Pantazis A and Olcese R (2012), Relative transmembrane segment rearrangements d...

XB-ART-46359

J Gen Physiol 2012 Aug 01;1402:207-18. doi: 10.1085/jgp.201210807.

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Relative transmembrane segment rearrangements during BK channel activation resolved by structurally assigned fluorophore-quencher pairing.

Pantazis A , Olcese R .

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Voltage-activated proteins can sense, and respond to, changes in the electric field pervading the cell membrane by virtue of a transmembrane helix bundle, the voltage-sensing domain (VSD). Canonical VSDs consist of four transmembrane helices (S1-S4) of which S4 is considered a principal component because it possesses charged residues immersed in the electric field. Membrane depolarization compels the charges, and by extension S4, to rearrange with respect to the field. The VSD of large-conductance voltage- and Ca-activated K(+) (BK) channels exhibits two salient inconsistencies from the canonical VSD model: (1) the BK channel VSD possesses an additional nonconserved transmembrane helix (S0); and (2) it exhibits a "decentralized" distribution of voltage-sensing charges, in helices S2 and S3, in addition to S4. Considering these unique features, the voltage-dependent rearrangements of the BK VSD could differ significantly from the standard model of VSD operation. To understand the mode of operation of this unique VSD, we have optically tracked the relative motions of the BK VSD transmembrane helices during activation, by manipulating the quenching environment of site-directed fluorescent labels with native and introduced Trp residues. Having previously reported that S0 and S4 diverge during activation, in this work we demonstrate that S4 also diverges from S1 and S2, whereas S2, compelled by its voltage-sensing charged residues, moves closer to S1. This information contributes spatial constraints for understanding the BK channel voltage-sensing process, revealing the structural rearrangements in a non-canonical VSD.

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Species referenced: Xenopus
Genes referenced: acta4 tbx2

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	Figure 1. BK channel topology and assembly. (A) Membrane topology of a BK α subunit (Wallner et al., 1996; Meera et al., 1997). Voltage-sensing charged residues D153 and R167 (S2), D186 (S3), and R213 (S4) (Ma et al., 2006) are indicated by their charge polarity. (B) The sequences of the human BK extracellular S1–S2 (S134–K146) and S3–S4 (N200–S202) linkers, which include positions pertinent to this work: cysteine substitutions for fluorescent labeling (in red: S135C and Y145C) and Trp introductions for fluorophore quenching (in blue: I138W and C141W; note that C141 is normally substituted to serine to prevent fluorophore conjugation). The native Trp at the extracellular tip of S4 (W203) is also in blue. (C) Top view of a potassium channel transmembrane domain (KV1.2–2.1 chimera; Protein Data Bank accession no. 2R9R; Long et al., 2007) with the addition of S0 ideal α helices to resemble BK channels. The pore domain (green) contains a K+ ion and is surrounded by four VSD helix bundles (orange). (D) Suggested packing arrangement of the BK VSD helix bundle (Liu et al., 2010).
	Figure 2. Voltage-dependent ΔF reported from the S1 extracellular flank is abolished by substitution of W203 at the extracellular flank of S4. (A) Illustration of the BK channel construct used; only the VSD transmembrane helices of one subunit are shown. A unique cysteine was substituted at the extracellular flank of S1 (S135C) and covalently labeled with fluorophore MTS-TAMRA to resolve conformational rearrangements from this region. (B) Voltage-pulse protocol, characteristic evoked K+ currents (black), and simultaneously recorded MTS-TAMRA fluorescence (red) from the BK channel construct illustrated above. ΔF voltage dependence: V0.5 = −81 ± 4 mV and z = 0.68 ± 0.04 e0. Conductance–voltage dependence: V0.5 = −29 ± 7 mV and z = 0.86 ± 0.03 e0; n = 11. (C and D) As in A and B, for channels with the additional mutation W203V to remove the native Trp at the extracellular portion of S4. MTS-TAMRA fluorescence traces are in blue. Note that the fluorescence scale is 10 times that for wild-type (WT) channels. ΔF voltage dependence: V0.5 = −97 ± 2 mV and z = 0.84 ± 0.04 e0. Conductance–voltage dependence: V0.5 = 32 ± 2 mV and z = 0.99 ± 0.06 e0; n = 5. (E) Mean fitted ΔFtotal/F0 signal, normalized for fitted maximal conductance for WT (red; ΔFtotal/F0/Gmax = 1.8 ± 0.42%/mS) and W203V (blue; ΔFtotal/F0/Gmax = 0.41 ± 0.09%/mS) channels. Mutation W203V attenuated the ΔF reported by MTS-TAMRA from position 135 by ≈85%. (F) Mean, normalized ΔF from WT (red squares) or W203V (blue diamonds) BK channels labeled at position 135 with MTS-TAMRA. Error bars represent SEM.
	Figure 3. W203, outside S4, is the principal quencher of ΔF reported from S2. (A) Illustration of the BK channel construct used; only the VSD transmembrane helices of one subunit are shown. A unique cysteine was substituted at the extracellular flank of S2 (Y145C) and covalently labeled with fluorophore TMRM to resolve conformational rearrangements from this region. (B) Voltage-pulse protocol, characteristic evoked K+ currents (black), and simultaneously recorded TMRM fluorescence (red) from the BK channel construct illustrated above. ΔF voltage dependence: V0.5 = −82 ± 5 mV and z = 0.74 ± 0.05 e0. Conductance–voltage dependence: V0.5 = −26 ± 10 mV and z = 1.2 ± 0.11 e0; n = 14. (C and D) As in A and B, for channels with the additional mutation W203V to remove the Trp residue at the extracellular portion of S4. TMRM fluorescence traces are in blue. Note that the fluorescence scale is 10 times that for WT channels. ΔF voltage dependence: V0.5 = −58 ± 9 mV and z = 0.57 ± 0.05 e0. Conductance–voltage dependence: V0.5 = 10 ± 2 mV and z = 0.86 ± 0.1 e0; n = 8. (E) Mean fitted ΔFtotal/F0 signal, normalized for fitted maximum conductance to normalize for channel expression for WT (red; ΔFtotal/F0/Gmax = 0.51 ± 0.17%/mS) and W203V (blue; ΔFtotal/F0/Gmax = 0.05 ± 0.007%/mS) channels. Mutation W203V attenuated the ΔF reported by TMRM from position 145 by ≈90%. (F) Mean, normalized ΔF from WT (red squares) or W203V (blue diamonds) BK channels labeled at position 145 with TMRM. Error bars represent SEM.
	Figure 4. Quenching the S1-conjugated fluorescent label with introduced tryptophans in the S1–S2 linker. (A) Illustration of the BK channel construct used; only the extracellular flanks of S1, S2, and S4 are shown. The intervals in the S1–S2 linker (S134–K146; Fig. 1 B) represent its residues. As in Fig. 2 A, a unique cysteine was substituted at the extracellular flank of S1 (S135C) and covalently labeled with fluorophore MTS-TAMRA. (B) Voltage-pulse protocol and characteristic evoked K+ currents (black) from the BK channel construct illustrated above, as in Fig. 2 B. (C) MTS-TAMRA fluorescence traces simultaneously recorded with the current traces above, which were shown in Fig. 2 to be dependent on W203 (at S4). (D) Mean, normalized ΔF, as in Fig. 2 F. (E–H) As in A–D, for S135C, MTS-TAMRA–labeled channels with the additional mutation C141W to introduce a Trp at the S1–S2 linker, near S2. Note the additional voltage-dependent quenching component. Conductance–voltage dependence: V0.5 = −6 ± 5 mV and z = 0.79 ± 0.04 e0; n = 6. Note that in all other constructs, C141 has been substituted by serine to prevent fluorophore labeling. (I–L) As in E–H, with the additional mutation W203V to remove the native Trp at the extracellular flank of S4. Note that the fluorescence transients observed in G are abolished. Conductance–voltage dependence: V0.5 = 61 ± 6 mV and z = 0.99 ± 0.04 e0; n = 13. The small unquenching component observed at hyperpolarized potentials is probably the same as that observed in the S135C–W203V channel (Fig. 2 D). (M–O) As in A–C, for S135C, MTS-TAMRA–labeled channels with the additional mutation I138W to introduce a Trp residue near the fluorophore position. The apparent lack of ΔF could indicate lack of MTS-TAMRA conjugation or static (voltage-independent) quenching of the fluorophore by the nearby W138. (P) An expansion of the time and fluorescence scales for the 120-mV depolarization in O to better demonstrate the transient, but consistently observed, unquenching of the fluorescence upon sufficient depolarization, confirming the fluorescent labeling of C135.
	Figure 5. A photochemical interpretation of the observed fluorescence deflections. (A) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4), as in Figs. 2 and 4 (A–D). At rest, the fluorophore is efficiently quenched by the Trp at S4 (W203), so that the reported fluorescence is relatively dim. Upon depolarization, the quenching efficiency of W203 decreases, so that overall fluorescence emission is increased and a positive ΔF is observed. The same interpretation was previously applied for TMRM fluorophore labeling the extracellular portion of S0 (Pantazis et al., 2010b) and can also account for the ΔF signals from TMRM-labeling S2, as their amplitude is also strongly attenuated by mutation W203V (Fig. 3). (B) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the native W203 (S4) and introduced W141 (near S2; Fig. 4, E–H). At rest, W203 quenches the fluorophore (as in A), whereas W141 quenches the fluorophore relatively less, so that the overall fluorescence detected is at an intermediate level. Upon depolarization, W203 quenching is lifted, generating positive ΔF, as in A. At the same time, W141 approaches and quenches the fluorophore. It seems that the W141 is a more efficient quencher than W203, as the overall fluorescence level at the depolarized state is dimmer than when at rest. However, W203 departure appears to have faster kinetics than W141 quenching, so it is observed as an upward transient deflection. Upon membrane repolarization, the two quenching processes are reversed: W203 returns to quench the label, so that overall fluorescence becomes transiently dimmer, whereas W141 departs, reducing its quenching efficiency. (C) A representative fluorescence trace recorded upon a 50-ms depolarization to 60 mV from channels labeled with MTS-TAMRA at position 135, at the S1 extracellular flank, in the presence of the introduced W141 (near S2), with additional mutation W203V to remove the native W203 (S4; as in Fig. 4, I–L). The fluorescent label is apparently unquenched at rest, as W203 has been substituted. Upon depolarization, W141 approaches and quenches the fluorophore, a process reported as a negative ΔF. Upon membrane repolarization, W141 departs, reducing its quenching efficiency. In agreement with the ΔF interpretation in A and B, the substitution of W203 resulted in the lack of observable depolarization-induced positive ΔF.
	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).

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