ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: Evidence for a three-state activation mechanism

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 (P(o)) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 μM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum P(o) 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 (f(c)) of this Lorentzian revealed that ATP increased 2πf(c) as a Michaelis- Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 μM), τ(open) (154 ms), and the ATP- sensitive τ(close) [(185 ms) (44 μM/[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 P(o) with measures of P(o) 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.