Effect of ATP concentration on CFTR Cl- channels: a kinetic analysis of channel regulation

Abstract

Phosphorylated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels require nucleoside triphosphates, such as ATP, to open. As the concentration of intracellular ATP increases, the probability of the channel being open (Po) increases. To better understand how ATP regulates the channel, we studied excised inside-out membrane patches that contained single, phosphorylated CFTR Cl- channels and examined the kinetics of gating at different concentrations of ATP. As the ATP concentration increased from 0.1 to 3 mM the mean closed time decreased, but mean open time did not change. Analysis of the data using histograms of open- and closed-state durations, the maximum likelihood method, and the log-likelihood ratio test suggested that channel behavior could be described by a model containing one open and two closed states (C1<==>C2<==>O). ATP regulated phosphorylated channels at the transition between the closed states C1 and C2: as the concentration of ATP increased, the rate of transition from C1 to C2 (C1-->C2) increased. In contrast, transitions from C2 to C1 and between C2 and the open state (O) were not significantly altered by ATP. Addition of ADP in the presence of ATP decreased the transition rate from C1 to C2 without affecting other transition rates. These data suggest that ATP regulates CFTR Cl- channels through an interaction that increases the rate of transition from the closed state to a bursting state in which the channel flickers back and forth between an open and a closed state (C2). This transition may reflect ATP binding or perhaps a step subsequent to binding.

ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator

“…For subsequent burst analysis, we used a value of 60 ms as the burst delimiter (tc). tc was determined according to the method described by Winter et al (26). Briefly, tc was derived from the function describing the closed dwell time histogram as the nadir between the peak values that define the mean short closed (intraburst) times and the long closed (interburst) times.…”

“…Further, combination of data from multiple experiments permits the generation of burst duration histograms. Burst analysis was performed using tc ϭ 60 ms. tc, the time that separates short, intraburst closures from long, interburst closures, was estimated using several approaches (25,26). The latter approach described by Winter et al for CFTR in biological membranes yielded a value for tc which led to the optimal separation of intraburst and interburst closed times.…”

The role of cystic fibrosis transmembrane conductance regulator phenylalanine 508 side chain in ion channel gating

“…Wild-type CFTR ion channel mean open time (τ o ) was independent of the nucleotide concentration (Winter et al 1994). However, different nucleotides (ATP, dATP, 8BrATP and 8N 3 ATP) supported CFTR ion channel gating that resulted in τ o characteristic for each particular nucleotide (Fig.…”

Dynamics Intrinsic to Cystic Fibrosis Transmembrane Conductance Regulator Function and Stability

“…Singlechannel recordings of CFTR indicate that the P.A. Chong et al channel has two phases, a burst interval, during which the channel is open but flickers rapidly through a closed state, and an interburst interval, during which the channel is closed (Winter et al 1994) As a large polytopic membrane protein containing multiple domains and disordered segments, full-length CFTR has resisted efforts to directly resolve the structure of any of these conformations by X-ray crystallography. …”