Figure 6. A possible model-independent structural interpretation of the fluorescence data. (Left) A hypothetical model of the BK VSD helices at rest. It is based on our previous illustration of BK VSD helices S0, S3, and S4 (Pantazis et al., 2010b), with the addition of S1 and S2. Helices S1–S4 are arranged in a counterclockwise bundle with the structure of their homologous helices in the KV1.2–2.1 crystal structure (Protein Data Bank accession no. 2R9R; Long et al., 2007). S0, modeled as an ideal α helix, is positioned according to the most recent information from disulfide cross-linking efficiency (Liu et al., 2010). S3 and S4 are connected by a short extracellular helix–loop–helix structure as inferred previously by bimane fluorescence scanning (Semenova et al., 2009). The native S4 Trp, W203, is shown in violet, and hypothetical positions for fluorophore conjugation cysteines in S1 (135) and S2 (145) are also indicated. The extracellular flank of S4 is relatively close to those of S0, S1, and S2, so that fluorophores labeling S0 (Pantazis et al., 2010b), S1 (Fig. 2), and S2 (Fig. 3) are efficiently quenched by W203 (Fig. 5 A). (Right) Membrane depolarization induces voltage-sensing residue R213 (S4) to move outward and D186 (S3) inward (Ma et al., 2006), causing the rearrangement of S3 and S4 with respect to the electric field (Savalli et al., 2006; Pantazis et al., 2010a). This results in a relative rearrangement between the voltage-sensing S3/S4 and S0 and S1, lifting the quenching effect of W203 to fluorophores labeling S0 (Pantazis et al., 2010b) or S1 (Figs. 2 and 5 A). S2 is shown to undergo a tilting motion upon depolarization, hypothetically induced by its voltage-sensing residues D153 and R167 (Ma et al., 2006; Pantazis et al., 2010a), causing its extracellular flank to diverge from W203 (S4) and move toward S1, consistent with the fluorescence data in this work, whereby an S2-conjugated fluorophore (position 145) is quenched by W203 (S4; violet) at rest (Figs. 3 and 5 A), whereas an S1-conjugated fluorophore (position 135) is quenched by an introduced Trp near S2 upon activation (Figs. 4, E–L, and 5, B and C). The image was made with PyMOL 0.99 (DeLano Scientific).
Figure 6.

A possible model-independent structural interpretation of the fluorescence data. (Left) A hypothetical model of the BK VSD helices at rest. It is based on our previous illustration of BK VSD helices S0, S3, and S4 (Pantazis et al., 2010b), with the addition of S1 and S2. Helices S1–S4 are arranged in a counterclockwise bundle with the structure of their homologous helices in the KV1.2–2.1 crystal structure (Protein Data Bank accession no. 2R9R; Long et al., 2007). S0, modeled as an ideal α helix, is positioned according to the most recent information from disulfide cross-linking efficiency (Liu et al., 2010). S3 and S4 are connected by a short extracellular helix–loop–helix structure as inferred previously by bimane fluorescence scanning (Semenova et al., 2009). The native S4 Trp, W203, is shown in violet, and hypothetical positions for fluorophore conjugation cysteines in S1 (135) and S2 (145) are also indicated. The extracellular flank of S4 is relatively close to those of S0, S1, and S2, so that fluorophores labeling S0 (Pantazis et al., 2010b), S1 (Fig. 2), and S2 (Fig. 3) are efficiently quenched by W203 (Fig. 5 A). (Right) Membrane depolarization induces voltage-sensing residue R213 (S4) to move outward and D186 (S3) inward (Ma et al., 2006), causing the rearrangement of S3 and S4 with respect to the electric field (Savalli et al., 2006; Pantazis et al., 2010a). This results in a relative rearrangement between the voltage-sensing S3/S4 and S0 and S1, lifting the quenching effect of W203 to fluorophores labeling S0 (Pantazis et al., 2010b) or S1 (Figs. 2 and 5 A). S2 is shown to undergo a tilting motion upon depolarization, hypothetically induced by its voltage-sensing residues D153 and R167 (Ma et al., 2006; Pantazis et al., 2010a), causing its extracellular flank to diverge from W203 (S4) and move toward S1, consistent with the fluorescence data in this work, whereby an S2-conjugated fluorophore (position 145) is quenched by W203 (S4; violet) at rest (Figs. 3 and 5 A), whereas an S1-conjugated fluorophore (position 135) is quenched by an introduced Trp near S2 upon activation (Figs. 4, E–L, and 5, B and C). The image was made with PyMOL 0.99 (DeLano Scientific).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal