Direct activation of cystic fibrosis transmembrane conductance regulator channels by 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX)

Abstract

8-Cyclopentyl-1,3-dipropylxanthine (CPX) and 1,3-diallyl-8-cyclohexylxanthine (DAX) are xanthine adenosine antagonists which activate chloride efflux from cells expressing either wild-type or mutant (DeltaF508) cystic fibrosis transmembrane conductance regulator (CFTR). These drugs are active in extremely low concentrations, suggesting their possible therapeutic uses in treating cystic fibrosis. However, knowledge of the mechanism of action of these compounds is lacking. We report here that the same low concentrations of both CPX and DAX which activate chloride currents from cells also generate a profound activation of CFTR channels incorporated into planar lipid bilayers. The process of activation involves a pronounced increase in the total conductive time of the incorporated CFTR channels. The mechanism involves an increase in the frequency and duration of channel opening events. Thus, activation by these drugs of chloride efflux in cells very likely involves direct interaction of the drugs with the CFTR protein. We anticipate that this new information will contribute fundamentally to the rational development of these and related compounds for cystic fibrosis therapy.

Fig. 1.. Characteristic ion channel activity from CFTR channels expressed in HEK293 cells.

A, representative current records for CFTR. Three representative current records, taken at −30, −50, and −80 mV, demonstrate the most common types of current events observed after fusion of microsomal membrane vesicles from HEK293 cells to the lipid bilayer. At least two channels of about 7 pS conductance are observed in this example. The bilayer system is a KCl gradient (cis, 200 mm; trans, 50 mm), with ATP and PKA in the cis compartment. The voltage is given relative to the cis compartment. B, expanded time scale of segments of a CFTR current record taken at a membrane potential of −80 mV. a, the data demonstrate the simultaneous incorporation of two channels of identical conductance. The ionic conditions are the same as for a. b, the data demonstrate current events of smaller amplitude than those shown in part a. The ionic conditions are the same as for A. c, the data demonstrate interspersed current events from both the large and small CFTR channel. The ionic conditions are that same as for A. C, current-voltage relationship for large and small CFTR channels. Each point represents the arithmetic mean ± S.E. of at least 500 events measured at each potential. The slopes of the lines, calculated from regressions fit to the points, indicate conductances of 6.7 and 2.5 pS to the large and small currents events, respectively. The slope conductances are calculated as the regression fit of the points. Both regression lines intersect on the horizontal axis at the equilibrium potential of about 22 mV.

Fig. 2.. Effect of CPX on CFTR channel activity.

Upper panel, control. Two minutes of continuous recording of a modestly activated CFTR channel, driven by a 50 mV membrane potential and a KCl gradient (200 mm cis and 50 mm trans). Middle panel, 2 min after the addition of CPX (500 nm) to the cis compartment. The channel activation is observed as an increase in the current activity both in number of opening events and multiple current levels. Bottom panel, further addition of CPX (up to 2000 nm CPX) to the cis compartment maintains the overall increased current activity.

Fig. 3.. CPX effects on CFTR channels.

A, influence of CPX on the I-V relationship of CFTR channels in a KCl gradient (200 mm cis and 50 mm trans). The relation between mean amplitude of unitary current events and the membrane voltage in control and CPX treated conditions is fitted by linear regression. The slope conductances are both 8.2 pS, and the intersections on the horizontal axis indicate current equilibrium potentials around 12 mV. Symbols are as indicated. B, current events amplitude histograms from records at −50 mV membrane potential. Left panel, amplitude distribution of unitary current events during control conditions. Right panel, amplitude distribution of the all recorded current events in the presence of 500 nm CPX in the cis compartment. Gaussian fit indicates that mean amplitude of the unitary events is 0.48 pA for −50 mV holding potential, in control and in the presence of the drug. C, scatter plot of the amplitude of the events in both control (filled circles) and CPX (cross symbols) as a function of duration events. Currents events of any open time duration, both in control and in the presence of the drug, are positioned around a mean current amplitude (or multiple) of 0.48 pA. Except for current events of very short duration (less than 20 ms) whose amplitudes are affected by the filtering conditions, no intermediate current amplitude events are observed.

Fig. 4.. Influence of CPX on small CFTR conductance.

A, effect of CPX on the activity of the 2.5 pS conductance associated with the expression of CFTR. Upper panel, control channel current events generated by −100 mV potential. Lower panel, same channels as in the upper panel, after addition of CPX (500 nm). B, influence of CPX on open time distributions of current events from the 2.5 pS ionic conductance associated with expression of CFTR. Upper panel, in control conditions the current events are distributed into two population. Two Gaussian fit to the data indicates peak durations of 8 and 24 ms. Lower panel, distribution of channel current events in the presence of 500 nm CPX, fitted to three Gaussian distributions. The peak durations are 9, 27, and 44 ms.

Fig. 5.. Influence of DAX on CFTR channels.

Upper panel, control. Two minutes of continuous recording of a modestly activated CFTR channel, driven by a 50 mV membrane potential and a KCl gradient (200 mm cis and 50 mm trans). Middle panel, after the addition of DAX (500 nm) to the cis compartment, a potent activation is observed as an increase in the current activity both in number of opening events and multiple current levels. Lower panel, further addition of DAX (up to 2000 nm) to the cis compartment maintains the overall increased current activity.

Fig. 6.. Concentration dependence for CPX and DAX on CFTR channel activation.

A, influence of CPX and DAX on open time of CFTR channels. Drugs were added to the cis compartment in a stepwise manner. Data were collected 3 min after drug addition. Open time probability was estimated from 2 min of subsequent data. B, influence of CPX and DAX on negative charge conducted by CFTR. Experiment was conducted exactly as in part A. Average charge conducted was calculated by integrating serial 15-s intervals over a 2-min period of current recording.

Fig. 7.. Effect of CPX on mean open time of CFTR channels.

A, continuous recording of CFTR channel activity after removal of PKA and ATP from the chamber. B, CFTR channel activity from part A after equilibrium activation by CPX (250 nm). Conditions include a KCl gradient as described under “Materials and Methods,” and a −80 mV electrical potential in cis. C, histograms of mean open time for CFTR channel under the conditions for A and B. Fitted lines give values of τ_o. The control value is 17.4 ms; the CPX value is 121 ms.