under this condition, the rotation of S6 required for long-lasting closings of the channel might not occur, not even at potentials as negative as −160 mV.


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)

S5-Pore-S6 alignment and pore model of EXP-2. (A) Sequence alignment for the S5-pore-S6 region of several K+ channels from different Kv subfamilies. The side chains highlighted by asterisks above the EXP-2 sequence are drawn as sticks in B. (“-” indicates identity to the sequence of Kv2.1; “.” indicates a gap required to maintain alignment. Conserved amino acids are shown by letters on the top; residues that are absolutely conserved in Kv channels are in bold.) (B) Top and side views of the S5-pore-S6 region of EXP-2. The four conserved side chains in S5 that are marked by asterisks (M414, L418, G421, and F425) are on the same side of the S5 helix and project toward S6. The side chain at position 480 in S6 points toward the conserved G421 that is flanked by L418 and F425 in S5. The model suggests possible interaction between G421 in S5 and the side chain at position 480 in S6. In case of an aromatic side chain at position 480, there may be aromatic–aromatic interaction with the invariant F425 in S5.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: S5-Pore-S6 alignment and pore model of EXP-2. (A) Sequence alignment for the S5-pore-S6 region of several K+ channels from different Kv subfamilies. The side chains highlighted by asterisks above the EXP-2 sequence are drawn as sticks in B. (“-” indicates identity to the sequence of Kv2.1; “.” indicates a gap required to maintain alignment. Conserved amino acids are shown by letters on the top; residues that are absolutely conserved in Kv channels are in bold.) (B) Top and side views of the S5-pore-S6 region of EXP-2. The four conserved side chains in S5 that are marked by asterisks (M414, L418, G421, and F425) are on the same side of the S5 helix and project toward S6. The side chain at position 480 in S6 points toward the conserved G421 that is flanked by L418 and F425 in S5. The model suggests possible interaction between G421 in S5 and the side chain at position 480 in S6. In case of an aromatic side chain at position 480, there may be aromatic–aromatic interaction with the invariant F425 in S5.
Mentions: To understand the molecular mechanism that may be responsible in keeping mutant EXP-2 channels from closing at negative membrane potentials, we used the crystal structure of the KcsA K+ channel (Doyle et al. 1998) as a scaffold to generate a 3-D model of the EXP-2 pore, including the transmembrane segments S5 and S6 (Davis et al. 1999). In this model, the large tyrosine side chain at position 480 in the C480Y mutant protrudes from S6 and projects toward S5 (see Fig. 1 B). In contrast, the much smaller cysteine side chain in the wild-type is buried close to S6 and is not in contact with S5. It was very interesting in this context that Perozo et al. 1999 had suggested that S6 may rotate counterclockwise (viewed from the outside) on KcsA channel opening. If the EXP-2 channel underwent a similar conformational change, then, in analogy to KcsA, the clockwise rotation of S6 that would be required for channel closing might be impaired due to steric hindrance in the presence of a bulky residue like tyrosine. Under this condition, the rotation of S6 required for long-lasting closings of the channel might not occur, not even at potentials as negative as −160 mV.

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