Monks SA et al. (1999), Helical structure and packing orientation of th...

XB-ART-13450

J Gen Physiol 1999 Mar 01;1133:415-23.

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Helical structure and packing orientation of the S2 segment in the Shaker K+ channel.

Monks SA , Needleman DJ , Miller C .

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Six transmembrane segments, S1-S6, cluster around the central pore-forming region in voltage-gated K+ channels. To investigate the structural characteristics of the S2 segment in the Shaker K+ channel, we replaced each residue in S2 singly with tryptophan (or with alanine for the native tryptophan). All but one of the 23 Trp mutants expressed voltage-dependent K+ currents in Xenopus oocytes. The effects of the mutations were classified as being of low or high impact on channel gating properties. The periodicity evident in the effects of these mutations supports an alpha-helical structure for the S2 segment. The high- and low-impact residues cluster onto opposite faces of a helical wheel projection of the S2 segment. The low-impact face is also tolerant of single mutations to asparagine. All results are consistent with the idea that the low-impact face projects toward membrane lipids and that changes in S2 packing occur upon channel opening. We conclude that the S2 segment is a transmembrane alpha helix and that the high-impact face packs against other transmembrane segments in the functional channel.

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Species referenced: Xenopus laevis
Genes referenced: kcnj1 tbx2

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	Figure 2. Gating of tryptophan-substituted S2 mutants. (A) Two-electrode voltage clamp recordings of wild-type channels expressed in Xenopus oocytes. Currents in response to a standard pulse protocol were recorded in KD98 medium. (B and C) Similar recordings taken for mutations I287W and E293W. Because of the left shift in activation and slowed kinetics of E293W, test potentials ranged from –80 to +30 mV, and pulse duration was increased to 100 ms. (D) Voltage-activation curves for wild-type, I287W, and E293W mutant channels, calculated from tail-current analysis. Solid curves are Boltzmann fits to the equilibrium activation data. Scale bars represent 10 ms, 1 μA in all data panels.
	Figure 3. Gating parameters of S2 tryptophan scan. Changes of empirical gating parameters brought about by point Trp mutations are displayed vs. residue number. (A) Intrinsic free energy of opening with respect to wild type, ΔΔGo. The shaded region (\|ΔΔGo\| < 1 kcal/mol) represents the range of values defining tolerant residues. (B) Mutant-to-wild type ratio of activation times, to, determined at the half point on the activation curve, Vo. (C) Mutant-to-wild type ratio of deactivation time constant at −70 mV, τd.
	Figure 4. Small polar residues are tolerated on the low-impact face. Two-electrode voltage clamp recordings (A) and activation curves (B) of channels carrying the Asn substitutions shown. Conditions and scale bars are as in Fig. 2. Electrophysiological parameters are reported in Table II.
	Figure 5. Structural verification of a previous tryptophan scan. The KcsA structure is represented in RASMOL images, showing residues identified as tryptophan tolerant in ROMK1 (Choe et al., 1995) mapped onto equivalent positions in KcsA. (Left) Ribbon diagrams, with the second transmembrane helix, M2, in red and the selectivity filter, GYG, in cyan. (Upper image) Longitudinal view, with external side of the pore oriented towards the top of the figure. (Lower image) View along the pore axis from the cytoplasmic side. Yellow spheres represent β carbons of residues equivalent to those scored as tolerant of tryptophan substitution in ROMK1. (Right) Space-filling image in longitudinal view, showing Trp-tolerant M2 side chains in yellow; natural tryptophan and tyrosine residues are colored violet to indicate the lipid bilayer-water interface. Residues in ROMK1 corresponding to KcsA residues were lined up according to Doyle et al. (1998).
	Figure 6. Lateral asymmetry between high-impact and tolerant residues. A helical wheel projection of the S2 segment is shown with residues scored as either not expressed (shaded triangle), high impact (shaded circles), or tolerant (open circles), according to criteria described in the text.
	Figure 7. Sequence variability in S1 and S3. BLAST searches were carried out on S1 and S3, as for S2 (Fig. 1 C), using Shaker S1 and S3 as query sequences. Conserved (circled, bold) and variable residues are distinguished as in Fig. 1 C.

References [+] :

Altschul, Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. 1997, Pubmed