TABLE I.
Kinetic Parameters of WT and Mutant CFTR Channels
| WT
|
K464A
|
D1370N
|
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| mean ± SEM | n | mean ± SEM | n | mean ± SEM | n | |||||||
| (A) 5 mM MgATP + 300 nM PKA | ||||||||||||
| τb | 644 ± 63 | 30 | 620 ± 58 | 21 | 3,768 ± 499 | 21 | ||||||
| τib | 1,671 ± 172 | 30 | 2,760 ± 439 | 21 | 3,588 ± 414 | 21 | ||||||
| 1,552 ± 170 | 19 | 2,438 ± 483 | 12 | 2,849 ± 491 | 12 | |||||||
| τF | 19.3 ± 2.0 | 30 | 20.8 ± 2.1 | 21 | 49.9 ± 4.3 | 21 | ||||||
| nF | 0.57 ± 0.06 | 30 | 0.50 ± 0.06 | 21 | 2.76 ± 0.25 | 21 | ||||||
| rCO | 0.75 ± 0.06 | 30 | 0.50 ± 0.05 | 21 | 0.38 ± 0.05 | 21 | ||||||
| 0.77 ± 0.08 | 19 | 0.54 ± 0.08 | 12 | 0.47 ± 0.07 | 12 | |||||||
| rOC | 1.95 ± 0.15 | 30 | 1.92 ± 0.15 | 21 | 0.43 ± 0.07 | 21 | ||||||
| (B) 5 mM MgATP | ||||||||||||
| τb | 338 ± 22 | 18 | 309 ± 23 | 8 | 1,748 ± 215 | 17 | ||||||
| τib | 4,506 ± 497 | 18 | 6,752 ± 1314 | 8 | 9,503 ± 1440 | 17 | ||||||
| 4,454 ± 1382 | 5 | 6,928 ± 1666 | 6 | 7,584 ± 1967 | 9 | |||||||
| τF | 23.5 ± 3.2 | 18 | 16.1 ± 2.2 | 8 | 51.5 ± 6.0 | 17 | ||||||
| nF | 0.42 ± 0.05 | 18 | 0.39 ± 0.06 | 8 | 1.40 ± 0.13 | 17 | ||||||
| rCO | 0.27 ± 0.03 | 18 | 0.18 ± 0.03 | 8 | 0.16 ± 0.03 | 17 | ||||||
| 0.33 ± 0.09 | 5 | 0.18 ± 0.03 | 6 | 0.22 ± 0.05 | 9 | |||||||
| rOC | 3.28 ± 0.21 | 18 | 3.43 ± 0.25 | 8 | 0.75 ± 0.09 | 17 | ||||||
| (C) 50 μM MgATP | ||||||||||||
| τb | 355 ± 44 | 12 | 323 ± 136 | 4 | 1,433 ± 381 | 4 | ||||||
| τF | 27.3 ± 5.2 | 12 | 22.1 ± 4.4 | 4 | 46.2 ± 10.8 | 4 | ||||||
| nF | 0.38 ± 0.05 | 12 | 0.45 ± 0.11 | 4 | 1.91 ± 0.34 | 4 | ||||||
| (D) 5 mM MgAMPPNP | ||||||||||||
| τb | 1,619 ± 232 | 32 | 271 ± 52 | 8 | ||||||||
| τF | 59.5 ± 6.6 | 32 | 26.8 ± 7.7 | 8 | ||||||||
| nF | 2.40 ± 0.26 | 32 | 0.38 ± 0.10 | 8 | ||||||||
Kinetic parameters were obtained using a maximum likelihood simultaneous fit to dwell-time histograms at all conductance levels (Csanády, 2000). Rate constants (rCO, rOC, rOF, rFO) were extracted applying the three-state “C-O-CF” model (see materials and methods) and derived parameters were calculated as follows: burst duration, τb = (1/rOC)*[1 + (rOF/rFO)]; interburst duration, τib = 1/rCO; flicker duration, τF = 1/rFO; number of flickers per burst, nF = rOF/rOC. Durations are given in ms, rates in s−1. For τib and rCO values, the top rows give “total” estimates and the bottom ones “best” estimates (as defined in materials and methods). The values given in C (obtained in experiments as in Fig. 2, A–C) show that frequency and duration of flickery closures do not depend on [MgATP]. The significance of the slight prolongation of τF for the D1370N mutant and for WT in 5 mM MgAMPPNP is unknown, but the rate rOF remained 1–2 s−1 for all conditions and mutants tested.