Lee SY et al. (2009), Two separate interfaces between the voltage sen...

XB-ART-39810

PLoS Biol 2009 Mar 03;73:e47. doi: 10.1371/journal.pbio.1000047.

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Two separate interfaces between the voltage sensor and pore are required for the function of voltage-dependent K(+) channels.

Lee SY , Banerjee A , MacKinnon R .

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Voltage-dependent K(+) (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate.

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Species referenced: Xenopus laevis
Genes referenced: kcna2 kcnb1 kcnh2 vsig1

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	Figure 1. Statistical Coupling Analysis of the Kv Family(A) A representative alignment of Kv subfamilies out of the full sequence alignment of the Kv family that was used for the analysis. The sequences include Kv 1.2 (NCBI Entrez Protein Database [http://www.ncbi.nlm.nih.gov/sites/entrez?db=protein] GI: 52000923), Kv 2.1 (GI: 47523520), Kv 3.1 (GI: 5817540), Kv 4.3 (GI: 2935434), Kv 5.1 (GI: 20070166), Kv 6.2 (GI: 82997534), Kv 7.5 (GI: 8132997), Kv 8.1 (GI: 20381121), Kv 9.1 (GI: 112821679), Kv 10.1 (GI: 22164088), BK (GI: 157776), HERG (GI: 4557729), HCN (GI: 5734516), KvAP (GI:14601099). Conserved residues that were used to guide the alignment are highlighted in yellow, and their degrees of conservation are shown as frequency (%) in red (the frequency of the most common amino acid at the indicated position in the full sequence alignment) at the bottom of the alignment. The residue number corresponds to that of Kv 1.2.(B) A matrix representation of all pair-wise coupling values (termed ΔΔGstat [14]) for 95 perturbations. The column and row show positions and perturbations from N to C terminus, respectively. The coupling values range from 0.5 kT* (blue) to 2 kT* (red) in units of “statistical energy” [12].(C) Two-dimensional clustering of the matrix shows a group of positions and perturbations that are clustered together by the similarity of coupling patterns.(D) Focused clustering of an initially clustered group (box in (C)) reveals the final cluster of 49 residues and 22 perturbations.
	Figure 2. Mapping SCA-Derived, Co-Evolved Residues onto the Structure of the Paddle Chimera (PDB ID: 2R9R)The co-evolved residues identified in Figure 1D are shown in van der Waals surface (light brown). K+ ions are shown as green spheres. Stereo-views of the paddle chimera are shown in (A): looking from the extra-cellular side; in (B): from the side;, and in (C): from the intra-cellular side. The co-evolved residues form a structurally connected network where the inner membrane leaflet part of the voltage sensor is coupled to the pore via the S4-S5 linker. The co-evolved residues that are involved in the interfaces between the voltage-sensor and the pore are shown in red in (A), (B), and (C).(D) Surface representations of the interfaces between the voltage sensor and the pore in the structure of the paddle chimera. Shown separately are the voltage sensor from a single subunit and the tetrameric pore domain. The contact surfaces corresponding to the co-evolved interfaces between the S1 and the pore helix and the S4-S5 linker and S6 are colored red. The contact surfaces between the S4 and S5 are colored blue. The contact surfaces comprise residues that are within 4.0 Å distance.
	Figure 3. Comparison of Mutational Data with SCA ResultsHighly conserved residues (over 80 % identity in the multiple sequence alignment) are highlighted in yellow and are likely SCA-insensitive (see text). Moderately conserved residues (40–80% identity) are highlighted in gray. The impacts of mutations to Ala [17], Trp [22,23], or other residues within the voltage sensor are shown under the alignment. The mutations in the “Other” row are as follows: Arg or Leu or Pro on I173 and S176 on S1 [40], Val on T184 on S1; Asn on S2 and S3 [22,23]; Gln on S4 [41,42]; Val on L314 (S4) [43]. Blue filled circles represent mutations that cause a large perturbation in gating energy (>1 kcal/mol), whereas open circles indicate small perturbation in gating energy (<1 kcal/mol). Red filled circles represent no expression and green filled circles indicate expressed but non-conducting mutations. Half-red circle represents no expression in Shaker but small perturbation in Kv2.1. The cyan bar represents the Kv2.1 part of the paddle chimera. Mutations that are enclosed with dotted lines were produced and analyzed in this study. The numbering is based on rat Kv1.2.
	Figure 4. Voltage-Dependent Gating Properties of S1–Pore Interface Tryptophan Mutants(A) Close-up view of the S1–pore interface in the structure of paddle chimera (PDB ID: 2R9R). Pore helices are shown in gray and S1 helices are shown in cyan. Co-evolved residues (green) are shown in ball-and-stick representation. The residues corresponding to the ones mutated in Shaker have been labeled in blue.(B) Shown are mean ionic currents at 5-mV increments, normalized and fitted to a two-state Boltzmann (see Materials and Methods). The corresponding residues in Kv1.2 are given in parentheses.
	Figure 5. Chemistry of Interaction at the S1–Pore Interface(A and B) Comparison of the S1–pore interface between paddle chimera (PDB ID: 2R9R) and MlotiK1 (PDB ID: 3BEH). (A) Stereoview of superposition of the paddle chimera (cyan) and MlotiK1 (red) structures viewed from the extracellular side. Superposition was done using residues 337–344, 359–389 in paddle chimera, and the corresponding residues 142–149 and 164–194 in MlotiK1.(B) Close-up view of the S1–pore interfaces. Pore helices are shown in gray and S1 helices are shown in cyan. Co-evolved residues (green) are shown in ball-and-stick representation.(C) Voltage-dependent gating properties of T248S Shaker. The corresponding residue in Kv1.2 is denoted in parentheses. On the left are shown families of ionic currents at 5-mV increments, and on right, the normalized currents are fitted to a two-state Boltzmann. The holding voltage was −80 mV, tail voltage was −60 mV.
	Figure 6. Representative Experiments Showing the Functional Effects of Disulfide Cross-Linking of S1–Pore Interface in KvAPCurrent traces were elicited after depolarization to positive voltages before (black) or after (blue) the addition of BaCl2 to the internal side, or before (black) and after (red) addition of CTX to the external side.(A and B) Air-oxidized 47Cys/183Cys KvAP.(C and D) DTT reduced 47Cys/183Cys (See Materials and Methods). Every experiment is from a separately painted membrane.
	Figure 7. Model of Force Transmission in Kv ChannelsShown is a complete monomeric subunit of the paddle chimera (PDB ID: 2R9R) and the pore region only from the adjacent subunit with which the S1–pore interface is formed. Components of the voltage sensor that are mobile along the transmembrane axis are shown in red, and components that are static along the transmembrane axis are shown in blue. The pore regions are shown in gray. The side chains of the residues forming the S1–pore interface are shown in blue ball and stick rendition, and the potassium ions lining the filter region are shown as cyan spheres. The blue arrow denotes the location of the S1–pore interface with respect to the entire protein structure. The red arrows indicate the putative direction of force transmission from the voltage sensor onto the S4-S5 linker and from the S4-S5 linker to the pore.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase