The calcium-activated chloride (CLCA) channel is expressed in tissues that are affected by cystic fibrosis (CF) and has been suggested as a possible alternative target for pharmacotherapy in the treatment of CF. To be able to develop compounds that activate this protein we need information on its cellular distribution and molecular regulation. Here we report on initial studies that are investigating one of these cloned CLCA channel proteins, GOB5 (or mCLCA3), as identified by Komiya et al. (1999).

An anti-GOB5 selective antibody was generated in rabbits to a peptide corresponding to the sequence KLETFKNAD (95-103) from the GOB5 amino acid sequence (GenBank Accession No. AB017156), following previously successful strategies (Chazot et al. 1994). All animal procedures were performed in accordance with UK legislation.

Human embryonic kidney cells (HEK293 cell line) were utilised to transiently co-express the GOB5 cDNA and the reporter gene, Green Fluorescent Protein (GFP), using a well-established calcium phosphate methodology (Chazot et al. 1994). In order to confirm the expression of the GOB5 protein in the HEK cells, transfected cell homogenates, prepared 24 h post-transfection, were probed using immunoblotting with the affinity-purified anti-GOB5 antibody. The anti-GOB5 antibody recognised a protein species (M_r 90 000) in HEK cells transfected with the cDNA encoding the GOB5 gene, which was not present in parent HEK cells, or HEK cells transfected in parallel with a control cDNA plasmid. This immunoreactive species corresponds well with the predicted size of the GOB5 gene product.

Transiently transfected HEK cells were also used for electrophysiological characterisation of GOB5. Positive transfectants were selected by their GFP fluorescence. In control HEK cells basal whole-cell current densities at E_rev ± 60 mV were 59 ± 17 and 56 ± 16 pA pF^-1 and the E_rev was -21.9 ± 2.9 mV (mean ± S.E.M.; n = 13); bath addition of 1 µM ionomycin had no effect. In GOB5-transfected HEK cells whole-cell current densities at E_rev ± 60 mV were 230 ± 47 and 190 ± 43 pA pF^-1 and the E_rev was -5.5 ± 0.9 mV (n = 7, P ▓le│ 0.003, Mann-Whitney); again bath addition of 1 µM ionomycin had no effect. Furthermore, with 10 mM EGTA in the pipette solution, these whole-cell currents were reduced to 82 ± 23 and 86 ± 25 pA pF^-1 (n = 5).

These initial data suggest that the GOB5 cDNA results in the expression of a calcium-dependent chloride current.

This work was supported by The Wellcome Trust and Royal Society.