The molecular correlate of hypertonicity-induced cation channels (HICCs) and their role in proliferation vs. apoptosis is a matter of debate. We report here that, in whole-cell patch-clamp recordings, hypertonic stress (340 ® 450 mosM) reversibly increased the Na⁺ conductance of HepG2 cells from 0.8 to 5.8 nS. The effect was dose-dependently inhibited by flufenamate and amiloride, known blockers of HICCs, with some 50% efficiency at 300 mM. In parallel, both drugs decreased HepG2 cell proliferation (in MTT assays and with automatic cell counting). siRNA silencing of the a-subunit of the epithelial Na⁺ channel (ENaC) reduced hypertonicity-induced Na⁺ currents to 60% whereas the rate of HepG2 cell proliferation was approx. half of that of control. Moreover, a-ENaC siRNA inhibited the regulatory volume increase (RVI) of HepG2 cells (measured with scanning acoustic microscopy) by 60%. In FACS measurements, silencing of a-ENaC led to a significant decrease in the G1 and an increase in the G2/M phase of the cell cycle whereas the S phase was not changing. Finally (determined by a caspase 3/7 assay), HICC inhibition by flufenamate and Gd³⁺ as well as siRNA silencing of a-ENaC increased the rate of apoptosis in HepG2 cells. These data strongly suggest that a-ENaC is one of the functional elements of the HICC in HepG2 cells and that this channel subunit is an important mediator of cell proliferation; likewise, HICC blockage shifts the system from a proliferative into an apoptotic one.

The molecular partners of a-ENaC in completing the architecture of the HICC are currently determined by use of the split-ubiquitin yeast two-hybrid membrane system. As a further approach, a high-throughput screening of siRNA libraries is performed employing scanning acoustic microscopy, thus functionally relating the process of regulatory volume increase to various ion transporters, channels and putative regulators.