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Figure 1. Mechanism of bimane fluorescence quenching by tryptophan. (A) Schematic of the electron transfer from tryptophan to bimane upon bimane excitation. The arrow represents donation of an electron from tryptophan to excited bimane. (B) Emission spectra of a 100 μM solution of bimane in increasing concentrations of tryptophan, indicated by the arrow. The reduction of the peak intensity is caused by tryptophan quenching. (C) Stern-Volmer analysis of the data in B. The straight line is a fit to the data with Eq. 3; the quenching constant K = 0.083 mMâ1.
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Figure 2. Characteristics of the C481 mutation. (A) Location of residues C481 and I600 in a homology model of the CNGA1 carboxy-terminal region based on the x-ray crystal structure of HCN2. (B) Cyclic nucleotide doseâresponse relations of unmodified and modified channels. Open squares are the cGMP doseâresponse data before modification. The smooth curve is a fit of Eq. 1 with Kd = 40 μM and n = 2.2. The open circle is the response to 16 mM cAMP, before modification. Closed squares are the cGMP doseâresponse data of the same patch after complete modification with 250 μM bimane maleimide in the presence of 2 mM cGMP. The data were fit with Eq. 1, with Kd = 4.0 μM and n = 0.84. The closed circle is the response to 16 mM cAMP after modification. (C) Time course of C481 modification by 400 μM bimane maleimide. Application of bimane maleimide in the absence or in the presence of 2 mM cGMP is indicated by the gray and black bars, respectively. Modification, as measured by the potentiation of the current in 16 mM cAMP relative to the current in 2 mM cGMP, occurs exceedingly slowly in closed channels (open squares), but proceeds with a rate constant of 29 ± 3.9 Mâ1sâ1 (n = 3) in open channels (solid squares). The smooth line is a fit of Eq. 2 with a rate k = 0.0098 sâ1 and m = 2.
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Figure 3. Spectroscopy of bimane-labeled C481 channels in patches. (A) Fluorescence image of an inside-out patch expressing C481 channels after modification with bimane maleimide. The fluorescence image is superimposed on a transmitted light image of the patch pipette. This patch contained 11 nA of cGMP-activated current, at 30 mV. (B) Emission spectrum of bimane in a patch similar to A (continuous black trace). The continuous red trace is the normalized emission spectrum of a solution of bimane maleimideâmodified BSA. The dashed trace represents the emission spectrum of bimane maleimide in aqueous solution. (C) Time course of the amplitude of current elicited by 30-mV depolarizing pulses during application of 2 mM cGMP. Application of tryptophan produces a 12.7 ± 0.2% (n = 3) reversible blockade of current. (D) Emission spectra of the patch in C acquired at the times indicated by numbers. Tryptophan produces a 40% reduction in fluorescence that is completely reversible (trace 3, dotted line). There is no difference in the fluorescence of open channels (traces at times 2 and 4) and closed channels (traces at times 1 and 5).
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Figure 4. Quantification of bimane fluorescence quenching by applied tryptophan in solution. (A) Stern-Volmer plot analysis of a bimane maleimideâmodified patch expressing C481 channels. Tryptophan quenching is more effective in the open state than in the closed state. The curves are fits of Eq. 4 with values: (closed) f = 0.15, K1 = 1.72 mMâ1, and K2 = 0.026 mMâ1; (open) f = 0.15, K1 = 3.73 mMâ1, and K2 = 0.025 mMâ1. The inset shows the patch emission spectra at different concentrations of tryptophan. (B) Quenching characteristics of I600C channels. Quenching is more effective in the closed state than in the open state. The curves are fits of Eq. 4 with values: (closed) f = 0.20, K1 = 1.092 mMâ1, and K2 = 0.0052 mMâ1; (open) f = 0.20, K1 = 0.55 mMâ1, and K2 = 0.0061 mMâ1. (C) Tryptophan quenching of a bimane-labeled inside-out patch containing cysteineless CNGA1 channels in the presence of 2 mM cGMP (open circles) and in the absence of nucleotides (closed circles). Smooth curves are fits of Eq. 3 with values: (closed) K = 0.11 mMâ1; (open) K = 0.096 mMâ1. Quenching of bimane maleimide in solution is represented by the triangles. The quenching constant K is 0.083 mMâ1.
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Figure 5. Components of patch fluorescence. (A) Fluorescence emission image of a patch expressing C481 channels modified with bimane maleimide without NEM block. The yellow line indicates the region used in the scan in C. The exposure to the 405-nm laser was 300 ms. (B) A patch expressing C481 channels modified after cysteine block with 1 mM NEM for 4 min in the closed state. The exposure was 1 s, with eight times the laser intensity used in A. (C) Line scans of the images in A and B.
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Figure 6. Location and quenching effects of tryptophan mutants. (A) Homology model of one subunit of the CNGA1 channel, showing the location of residues that were mutated to tryptophan. The numbering is that of the CNGA1 sequence. Distances to the α-carbon of C481 are indicated by green lines and are given in angstroms. (B) Quantification of the degree of state-dependent quenching of bimane in C481 produced by individual tryptophan residues. The bars represent the mean. The error bars are the SEM.
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Figure 7. Fluorescence changes in tryptophan mutants. (A) Currents from D588W channels after bimane modification upon application of 2 mM cGMP elicited by pulses to 30 mV. (B) Spectra taken at the times indicated by the numbers in A. (C) Currents from A461W channels after bimane modification upon application of 2 mM cGMP elicited by pulses to 30 mV. (D) Spectra taken at the times indicated by the numbers in C. Red traces are the open channel emission spectra, and black traces are the closed channel emission spectra. Quenching of peak fluorescence in A461W channels upon opening is â¼18%.
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Figure 8. The cyclic nucleotide dependence of bimane quenching suggests that fluorescence reports a conformational change associated with channel opening. (A) Average cyclic nucleotide doseâresponse relations of unmodified and modified channels. Open squares are the cGMP doseâresponse data before modification. The smooth curve is a fit of Eq. 1 with Kd = 30 μM and n = 2.0. The open circle is the response to 16 mM cAMP, before modification. Closed squares are the cGMP doseâresponse data after modification. The data were fit with Eq. 1, with Kd = 15 μM and n = 1.0. The closed circle is the response to 16 mM cAMP after modification. (B) cGMP doseâresponse relation of bimane quenching in modified A461W channels. The data were fit with Eq. 1 with a maximum value of 0.15, and the same parameters used for the cGMP doseâresponse relation of modified channels in A. The red symbol is the quenching observed when the channels are opened by 16 mM cAMP and was corrected for the 3.1 ± 0.6% (n = 3) quenching of bimane produced by 16 mM cAMP alone.
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Figure 9. Fluorescence changes in A461W-C481 channels modified with 250 μM monobromobimane. After modification, spectra were acquired in the absence of cyclic nucleotide (closed state, black traces) or in the presence of 2 mM cGMP (open state, red traces) in the order indicated by the numbers in boxes. The amount of quenching in this experiment was 14.8%.
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