The electroneutral KCl cotransporter (KCC) plays a role in cell volume regulation, transepithelial transport, and in the regulation of intracellular chloride concentration. Four KCC isoforms (KCC1-4) have been cloned. Human cervical carcinogenesis is accompanied by the upregulation of mRNA transcripts in KCC1, KCC3 and KCC4 (Shen et al. 2000). The up-regulation of volume-sensitive KCC activity is also associated with human cervical carcinogenesis. KCC works synergistically with ion channels for volume regulation of human cervical cancer cells. Truncation of the N-terminal 117 amino acids of KCC1 diminished KCC function and exhibited dominant negative phenotypes for KCC1, KCC3 and KCC4 (Casula et al. 2001). To study the physiological roles of KCC, we developed the stable transfection of N-terminal truncation of KCC1 mutant in human cervical cancer SiHa cell lines. KCC activities are studied using ⁸⁶Rb⁺ as a congener of K⁺. In the wild-type cervical cancer SiHa cells, the KCC activity is nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge, 1 mM staurosporine and 1 mM N-ethylmaleimide (NEM). In contrast, KCC1 mutant cervical cancer cells have KCl cotransport which is also quiescent in normal physiological conditions and is relatively refractory to hypotonic stimulation, and treatment with NEM or staurosporine. Basal intracellular Cl^– concentration in wild-type cervical cancer cells is 30 ± 2 mM (mean ± S.E.M., n = 5) estimated by ³⁶Cl^– equilibrium, and KCC1 mutant transfection significantly increases the intracellular Cl^– concentration to 46 ± 3 mM (n = 5, P < 0.05, Student’s unpaired t test). Surprisingly, for wild-type cervical cancer cells, the swelling-activated Cl^– current is 70 ± 3 pA pF^-1 (n = 30) at +100 mV and the current density is significantly decreased to 30 ± 2 pA pF^-1 (n = 20; P < 0.01) for KCC1 mutant cervical cancer cells. In addition to altering the current amplitude, the activation rate of the swelling-activated Cl^– channel is also significantly decreased (wild-type: 0.8 ± 0.1 pA pF^-1 s^-1 at +100 mV, n = 30 vs. KCC1 mutant: 0.35 ± 0.08 pA pF^-1 s^-1, n = 20, P < 0.01). Consistently, the swelling-activated taurine efflux rate constant decreases significantly from 0.06 ± 0.002 min^-1 (n = 6) in wild-type cervical cancer cells to 0.032 ± 0.001 min^-1 (n = 6) in KCC1 mutant cells (P < 0.05). KCC1 mutant cervical cancer cells display a slower growth curve. More importantly, the active forms of Rb and cdc2 kinase, two key regulators controlling cell cycle progression, are decreased in the KCC1 mutant cells. Considering the results overall, KCC plays an important role in the volume regulation and cell proliferation of human cervical cancer cells. Interestingly, reducing regulatory volume decrease (RVD) via inhibition of KCC1 also lowers the swelling-activated Cl^– currents, suggesting an interaction at either the signallings or membrane activity level for the process of RVD. In addition, overexpression of N-terminal mutant offers a unique non-pharmacological tool for functional inhibition of KCC gene products.

Meng-Ru Shen thanks the Swire Group for the scholarship.