CLUSTERS OF CL- CHANNELS IN CFTR-EXPRESSING SF9 CELLS SWITCH SPONTANEOUSLY BETWEEN SLOW AND FAST GATING MODES

The Sf9 insect Spodotora frugiperda cell line was used for heterologou s expression of the cloned human cystic fibrosis transmembrane conduct ance regulator (CFTR) cDNA, or the cloned beta-galactosidase gene, usi ng the baculovirus Autographa califonica as the infection vector. Usin g application of the patch-clamp technique, evidence for functional ex pression of CFTR was obtained according to the following three criteri a. Firstly, whole-cell currents recorded 2 days after infection with C FTR revealed a statistically significant Increase of membrane conducta nce, approximate to 25 times above that of mock-infected control cells , with the reversal potential of the major current component being gov erned by the chloride equilibrium potential (E(Cl)). Secondly, in cont rast to uninfected cells and cells infected with beta-galactosidase, t he membrane conductance to chloride of CFTR-injected cells wa stimulat ed by cytosolic adenosine 3',5'-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 mu M forskolin. Thirdly, recording s of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly di fferent from the native insect chloride channel. Excised outside-out p arches of CFTR-infected and forskolin-stimulated cells exhibited wave- like gating kinetics of well-resolved current transitions. All-point G aussian distributions revealed contributions from Several (live to nin e) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl-] = 40 mM and a bath [Cl-]- 150 mM, rectifi ed the current in agreement with simple electrodiffusion and with a si ngle-channel Goldman-Hodgkin-Katz permeability, P-Cl = 1.34 . 10(-14) +/- 0.23 . 10(-14) cm(3)/s (n = 5), corresponding to a physiological s ingle-channel conductance of 2.8 +/- 0.5 pS (V-M = E(Cl)) and a limiti ng conductance, gamma(150/150), = 7.7 +/- 1.3 pS ([Cl-](Bach) = [Cl-]( Cell) = 150 mM). Currents recorded from multichannel excised outside-o ut patches could shift from the above mode of resolvable unitary condu ctance transitions to one which was too fast to reveal the dwell-limes of closed and open states. During periods characterized by noisy curr ents, the variance (sigma(2)) of current fluctuations about their stat ionary mean value depicted a U-shaped function of membrane potential, with a minimum value at a pipette potential where the chloride current was shown to be zero. Thus, it can be concluded that the current fluc tuations are caused by fast gating: of channels specific for chloride ions. Switching back and forth between the two gating modes of cluster s of chloride channels occurred from moment to moment in excised patch es when the membrane potential was held at a constant value indicating cooperative gating asa result of interaction between neighbouring chl oride channels.