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The lipid-protein interface of a Shaker K(+) channel.

Hong KH, Miller C - J. Gen. Physiol. (2000)

Bottom Line: A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence.These results, combined with an analogous study of S2 (Monks, S., D.J.Miller. 1999.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Tryptophan-substitution mutagenesis was applied to the first and third transmembrane segments (S1 and S3) of a Shaker-type K(+) channel for the purpose of ascertaining whether these sequences are alpha-helical. Point mutants were examined for significant functional changes, indicated by the voltage-activation curves and gating kinetics. Helical periodicity of functional alteration was observed throughout the entire S1 segment. A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence. In both helical stretches, tryptophan-tolerant positions are clustered on approximately half the alpha-helix surface, as if the sidechains are exposed to the hydrocarbon region of the lipid bilayer. These results, combined with an analogous study of S2 (Monks, S., D.J. Needleman, and C. Miller. 1999. J. Gen. Physiol. 113:415-423), locate S1, S2, and S3 on the lipid-facing periphery of K(v) channels.

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Recordings from tryptophan-substituted Shaker channels. (A) Two-electrode voltage-clamp recordings of wild-type channels expressed in Xenopus oocytes. Currents were recorded in KD98 medium with a holding potential of −90 mV, test pulses (30-ms duration) from −60 to +50 mV in 10-mV increments, and a tail potential of −70 mV. (B and C) Similar recordings taken for mutations I243W and L238W. For L238W, test pulses were from −80 to +20 mV with duration of 200 ms and each test pulse was followed by a fixed tail voltage −80 mV. (D) Voltage-activation curves for wild-type, I243W, and L238W mutant channels, calculated from tail-current analysis. Solid curves are Boltzmann fits to the equilibrium activation data. Scale bars in all data panels represent 1 μA, 10 ms.
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Figure 3: Recordings from tryptophan-substituted Shaker channels. (A) Two-electrode voltage-clamp recordings of wild-type channels expressed in Xenopus oocytes. Currents were recorded in KD98 medium with a holding potential of −90 mV, test pulses (30-ms duration) from −60 to +50 mV in 10-mV increments, and a tail potential of −70 mV. (B and C) Similar recordings taken for mutations I243W and L238W. For L238W, test pulses were from −80 to +20 mV with duration of 200 ms and each test pulse was followed by a fixed tail voltage −80 mV. (D) Voltage-activation curves for wild-type, I243W, and L238W mutant channels, calculated from tail-current analysis. Solid curves are Boltzmann fits to the equilibrium activation data. Scale bars in all data panels represent 1 μA, 10 ms.

Mentions: Of the 22 tryptophan mutants in S1, 21 expressed voltage-dependent, K+-selective currents, the single exception being I237W. Responses to families of depolarizing pulses are shown in Fig. 3 for wild-type Shaker and two mutants—L238W, which is left-shifted by 21 mV, and I243W, which is right-shifted by 27 mV. The activation and deactivation kinetics of L238W are slowed substantially, while those of I243W are similar to wild type. All S1 mutants were analyzed in this way, and gating parameters are reported in Table .


The lipid-protein interface of a Shaker K(+) channel.

Hong KH, Miller C - J. Gen. Physiol. (2000)

Recordings from tryptophan-substituted Shaker channels. (A) Two-electrode voltage-clamp recordings of wild-type channels expressed in Xenopus oocytes. Currents were recorded in KD98 medium with a holding potential of −90 mV, test pulses (30-ms duration) from −60 to +50 mV in 10-mV increments, and a tail potential of −70 mV. (B and C) Similar recordings taken for mutations I243W and L238W. For L238W, test pulses were from −80 to +20 mV with duration of 200 ms and each test pulse was followed by a fixed tail voltage −80 mV. (D) Voltage-activation curves for wild-type, I243W, and L238W mutant channels, calculated from tail-current analysis. Solid curves are Boltzmann fits to the equilibrium activation data. Scale bars in all data panels represent 1 μA, 10 ms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887780&req=5

Figure 3: Recordings from tryptophan-substituted Shaker channels. (A) Two-electrode voltage-clamp recordings of wild-type channels expressed in Xenopus oocytes. Currents were recorded in KD98 medium with a holding potential of −90 mV, test pulses (30-ms duration) from −60 to +50 mV in 10-mV increments, and a tail potential of −70 mV. (B and C) Similar recordings taken for mutations I243W and L238W. For L238W, test pulses were from −80 to +20 mV with duration of 200 ms and each test pulse was followed by a fixed tail voltage −80 mV. (D) Voltage-activation curves for wild-type, I243W, and L238W mutant channels, calculated from tail-current analysis. Solid curves are Boltzmann fits to the equilibrium activation data. Scale bars in all data panels represent 1 μA, 10 ms.
Mentions: Of the 22 tryptophan mutants in S1, 21 expressed voltage-dependent, K+-selective currents, the single exception being I237W. Responses to families of depolarizing pulses are shown in Fig. 3 for wild-type Shaker and two mutants—L238W, which is left-shifted by 21 mV, and I243W, which is right-shifted by 27 mV. The activation and deactivation kinetics of L238W are slowed substantially, while those of I243W are similar to wild type. All S1 mutants were analyzed in this way, and gating parameters are reported in Table .

Bottom Line: A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence.These results, combined with an analogous study of S2 (Monks, S., D.J.Miller. 1999.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, Waltham, Massachusetts 02454-9110, USA.

ABSTRACT
Tryptophan-substitution mutagenesis was applied to the first and third transmembrane segments (S1 and S3) of a Shaker-type K(+) channel for the purpose of ascertaining whether these sequences are alpha-helical. Point mutants were examined for significant functional changes, indicated by the voltage-activation curves and gating kinetics. Helical periodicity of functional alteration was observed throughout the entire S1 segment. A similar result was obtained with the first 14 residues of S3, but this periodicity disappeared towards the extracellular side of this transmembrane sequence. In both helical stretches, tryptophan-tolerant positions are clustered on approximately half the alpha-helix surface, as if the sidechains are exposed to the hydrocarbon region of the lipid bilayer. These results, combined with an analogous study of S2 (Monks, S., D.J. Needleman, and C. Miller. 1999. J. Gen. Physiol. 113:415-423), locate S1, S2, and S3 on the lipid-facing periphery of K(v) channels.

Show MeSH
Related in: MedlinePlus