Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2.
We evaluated the suitability of the S2 foetal Drosophila cell line as an expression system for vertebrate anion channel proteins (e.g. cystic fibrosis transmembrane conductance regulator, CFTR) in patch-clamp studies of the endogenous ion channels. In the inside-out configuration (symmetric 150 mM Cl-) we found most frequently an inwardly rectifying Cl- channel with single-channel conductances (gamma) of 57, 45 and 17 pS at -80, 0 and 80 mV, respectively. Reduction of bath [Cl-] to 40 mM caused a shift in reversal potential (Vrev) to -22.5 mV indicating pronounced Cl- selectivity. In the outside-out configuration ([Cl-]pipette = 40 mM, [Cl-]bath = 150 mM) we observed a Cl- channel with a linear unitary current/voltage (i/V) relation for which gamma was 30 pS. The kinetics were quite slow in both configurations. Cl-selectivity was also observed in whole-cell experiments ([Cl-]pipette = 40 mM) in which a Vrev of -43.8 mV, i.e. close to the Cl- equilibrium potential, demonstrated that the membrane current was dominated by Cl-. We conclude that the important features making S2 cells suitable as an expression system for heterologous expressed anion channel proteins are: small total whole-cell currents (less than 100 pA), single-channel and whole-cell currents that, unlike those of CFTR, cannot be described by the Goldman-Hodgkin-Katz regime, and slow kinetics distinctly different from those of CFTR.
Asmild
M
M
August Krogh Institute, University of Copenhagen, Denmark.
Willumsen
N J
NJ
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D004330:Drosophila / Q000166:cytology / Q000196:embryology*; D004553:Electric Conductivity; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000502:physiology; D018408:Patch-Clamp Techniques
D018118:Chloride Channels