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 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.

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