CHARACTERIZATION OF 2 DISTINCT CL- CONDUCTANCES IN FUSED HUMAN RESPIRATORY EPITHELIAL-CELLS .2. RELATION TO CYSTIC-FIBROSIS GENE-PRODUCT

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at charact erizing the molecular basis of the Cl- conductances regulated by cycli c adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (V-m) and resistan ce (R(m)) were recorded as well as their response to substitution of 9 0% of bath Cl- by isethionate (Delta V-m,V-ISE), by I- (Delta V-m,V-I) , or by other halide anions. Fused CF cells had significantly (P < 0.0 5) higher control V-m values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than f used non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2 ionophore A23187 with an increase in the V-m response to Cl- substitu tion, but did not respond to forskolin. This indicates that CF cells e xpress only the Ca2+-stimulated Cl- conductance. Injection of the anti body M3A7 against a fusion protein containing amino acids 1195 to 1480 of the CF gene product into young, forskolin-stimulated or old non-CF cells decreased Delta V-m,V-ISE and Delta V-m,V-I within 15 min to va lues observed in CF cells. This indicates inhibition of the cAMP-stimu lated Cl- conductance and supports the molecular identity of this cond uctance with the CF gene product. However, the slow onset of inhibitio n does not allow secondary effects to be excluded and a slight fall in R(m) remains unexplained. Stimulation of the Ca2+-regulated Cl- condu ctance was not impaired. Injection of M3A7 into CF cells or of a contr ol antibody in non-CF cells had no effect. In the search for the singl e-channel equivalent of the Ca2+-stimulated Cl- conductance we injecte d a concentrated placental cytosol fraction containing a cytosolic inh ibitor of the outwardly rectifying intermediate conductance (ORIC) Cl- channel into fused non-CF cells stimulated either with A23187 or fors kolin. However, no effect was observed. This speaks against a role of the ORIC Cl- channel in the Ca2+-activated Cl- conductance, although i t cannot definitely be excluded.