RESUMO
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.
