ISSN:
1432-2013
Keywords:
Key words Cl
;
channels
;
CFTR
;
cAMP
;
Forskolin
;
Insect chloride channels
;
Sf9 cells
;
Patch clamp
Source:
Springer Online Journal Archives 1860-2000
Topics:
Medicine
Notes:
Abstract The Sf9 insect Spodoptora frugiperda cell line was used for heterologous expression of the cloned human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, or the cloned β-galactosidase gene, using the baculovirus Autographa califonica as the infection vector. Using application of the patch-clamp technique, evidence for functional expression of CFTR was obtained according to the following three criteria. Firstly, whole-cell currents recorded 2 days after infection with CFTR revealed a statistically significant increase of membrane conductance, ≈25 times above that of mock-infected control cells, with the reversal potential of the major current component being governed by the chloride equilibrium potential (E Cl). Secondly, in contrast to uninfected cells and cells infected with β-galactosidase, the membrane conductance to chloride of CFTR-injected cells was stimulated by cytosolic adenosine 3´,5´-cyclic monophosphate (cAMP), which was raised by exposing the cells to 10 μM forskolin. Thirdly, recordings of currents through single channels in excised outside-out membrane patches of CFTR-infected cells revealed channels which were clearly different from the native insect chloride channel. Excised outside-out patches of CFTR-infected and forskolin-stimulated cells exhibited wave-like gating kinetics of well-resolved current transitions. All-point Gaussian distributions revealed contributions from several (five to nine) identical channels. Such channels, in excised outside-out patches, studied with a pipette [Cl−] = 40 mM and a bath [Cl−] = 150 mM, rectified the current in agreement with simple electrodiffusion and with a single-channel Goldman-Hodgkin-Katz permeability, P Cl = 1.34⋅10−14 ± 0.23⋅ 10−14 cm3/s (n = 5), corresponding to a physiological single-channel conductance of 2.8 ± 0.5 pS (V M = E Cl) and a limiting conductance, γ150/150, = 7.7 ± 1.3 pS ([Cl−]Bath = [Cl−]Cell = 150 mM). Currents recorded from multichannel excised outside-out patches could shift from the above mode of resolvable unitary conductance transitions to one which was too fast to reveal the dwell-times of closed and open states. During periods characterized by noisy currents, the variance (σ2) of current fluctuations about their stationary 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 fluctuations are caused by fast gating of channels specific for chloride ions. Switching back and forth between the two gating modes of clusters of chloride channels occurred from moment to moment in excised patches when the membrane potential was held at a constant value indicating cooperative gating as a result of interaction between neighbouring chloride channels.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s004240050166
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