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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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Effects of Asn substitutions at certain Trp-tolerant positions. Two-electrode voltage clamp recordings (A) and voltage-activation curves (B) of the indicated mutants displayed as in Fig. 3. Electrophysiological parameters are reported in Table .
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Figure 6: Effects of Asn substitutions at certain Trp-tolerant positions. Two-electrode voltage clamp recordings (A) and voltage-activation curves (B) of the indicated mutants displayed as in Fig. 3. Electrophysiological parameters are reported in Table .

Mentions: We worried that this uninterrupted string of five low-impact residues might be an artifact arising from the nature of the wild-type residues in this sequence. Since four of these are large or aromatic, it is possible that substitution by Trp is inherently nondisruptive even at a protein-packed interface. Previous work on S2 had shown that in such a case, substitution by Asn, a small, polar residue could be used to buttress an argument for a lipid-exposed position (Monks et al. 1999). We therefore changed the four nonproline residues individually to Asn (Fig. 6, Table ). By this criterion, one of these residues, I321, scores as low impact and one, Y323, as high impact. The two remaining residues, I320 and F324, are ambiguously near the borderline and cannot be reliably scored. Taking these adjustments into account, the S3 scan identifies 2 ambiguous, 8 low-impact, and 12 high-impact positions.


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

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

Effects of Asn substitutions at certain Trp-tolerant positions. Two-electrode voltage clamp recordings (A) and voltage-activation curves (B) of the indicated mutants displayed as in Fig. 3. Electrophysiological parameters are reported in Table .
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Effects of Asn substitutions at certain Trp-tolerant positions. Two-electrode voltage clamp recordings (A) and voltage-activation curves (B) of the indicated mutants displayed as in Fig. 3. Electrophysiological parameters are reported in Table .
Mentions: We worried that this uninterrupted string of five low-impact residues might be an artifact arising from the nature of the wild-type residues in this sequence. Since four of these are large or aromatic, it is possible that substitution by Trp is inherently nondisruptive even at a protein-packed interface. Previous work on S2 had shown that in such a case, substitution by Asn, a small, polar residue could be used to buttress an argument for a lipid-exposed position (Monks et al. 1999). We therefore changed the four nonproline residues individually to Asn (Fig. 6, Table ). By this criterion, one of these residues, I321, scores as low impact and one, Y323, as high impact. The two remaining residues, I320 and F324, are ambiguously near the borderline and cannot be reliably scored. Taking these adjustments into account, the S3 scan identifies 2 ambiguous, 8 low-impact, and 12 high-impact positions.

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