|
Figure 1. Structural comparison of heterodimeric, TM287/288 (A–C), and homodimeric, Sav1866 (D–F), ABC transporters. (A and D) Cartoon (left) and surface (right) representations of TM287/288 (A; PDB accession no. 3QF4) and Sav1866 (D; PDB accession no. 2ONJ) viewed along expected membrane plane (boundaries indicated by gray lines). Transmembrane domains equivalent to CFTR’s transmembrane domain 1 and transmembrane domain 2 are forest green and blue, respectively; NBDs equivalent to CFTR’s NBD1 and NBD2 are lime and cyan, respectively. Walker A motifs are shown in red, Walker B in orange, ABC signature in purple, bound AMPPNP as yellow spheres, and target residue positions as CPK spheres. (B and E) Magnified (∼2×) surface views of the boxed cytoplasmic regions in A and D. Interfacial residues equivalent to CFTR’s ABC signature-sequence targets S549 and S1347 are exposed in TM287/288 (B) but buried in Sav1866 (E). (C and F) NBD dimer interfaces viewed from the membrane, after 90° rotation from B and E views and removal of transmembrane domains, with solid (left) and transparent (right) surfaces. In Sav1866 (F), a signature sequence and AMPPNP are buried in the tight NBD dimer interface in each composite site, whereas in TM287/288 (C), both signature sequences are exposed and a single AMPPNP is bound by the CFTR equivalent NBD1 Walker A motif.
|
|
Figure 2. Size comparison of MTS reagents, nucleotides, NEM, and MTS-biotin–avidin complex, shown as CPK-colored spheres, except for AMPPNP (yellow spheres; from TM287/288 structure; PDB accession no. 3QF4) and avidin tetramer (orange ribbon). MTSET+, MTSACE, and MTSES− all approximate cylinders ∼12 × ∼6-Å diameter, and NEM is ∼8 × ∼6-Å diameter. Minimum box sizes to fit low energy rotamers are shown: MTS-glucose, ∼16 × ∼8 × ∼7 Å; MTS-biotin, ∼19 × ∼7 × ∼6 Å; MTS-rhodamine, ∼22 × ∼14 × ∼8 Å; ATP, ∼19 × ∼9 × ∼8 Å; AMP-PNP, ∼19 × ∼9 × ∼6 Å; tetrameric avidin–MTS-biotin complex (PDB accession no. 1AVD) is ∼45-Å wide × ∼58-Å long.
|
|
Figure 3. Similarly rapid decay of current in S549C CFTR channels (containing a single target Cys in the active catalytic site) upon ATP washout (w/o) or modification by MTS reagents. (A and B) S549C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTSET+ (A; red bars below record) or 5 and 50 µM MTSACE (B; green bars below record), and modification was reversed by DTT (A, 10 mM; B, 20 mM; black bars above record). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSACE modification (dotted green, τATPw/o(mod)); colored numbers give fit time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.3 s (A) and 0.2 s (B). (C) Average τATPw/o with corresponding average τMTS from the same patches (left: gray bar, w/o, 1.6 ± 0.2 s; red bar, ≥50 µM MTSET+, 1.4 ± 0.1 s; n = 6 measurements in five patches; right: gray bar, w/o, 3.5 ± 0.4 s; green bar, ≥50 µM MTSACE, 4.0 ± 1 s; n = 6 measurements in four patches); slower washout and modification time courses in the MTSACE experiments likely reflected slower superfusion influenced by patch geometry, but comparisons were always made within the same patch. (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 0.9 ± 0.1; green open bar, τMTSACE/τATPw/o, 1.2 ± 0.3). (E) Average of washout time constant ratios, before and after MTSACE modification (green hatched bar, τATPw/o(mod)/τATPw/o, 0.9 ± 0.1; n = 5); absolute current amplitude of MTSACE-modified channels was 7–22 pA. Error bars represent mean ± SEM.
|
|
Figure 4. S549C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after 60-s applications of 5 µM MTSET+ (red trace and bar), 100 µM MTSACE (green trace and bar), or 100 µM MTSES− (blue trace and bar) to closed S549C channels in the absence of ATP, brief exposures to 3 mM ATP (black bars below record) assessed residual channel activity; the time constant of Ca2+-dependent Cl− current decay in this patch was 0.2 s. Exposures to 10 mM DTT (black bars above record) fully restored ATP-activated current by releasing adducts after each modification. (B) Relative amplitude of residual ATP-dependent current (Iresidual %) of modified S549C channels. For modification while channels were closed (left, 0 ATP), residual ATP-activated current (peak current a few seconds after adding ATP minus baseline current just before ATP addition) of modified channels was compared with the average of peak ATP-activated currents of the same channels after full recovery from modification, and just before modification by MTSET+ (red bar, 6 ± 1%; n = 8 measurements in six patches), by MTSACE (green bar, 18 ± 2%; n = 5 measurements in four patches), or by MTSES− (blue bar, 17 ± 3%; n = 4 measurements in three patches). For modification while channels were opening and closing in ATP (right, 3 mM ATP), residual ATP-dependent current (measured as the difference before and after ATP removal) of modified channels was compared with ATP-activated current of the same channels immediately before modification by ≥50 µM MTSET+ (red bar, 4 ± 1%; n = 10 measurements in five patches) or by ≥50 µM MTSACE (green bar, 18 ± 6%; n = 4 measurements in four patches). Error bars represent mean ± SEM.
|
|
Figure 5. Similarly rapid decay of current in S1347C CFTR channels (containing a single target Cys in the dead composite site) upon ATP washout (w/o) or modification by MTS reagents. (A and B) S1347C CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 1 mM MTSET+ or MTSACE (A and B, red and green bars below records, respectively). Colored lines show single-exponential fits to current decay time courses on modification (τMTS) by MTSET+ (red) or by MTSACE (green), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSET+ modification (dotted red, τATPw/o(mod)); colored numbers give time constants; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.1 s (A) and 0.3 s (B). (C) Average τATPw/o (gray bars) with corresponding average τMTS from the same patches (left: gray bar, w/o, 0.8 ± 0.2 s; red bar, ≥50 µM MTSET+, 0.8 ± 0.2 s; n = 8 measurements in six patches; right: gray bar, w/o, 1.3 ± 0.1 s; green bar, ≥50 µM MTSACE, 0.9 ± 0.2 s; n = 9 measurements in seven patches). (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 1.0 ± 0.1; green open bar, τMTSACE/τATPw/o, 0.8 ± 0.2). (E) Ratio of washout time constants before and after MTSET+ and MTSACE modification (τATPw/o(mod)/τATPw/o: red hatched bar, MTSET+, 1.5 ± 0.3, n = 8 measurements in six patches; green hatched bar, MTSACE, 1.0 ± 0.1; n = 8 measurements in seven patches); absolute current amplitudes were 5–64 pA for MTSET+-modified channels and 6–80 pA for MTSACE-modified channels. Error bars represent mean ± SEM.
|
|
Figure 6. S1347C CFTR channels are readily modified by MTS reagents when closed. (A) Immediately after ∼60-s applications of 50 µM MTSET+ (red trace and bar), MTSACE (green trace and bar), or MTSES− (blue trace and bar) to closed S1347C channels in the absence of ATP, brief exposures to 3 mM ATP (black bars below record) assessed residual channel activity. Exposures to 20 mM DTT (black bars above record) restored ATP-activated current by releasing adducts after each modification; the asterisk above the record marks brief activation of Ca2+-dependent Cl− current to monitor solution exchange speed (time constant = 0.3 s for this patch). (B) Amplitude of residual ATP-dependent current (Iresidual %), relative to ATP-activated current before modification, for S1347C channels modified, while closed (left, 0 ATP), by MTSET+ (red bar, 33 ± 8%; n = 4 measurements in four patches), by MTSACE (green bar, 24 ± 6%; n = 3 measurements in three patches), or by MTSES− (blue bar, 16 ± 5%; n = 3 measurements in three patches), or while opening and closing (right, 3 mM ATP), by ≥50 µM MTSET+ (red bar, 42.4 ± 4.5%, n = 8 measurements in four patches) or by ≥50 µM MTSACE (green bar, 19.5 ± 2.0%, n = 9 measurements in four patches). Error bars represent mean ± SEM.
|
|
Figure 7. Hydrolysis-impairing mutation, K1250R, of the conserved Walker A lysine in the active composite site similarly slows current decay after ATP washout and upon MTS modification of both S549C and S1347C channels. (A and B) ATP-activated (3 mM, black bars below records) currents of S549C-K1250R (A) and S1347C-K1250R (B) CFTR channels with single-exponential fits to current decline upon ATP removal (gray, τATP w/o) or modification (τMTS) by 50 µM MTSET+ (red) or MTSACE (green); 20 mM DTT (black bars above records) restored activation of currents by ATP; asterisks above the records mark brief activations of Ca2+-dependent Cl− currents to monitor speed of solution exchange (0.3 s in A and 0.2 s in B). (C) Average τATPw/o (gray bars) with corresponding average τMTS from the same patches (left, S549C-K1250R: gray bar, w/o, 17 ± 3.8 s; red bar, MTSET+, 20.9 ± 7.6 s; n = 3 measurements in three patches; right, S1347C-K1250R: gray bar, w/o, 15.4 ± 2.0 s; green bar, MTSACE, 18.6 ± 3.5 s; n = 9 and 7 measurements, respectively, in three patches). (D) Averages of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, S549C-K1250R, τMTSET/τATPw/o, 1.2 ± 0.2; green open bar, S1347C-K1250R, τMTSACE/τATPw/o, 1.2 ± 0.1). Error bars represent mean ± SEM.
|
|
Figure 8. Larger MTS reagents also readily modify S549C CFTR channels when they are closed. (A and B) ATP-activated current (3 mM, black bars below record) was strongly diminished after modification of closed S549C CFTR channels by ≤60-s exposures to larger MTS reagents, 20 µM MTS-glucose (A; orange bar), 5 µM MTS-rhodamine (A; magenta bar), 5 µM MTS-biotin (B; dark yellow bar), and MTS-biotin–avidin complex (B; 5 µM biotin plus 5 µM avidin, cyan bar), all in the absence of ATP; 10 mM DTT (black bars above records) released adducts after each modification; the time constants of Ca2+-dependent Cl− current decays in these patches were 0.5 s (A) and 0.2 s (B). (C) Amplitude of residual ATP-activated current (Iresidual %), relative to ATP-activated current before modification, for S549C channels modified, while closed (in 0 ATP), by MTS-biotin (dark yellow bar, 15 ± 3%, n = 7 measurements), by MTS-glucose (orange bar, 7 ± 1%, n = 3 measurements), by MTS-rhodamine (magenta bar, 5 ± 2%, n = 5 measurements), or by MTS-biotin–avidin complex (cyan bar, 90 ± 8%, n = 5 measurements). Error bars represent mean ± SEM.
|
|
Figure 9. Larger MTS reagents relatively rapidly modify S1347C CFTR channels in the presence of ATP. (A and B) S1347C channels were activated by 3 mM ATP (black bars below records) and modified by 50 µM MTS-glucose (A and B; orange traces and bars), 50 µM MTSACE (A; green trace and bar), 50 µM MTS-biotin (A; dark yellow trace and bar), or 50 µM MTS-rhodamine (A; magenta trace and bar), but not by the MTS-biotin–avidin complex (B; 50 µM biotin plus 60 µM avidin, cyan trace and bar). Each modification was reversed by 20 mM DTT (A and B; black bars above record). Asterisks above the record in A mark brief activations of Ca2+-dependent Cl− currents to monitor speed of solution exchange (here, 0.3 s and 0.2 s); the gaps in A omit 30 and 450 s, and during the second gap the channels were rephosphorylated with PKA catalytic subunit. (C) Amplitude of residual ATP-activated current (Iresidual %), as a fraction of ATP-dependent current before MTS treatment, for S1347C channels modified, while opening and closing (in 3 mM ATP), by MTS-biotin (dark yellow bar, 16 ± 2%, n = 6 measurements in six patches), by MTS-glucose (orange bar, 14 ± 3%, n = 6 measurements in three patches), by MTS-rhodamine (magenta bar, 18 ± 6%, n = 6 measurements in five patches), and MTS-biotin–avidin complex (cyan bar, 119 ± 2%, n = 3 measurements in two patches). Error bars represent mean ± SEM.
|
|
Figure 10. Similarly rapid decay of current in S605C CFTR channels (containing a mid-interface target Cys—in the NBD1 H loop—between the two composite sites) upon ATP washout (w/o) or modification by MTSET+. (A) S605C-(C832S-C1458S) CFTR channels were activated by 3 mM ATP (black bars below records) and modified by 1 mM MTSET+ (red bar). 10 mM DTT (black bars above record) kept the thiol reactive but could not remove the MTSET+ adduct. Colored lines show single-exponential fits to current decay time courses on modification by MTSET+ (red, τMTS), or on ATP removal (gray, τATPw/o) before modification, or on ATP removal after MTSET+ modification (dotted red, τATPw/o(mod)); colored numbers give time constants. (B) Amplitude of residual current (Iresidual %), relative to ATP-activated current before modification, for S605C channels modified in 3 mM ATP by 1 mM MTSET+ (red bar, 21 ± 4%, n = 4 measurements in four patches). (C) Average τATPw/o (gray bar, w/o, 0.9 ± 0.2 s; n = 4 measurements in four patches) with corresponding average τMTS (red bar, MTSET+, 1.1 ± 0.1 s) from the same patches. (D) Average of individual ratios of washout and modification time constants determined for each pair of measurements (from experiments of C; red open bar, τMTSET/τATPw/o, 1.5 ± 0.3). (E) Ratio of washout time constants before and after MTSET+ modification (τATPw/o(mod)/τATPw/o: red hatched bar, 0.9 ± 0.2, n = 4 measurements in four patches); absolute current amplitudes were 13–32 pA for MTSET+-modified channels. Error bars represent mean ± SEM.
|
|
Figure 11. Rapid decay of current in A1374C CFTR channels (containing a mid-interface target Cys—in the NBD2 D loop—between the two composite sites) upon ATP washout (w/o) or modification by MTSET+. (A and B) A1374C-(C832S-C1458S) CFTR channels were modified by 1 mM MTSET+ (red trace bar) either while closed in the absence of ATP (A), as assessed by subsequent diminished ATP-activated current (3 mM, black bars below records), or in the presence of ATP (B). 10 mM DTT (black bars above record) removed the MTSET+ adduct after each modification. (B) Colored lines show single-exponential fits to current decay time courses on modification by MTSET+ (red, τMTS), or on ATP removal (gray, τATPw/o); colored numbers give time constants. (C) Amplitude of residual current (Iresidual %), relative to ATP-activated current before modification, for A1374C channels modified, while closed (left, 0 ATP), by 5 µM–1 mM MTSET+ (red bar, 9 ± 2%; n = 8 measurements in six patches), or while opening and closing (right, 3 mM ATP), by 10 µM–1 mM MTSET+ (red bar, 17 ± 6%, n = 3 measurements in two patches). (D) Average τATPw/o (gray bar, w/o, 1.2 ± 0.6 s; n = 4 measurements in three patches) with corresponding average τMTS (red bar, MTSET+, 2.5 ± 0.9 s) from the same patches. Error bars represent mean ± SEM.
|