Specific small interfering RNA (siRNA) constructs were used to test for the functional relation of subunits α, β, and γ of the epithelial Na + channel (ENaC) to the hypertonicity-induced cation channel (HICC) in confluent rat hepatocytes. In current-clamp recordings, hypertonic stress (300 → 400 mosM) increased membrane conductance from 75.4 ± 9.4 to 91.1 ± 11.2 pS ( p < 0.001). The effect was completely blocked by 100 μM amiloride and reduced to 46, 30, and 45% of the control value by anti-α-, anti-β-, and anti-γ-rENaC siRNA, respectively. Scanning acoustic microscopy revealed an initial shrinkage of cells from 6.98 ± 0.45 to 6.03 ± 0.43 pl within 2 min. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 ± 0.05 pl ( p < 0.001). With anti-α-, anti-β-, and anti-γ-rENaC siRNA, the volume response was reduced to 31, 31, and 36% of the reference level, respectively. It is concluded that all three subunits of the ENaC are functionally related to RVI and HICC activation in rat hepatocytes.

Hypertonicity-induced cation channels (HICCs) are a substantial element in the regulatory volume increase (RVI) of osmotically shrunken cells. Under isotonic conditions, they are key effectors in the volume gain preceding proliferation; HICC repression, in turn, significantly increases apoptosis rates. Despite these fundamental roles of HICCs in cell physiology, very little is known concerning the actual molecular architecture of these channels. Here, an siRNA screening of putative ion channels and transporters was performed, in HepG2 cells, with the velocity of RVI as the read-out; in this first run, δENaC, TRPM2 and TRPM5 could be identified as HICCs. In the second run, all permutations of these channels were tested in RVI and patch-clamp recordings, with special emphasis on the non-additivity and additivity of siRNAs - which would indicate molecular interactions or independent ways of channel functioning. At first sight, the HICCs in HepG2 cells appeared to operate rather independently. However, a proximity ligation assay revealed that δENaC was located in proximity to both TRPM2 and TRPM5. Furthermore, a clear synergy of HICC current knock-downs (KDs) was observed. δENaC, TRPM2 and TRPM5 were defined as mediators of HepG2 cell proliferation and their silencing increased the rates of apoptosis. This study provides a molecular characterization of the HICCs in human hepatocytes and of their role in RVI, cell proliferation and apoptosis.