Abstract
The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated Cl- channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition. (Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Cell. 67:775-784). The aim of this study was to quantify possible effects of ATP on CFTR gating. We analyzed multichannel records since only 1 of 64 patches contained a single channel. ATP increased the channel open probability (Po) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 microM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum Po was not 1, the simplest description of the effect of ATP on CFTR gating is the noncooperative three-state mechanism of del Castillo and Katz (1957. Proceedings of the Royal Society of London. B. 146:369-381). We analyzed current fluctuations to quantify possible changes in CFTR gating. The power density spectra appeared to contain a single Lorentzian in the range of 0.096-31 Hz. Analysis of the corner frequency (fc) of this Lorentzian revealed that ATP increased 2 pi fc as a Michaelis-Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 tau open (154 ms), and the ATP-sensitive tau close [(185 ms) (44 microM/[ATP] + 1)]. These results suggest that the binding reaction is rapid compared to the opening and closing rates. Assuming that there is a single set of closed-to-open transitions, it is possible to verify the outcome of fluctuation analysis by comparing fluctuation-derived estimates of Po with measures of Po from current records. The two values were nearly identical. Thus, noise analysis provides a quantitative description of the effect of ATP on CFTR opening. The noncooperative three-state model should serve as a basis to understand possible alterations in CFTR gating resulting from regulators or point mutations.
Highlights
Cystic fibrosis (CF) is an autosomal recessive genetic disease
Three types of evidence indicate that the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), functions as the low-conductance, cAMP-activated CI- channels that have been identified in the apical membranes of Cl--transporting epithelial cells (Gray, Greenwell, and Argent 1988; Tabcharani, Low, Elie, and Hanrahan 1990)
We found that measurements from current records and fluctuation analysis provide convergent descriptions of the effect of ATP on CVI'R, and they are consistent with a noncooperative three-state activation mechanism. We propose that this simple three-state mechanism is the central element in a more elaborate model for CFTR gating that accounts for the possibility of additional open-shut transitions (Tabcharani et al, 1991; Haws, Krouse, Xia, Gruenert, and Wine, 1992; McCarty, McDonough, Cohen, Riordan, Davidson, and Lester, 1993)
Summary
Cystic fibrosis (CF) is an autosomal recessive genetic disease. The epithelial cells lining the airways, pancreatic ducts, intestines, and sweat ducts of CF patients lack a cAMP-activated apical membrane CI- conductance (Quinton, 1990). Three types of evidence indicate that the CF gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), functions as the low-conductance, cAMP-activated CI- channels that have been identified in the apical membranes of Cl--transporting epithelial cells (Gray, Greenwell, and Argent 1988; Tabcharani, Low, Elie, and Hanrahan 1990). Transfection of CFTR cDNA into CF cells (Rich, Anderson, Gregory, Cheng, Paul, Jefferson, McCann, Klinger, Smith, and Welsh 1990; Drumm, Pope, Cliff, Rommens, Marvin, Tsui, Collins, Frizzell, and Wilson 1990; Cliff, Schoumacher, and Frizzell, 1992) or cells not normally expressing CF-I'R (Berger, Anderson, Gregory, Thompson, Howard, Maurer, Smith, and Welsh 1991; Tabcharani, Chang, Riordan, and Hanrahan, 1991) resulted in the appearance of low-conductance, cAMP-regulated CIchannels. There is considerable evidence that the CF gene product CFTR functions as the lowconductance, regulated CI- channel that is defective in CF
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