|
Figure 1. . The 8-azido group slows CFTR Clâ channel opening and closing. (A) Outward currents (at +40 mV) activated by nucleotides indicated beneath traces in patches excised from oocytes expressing WT CFTR, after prior exposure to PKA plus 5 mM MgATP. (B and C) Mean rates of channel opening to a burst (rCO, B) and of closing from a burst (rOC, C) vs. [MgATP] (filled circles) or [Mg8-N3ATP] (red triangles), from analysis of records as in A (lower trace). For each test concentration (e.g., 15 μM Mg8-N3ATP, in A), kinetic parameters were normalized to the average of the values obtained from the same channels during the bracketing exposures to saturating nucleotide (e.g., 2 mM Mg8-N3ATP, in A). These ratios were then scaled by our estimates of absolute opening and closing rates at saturating [nucleotide] (see materials and methods): for MgATP, rCO(5 mM MgATP) = 0.27 ± 0.03 sâ1 and rOC(5 mM MgATP)= 3.3 ± 0.2 sâ1 (n = 18; Vergani et al., 2003). For Mg8-N3ATP we multiplied those rates by the factors (0.42 for rCO, and 0.19 for rOC) determined by direct comparison (as in A, top trace), assuming that 2 and 5 mM MgATP are both saturating concentrations. Curves in B show Michelis-Menten fits, with parameters (mean ± SEM; 2 ⤠n ⤠7) K0.5 = 55 ± 5 μM and 11 ± 3 μM and Vmax = 1.02 ± 0.02 and 1.01 ± 0.04, for MgATP and Mg8-N3ATP, respectively. The high apparent affinity for Mg8-N3ATP is directly evident in Fig. 1 A, bottom trace: thus, rCO at 15 μM Mg8-N3ATP averaged 60 ± 11% (n = 7) of rCO at saturating, 2 mM, Mg8-N3ATP, whereas rCO at 15 μM MgATP was only 17 ± 1% (n = 8) of rCO at saturating [MgATP] (Fig. 1 B).
|
|
Figure 2. . [α32P]8-N3ATP and [γ32P]8-N3ATP binding to Flag-CFTR in HEK293T cell membranes and inhibition by nucleotides. (A) Autoradiogram showing competition by cold MgATP for binding of 5 μM [α32P]8-N3ATP to CFTR at 0°C, followed by UV irradiation, washing, immunoprecipitation, and SDS-PAGE. (B) CFTR incubated with 5 μM [γ32P]8-N3ATP at 0°C was photolabeled only after UV irradiation, indicating lack of phosphorylation by endogenous kinases under these conditions. (C) Quantification of competition for binding of 5 μM [α32P]8-N3ATP by unlabeled MgATP (as in A), MgADP, or MgAMPPNP (all in Mg2+-containing buffer), or by unlabeled TrisATP (in Mg2+-free buffer). Photolabeling was normalized to the signal obtained in the absence of competing nucleotide (note broken abscissa), and the data (error bars show ±SD) fitted assuming simple competition and a Kd of 5 μM for Mg8-N3ATP, or 8 μM for free 8-N3ATP. Ki values from the fits were 1 ± 1 μM for MgADP (filled triangles, n = 2), 6 ± 2 μM for MgAMPPNP (empty squares, n = 4), 16 ± 4 μM for MgATP (filled circles, n = 4), and 69 ± 20 μM for TrisATP (empty circles, n = 3). Given the finite duration of UV irradiation, these apparent binding affinities might be less accurate than the ratios of the values obtained for the different nucleotides.
|
|
Figure 3. . Binding of [α32P]8-N3ATP to split CFTR channels. (A) Membranes from oocytes coexpressing Flag-3â835 CFTR and Flag-837â1480 CFTR (+), or noninjected controls (â), incubated with 5 or 50 μM [α32P]8-N3ATP at 0°C were irradiated with UV before washing, immunoprecipitation, and SDS-PAGE. Arrow indicates photolabeled band in the autoradiogram corresponding to CFTR half containing NBD1. (B) Immunoblots of proteins immunoprecipitated and run in same SDS-PAGE gel used for A, before transfer to nitrocellulose and blotting with anti-R domain antibody (left) that recognizes Flag-3â835-containing NBD1, or with antiâCOOH-terminal antibody (right) that recognizes Flag-837â1480-containing NBD2.
|
|
Figure 4. . Vi locks open WT CFTR channels activated by PKA and either MgATP or Mg8-N3ATP. (AâC) Macroscopic current, in patches containing hundreds of channels excised from oocytes expressing WT CFTR, relaxed with double exponential time course (smooth fit lines, largely obscured by data) after removal of nucleotides. Nucleotides (0.5 mM in A and B; 5 mM in C) and Vi were applied in the presence of 300 nM PKA. Fit parameters for trace in A are, af = 25 pA, as = 24 pA, Ïf = 0.5 s, Ïs = 20 s; for trace in B, af = 14 pA, as = 7 pA, Ïf = 3 s, Ïs = 35 s; for first decay in C, af = 50 pA, Ïf = 0.5 s; second decay in C, af = 27 pA, as = 18 pA, Ïf = 0.6 s, Ïs = 21 s. Solution exchange took <1 s, estimated from current decay after brief activation of endogenous Ca2+-activated Clâ channels. (D and E) Summary of mean (±SEM, n = 6) time constants (D) and fractional amplitudes (E) of slow components of current decay after activation by PKA and 0.5 mM MgATP without (white bars), or with (black bars) 5 mM Vi, or by PKA and 0.5 mM Mg8-N3ATP with 5 mM Vi (gray bars). (F and G) Macroscopic current records from inside-out patches excised from HEK293T cells expressing WT CFTR; MgATP (5 mM) and Vi (5 mM) applications were in the presence of 300 nM PKA; the breaks indicate omission of 30 s (F) and 100 s (G) of record. (In C, F, and G, offset bars beneath traces signal different solution compositions.)
|
|
Figure 5. . (A) In split CFTR, tightly bound [α32P]8-azido nucleotide predominantly labels NBD1. (Left) Autoradiogram from membranes of oocytes coexpressing Flag-3â835 and Flag-837â1480 (+), or of noninjected oocytes (â) incubated for 15 min at 30°C with 5 μM [α32P]8-N3ATP and 10 mM Mg2+ ± 1 mM Vi, subjected to the standard wash procedure including 5-min postincubation at 30°C, irradiated with UV, immunoprecipitated, and subjected to SDS-PAGE. Arrow indicates photolabeled band corresponding to CFTR half containing NBD1. (Right) Proteins from the same membranes were immunoprecipitated and blotted with either antiâR-domain antibody, which recognizes the half of CFTR containing NBD1 (left two lanes), or with antiâCOOH-terminal antibody, which recognizes the half containing NBD2 (right two lanes). (B) Extremely slow release of [α32P]8-azido nucleotide during nucleotide-free postincubation at 30°C before UV irradiation. Initial incubation of membranes from HEK293T cells expressing Flag-CFTR was with 5 μM [α32P]8-N3ATP for 15 min at 30°C with (+Vi) or without (âVi) 1 mM Vi. After postincubation for time indicated, immunoprecipitation, and SDS-PAGE, the resulting autoradiogram signals were normalized to that obtained with Vi but without postincubation (postincubation time = 0) in each experiment (n = 6 for 0- and 5-min points; n = 2 for 1.5- and 15-min points; error bars give ± SD). Curves show simultaneous least-squares fits to both datasets with single time constant, yielding Ï = 15 ± 2 min.
|
|
Figure 6. . Tightly bound 8-N3 nucleotide at NBD1 is largely ATP. (A) Autoradiogram showing labeling of CFTR by [γ32P]8-N3ATP without UV (reflecting protein phosphorylation; right) and after UV irradiation (reflecting both photocrosslinking and phosphorylation; left). Prephosphorylated (with PKA; see Fig. 7) HEK293T membranes containing Flag-CFTR were incubated for 15 min at 30°C with 5 μM [γ32P]8-N3ATP plus 1 mM Vi, subjected to usual washes plus 5-min nucleotide-free postincubation at 30°C, exposed to UV (+) or kept in the dark (â), immunoprecipitated, run on SDS-PAGE, and exposed. (B and C) Both [γ32P]8-N3ATP and [α32P]8-N3ATP predominantly photolabel split CFTR (arrow) at NBD1. (B) Autoradiogram from (not prephosphorylated) membranes of oocytes coexpressing Flag-3â835 and Flag-837â1480 (+) or from noninjected oocytes (â), incubated for 15 min at 30°C with 1 mM Vi and either 5 μM [α32P]8-N3ATP (left two lanes) or 5 μM [γ32P]8-N3ATP (right three lanes), followed by washes and 5-min postincubation at 30°C and then exposure to UV light (+) or darkness (â), before immunoprecipitation and SDS-PAGE. (C) Immunoprecipitates from the same membranes were immunoblotted with antiâR-domain antibody (left) or antiâCOOH-terminal antibody (right).
|
|
Figure 7. . Little influence of phosphorylation with PKA on [α32P]8-N3ATP binding at 0°C (A) or tight binding/occlusion at 30°C (B and C). (A) Before the binding assay, HEK293T membranes expressing Flag-CFTR were incubated with (+PKA) or without (âPKA) PKA, washed twice with ice-cold nucleotide-free (and PKA-free) buffer, and then incubated for 5 min on ice with 0.5, 1, or 5 μM [α32P]8-N3ATP before UV irradiation without washes, immunoprecipitation, and SDS-PAGE to yield the autoradiogram shown. (B) Membranes were prephosphorylated with PKA (+) or not (â) and washed as in A, then incubated at 30°C for 15 min with 5 μM PKI and 5 μM [α32P]8-N3ATP ± 1 mM Vi (as indicated), washed as usual including a 5-min postincubation at 30°C, and then UV irradiated (except lane marked âââ) before immunoprecipitation, SDS-PAGE, and autoradiography. The right lane is a control using the same membranes but prephosphorylated with PKA and [γ32P]ATP, then washed and incubated with Vi for 15 min at 30°C like the other samples, but without [α32P]8-N3ATP, before washing, immunoprecipitation, SDS-PAGE, and autoradiography: the PKA-mediated phosphorylation of CFTR was retained throughout the incubation period with 8-N3 nucleotide. (C) PKA was included (+) or not (â) in the 10-min incubation of HEK293T-cell membranes expressing Flag-CFTR with 5 μM [α32P]8-N3ATP ± 1 mM Vi (as indicated) at 25°C, after which the membranes were washed as usual including a 5-min postincubation at 25°C, and then UV irradiated before immunoprecipitation, SDS-PAGE, and autoradiography.
|
|
Figure 8. . Mutation K464A in CFTR impairs occlusion at 30°C, but not binding at 0°C, at low [nucleotide], and disrupts Vi-induced stabilization of open burst state. (A, left) Membranes of oocytes expressing WT or K464A CFTR (without Flag tags) were run on SDS-PAGE gels, transferred to nitrocellulose membranes, and blotted with antiâR-domain antibody. WT CFTR was expressed at least twice as well as K464A CFTR in this batch of oocytes (arrow marks mature fully-glycosylated CFTR; lower, sharper band is core-glycosylated CFTR). (Right) Autoradiogram showing nucleotide occlusion in the same membranes as at left, after incubation at 30°C for 15 min with 5 μM [α32P]8-N3ATP ± 1 mM Vi, washing including 5-min postincubation at 30°C, irradiation with UV, solubilization, and SDS-PAGE. Twice as much membrane was used for K464A samples as for WT samples. (B, left) Immunoblots of membranes from Flag-WTâ and Flag-K464Aâexpressing oocytes (the Flag tags facilitated immunoprecipitation to enhanced signal-to-noise ratio, as membranes were not washed before photocrosslinking) blotted with antiâR-domain antibody as in A. The membranes contained about one third more WT than mutant K464A protein. (Right) Autoradiogram showing photolabeling of CFTR in the same membranes as at left after incubation with 5 or 50 μM [α32P]8-N3ATP for 5 min on ice, followed by UV irradiation, immunoprecipitation, SDS-PAGE, and autoradiography. Approximately 30% more membrane was used for Flag-K464A samples as for Flag-WT samples. (C) Macroscopic current in an oocyte patch containing hundreds of K464A CFTR channels. Current decays with comparable biexponential time course (superimposed fit lines largely obscured by data) after activation by 5 mM MgATP plus 300 nM PKA with 5 mM Vi (for trace shown: af = 23 pA, as = 7 pA, Ïf = 0.7 s, Ïs = 5.5 s) or without it (first decay af = 29 pA; as = 31 pA, Ïf = 0.5 s, Ïs = 1.4 s, second decay af = 57 pA; as = 5 pA, Ïf = 0.6 s, Ïs = 2.1 s). Solution exchange time <1 s. Bars beneath trace offset to signal different solution compositions. (D and E) Mean fit parameters for the time constant (D) and fractional amplitude (E) of the slow component of current decay after activation by 5 mM MgATP plus 300 nM PKA without (white bars) or with 5 mM Vi (black bars) for WT (n = 19) or K464A (n = 10).
|
