Characterization of two distinct Cl- conductances in fused human respiratory epithelial cells. II. Relation to cystic fibrosis gene product.

摘要

The present microelectrode experiments on fused respiratory epithelial cells of cystic fibrosis (CF) origin and non-CF origin aim at characterizing the molecular basis of the Cl- conductances regulated by cyclic adenosine monophosphate (cAMP) or respectively Ca2+, as described in the preceding publication. Cell membrane potential (Vm) and resistance (Rm) were recorded as well as their response to substitution of 90% of bath Cl- by isethionate (delta Vm,ISE), by I- (delta Vm,I), or by other halide anions. Fused CF cells had significantly (P < 0.05) higher control Vm values (-18.0 +/- 9.4 mV, +/- SD, n = 68) than fused non-CF cells (-12.5 +/- 6.6 mV, n = 69) and responded to the Ca2+ ionophore A23187 with an increase in the Vm response to Cl- substitution, but did not respond to forskolin. This indicates that CF cells express only the Ca(2+)-stimulated Cl- conductance. Injection of the antibody 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 Vm,ISE and delta Vm,I within 15 min to values observed in CF cells. This indicates inhibition of the cAMP-stimulated Cl- conductance and supports the molecular identity of this conductance with the CF gene product. However, the slow onset of inhibition does not allow secondary effects to be excluded and a slight fall in Rm remains unexplained. Stimulation of the Ca(2+)-regulated Cl- conductance was not impaired. Injection of M3A7 into CF cells or of a control antibody in non-CF cells had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)