WNK463 stimulates K-Cl cotransport independently of WNK kinase activity

K-Cl cotransport (KCC) mediates the Na⁺-independent, tightly coupled movement of K⁺ and Cl^- across biological membranes and is characteristically activated by cell swelling. Here, we show that swelling-activated KCC-mediated K⁺ influx is absent in eight transformed mammalian cell lines, including HEK293-T cells. Nevertheless, KCC function can be induced in all these cells by treatment with the with-no-lysine kinase (WNK) inhibitor WNK463. Activation of KCC-mediated K⁺ influx requires concentrations of WNK463 that are approximately two orders of magnitude higher than those needed to inhibit Na-K-2Cl cotransporter 1 (NKCC1). This marked difference suggests that the effect of WNK463 on KCC is not mediated by inhibition of WNK kinases themselves but rather through one or more alternative kinase targets. This conclusion is further supported by experiments performed in cells in which all four WNK kinase genes were disrupted. In these cells, WNK463 retained its ability to activate KCC, demonstrating that its effect is independent of WNK kinase expression. The WNK463 effect exhibits measurable activation and deactivation kinetics, with an activation time constant of approximately 18 minutes and an inactivation time constant of approximately 50 minutes. To identify the kinase(s) responsible for WNK463-induced activation of KCC, we screened a kinase inhibitor library containing approximately 1,100 compounds. Activation of KCC was PP121 sensitive and PI-103 insensitive, suggesting involvement of tyrosine kinases. Collectively, these results indicate that transformed mammalian cell lines possess a kinase-dependent mechanism that suppresses KCC activity. Both WNK463 and PP121 can relieve this suppression, but the responsible kinase appears to be distinct from WNK kinases and remains unidentified. Further studies will be required to define the signaling pathway that prevents K-Cl cotransport activity in these cells.NEW & NOTEWORTHY This study provides evidence that cells in culture that divide indefinitely and escape control of normal cellular growth (or "transformed" cells) fail to demonstrate swelling-activated K⁺ influx but exhibit the with-no-lysine kinase (WNK) inhibitor WNK463-induced K-Cl cotransport. As K-Cl cotransport activation by WNK463 still occurs in cells targeted for WNK kinase, the mechanism at play cannot be a WNK kinase. This work provides the framework and rationale for the discovery of a novel WNK463 target.