The inv mouse exhibits reversal of the left-right body plan and develops renal cysts (Yokayama et al. 1993). The protein encoded by the invs gene, inversin (Morgan et al. 1998), binds calmodulin and is highly expressed in the primary cilium of renal cells (Morgan et al. 2002a,b). Other proteins (e.g. polycystin-1 and polycystin-2), mutation of which also causes renal cystic disease, are also expressed in the primary cilium and are involved in Ca²⁺ signalling (Nauli et al. 2003). To establish whether Ca²⁺ signalling is disrupted in inv cells, we plan to use apically located Ca²⁺-activated Cl^– channels (CACC) as sensitive detectors of intracellular Ca²⁺ concentration. As a first step, it is necessary to exclude the possibility that expression and/or regulation of CACC per se is altered by the inv genotype.

Renal collecting ducts were isolated from wild-type (WT), heterozygote (Het) and homozygous (Hom) inv mice (which were humanely killed) and cultured in OPTIMEM 1 medium. Primary epithelial cells growing out from the ducts were used for patch clamping after 2-4 days. Capacitance measurements were (means ± S.E.M.): WT, 17.0 ± 4.2 pF (n = 3 cells); Het, 14.1 ± 0.7 pF (n = 4 cells); Hom, 12.4 ± 1.3 pF (n = 7 cells), indicating no genotypic related differences in cell surface area. Baseline currents were normally small (< 15 pA pF^-1 at ± 60 mV) in all three genotypes. Ionomycin (1 µM) evoked large Cl^–-selective currents with time-dependent kinetics typical of CACC (WT, 166.5 ± 40.2 pA pF^-1 and -111.2 ± 26.9 pA pF^-1; Het, 274.6 ± 87.5 pA pF^-1 and -186.3 ± 54.3 pA pF^-1; Hom, 161.2 ± 26.2 pA pF^-1 and -130.3 ± 21.6 pA pF^-1 at ± 60 mV respectively with n values as above). There were no significant differences in CACC density between the genotype groups (P > 0.05, ANOVA). In four paired experiments using homozygous inv cells, ionomycin increased currents to 112.9 ± 21.5 pA pF^-1 and -93.0 ± 18.7 pA pF^-1 at ± 60 mV respectively. Subsequent reduction of bath Ca²⁺ from 2 mM to 0.1 µM (maintaining ionomycin at 1 µM) caused current density to decline to 6.6 ± 2.8 pA pF^-1 and -7.9 ± 2.4 pA pF^-1 at ± 60 mV, respectively.

We conclude that homozygous inv cells have a normal complement of CACC and that the channels can be activated normally by the intracellular Ca²⁺ signals generated by 1 µM ionomycin.

This work was funded by the Newcastle University Hospitals Special Trustees.