Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel

Abstract

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.

FIGURE 1. A biochemical assay to determine the extra- and intracellular residues of KvAP expressed in live E. coli

A, selective radiolabelling of KvAP. Autoradiogram of [³⁵S]radiolabelled proteins extracted from cells harbouring KvAP-pET28a (lanes 1-3) or the empty vector (lanes 4-6), before (lanes 1 and 4) and after (lanes 2 and 5) induction with IPTG; lanes 3 and 6 contain proteins from rifampicin-treated cells. Position of the KvAP band is shown with an arrowhead. B, structures of reagents used in the accessibility assay. C, gel-shift assay validated with representative cysteine positions, depicted in the schematic. Cells expressing the indicated radiolabelled KvAP mutants were pre-treated with 5 mM AMS plus or minus 2 % chloroform, followed by mal-PEG reaction, as indicated above each gel (for details see Experimental Procedures). Each set of gel images represents samples from the same experiment and gel, edited together for clarity. Positions of unmodified (∼25 KDa) and mal-PEG modified KvAP (∼33 kDa) are indicated with arrowheads.

FIGURE 2. Extra- and intra-cellular accessibility of cysteines at the membrane-aqueous borders of S1 and S2

A and B, gel-shift assay was performed on the indicated cysteine mutants as described in Fig. 1C using AMS alone (A) or AMS plus chloroform (B); lanes T and C of each panel represent data from test (pre-treated with AMS or AMS/chloroform) and control (no pre-treatment) experiments; representative data from the same experiment and gel, edited together for clarity. The top band (indicated by arrowhead) on each gel corresponds to PEGylated KvAP. C, cartoon topology of KvAP residues 21 to 85 (helices based on the crystal structure of the isolated VSD (1ORS), summarising our accessibility data (see Supplementary Figure 6 for a full scan), compared with previous studies; filled circles, accessible; open circles, inaccessible; grey circles, untested. For Shaker accessibility data KvAP equivalents are shown.

FIGURE 3. Extra- and intra-cellular accessibility of cysteines engineered into the S3 segment and its flanking regions

A and B, gel-shift assay showing extra- and intra-cellular accessibilities (details are as described for Fig. 2A & B). (C) Summary of accessibility data, compared with other works, presented as in Fig. 2C. D, helical wheel diagram of the S3b helix showing the accessible residues (filled circles) on one face of the helix.

FIGURE 4. Extra- and intra-cellular accessibility of cysteines engineered into the S4 segment and its flanking regions

A and B, gel-shift assay showing extra and intra-cellular accessibilities (details are as described for Fig. 2A & B). C, summary of accessibility data, compared with other works, presented as in Fig. 2C. For Shaker accessibility data (last cartoon) KvAP equivalents are shown; triangles represent accessible (▲, extracellular) and inaccessible (△, extracellular; ▽, intracellular; ○, both sides) residues where tested.

FIGURE 5. Summary of aqueous accessibility study on the KvAP voltage-sensing domain

A, cartoon topology of KvAP residues 21 to 148 based on the crystal structure of the isolated KvAP voltage-sensor (1ORS), depicting the water accessible residues (data from Figs. 2 to 4); red circles, extracellular; blue circles, cytosolic; white circles, inaccessible; grey circles, untested. B, structure of the VSD, showing the water inaccessible membrane region (grey) separating the extracellular (red) and intracellular (blue) solutions; the dashed lines represent the approximate boundaries. Accessible regions of the protein are colour coded: red, extracellular; blue, intracellular. C, surface representation of S3 showing highly accessible regions of S3a and S3b, with the helices superimposed; colour code and orientation is as for B.