Investigation of Protein kinase C-mediated internalization of the Na⁺K⁺2Cl^-cotransporter 1 in Madin-Darby Canine Kidney Cells

In the colon, the Cl - -driven fluid secretion protects the epithelium by expanding the mucus layer and flushing away pathogens or toxins. Dysregulation of Cl - secretion is the hallmark of diseases such as diarrhea or cystic fibrosis. The vectorial transport of Cl - across the epithelium depends on the basolateral Na + -K + -2Cl - cotransporter 1 (NKCC1) and apical chloride channels. NKCC1 is an important and independent site that regulates Cl - secretion. Previous work has shown that activation of protein kinase C (PKC) causes NKCC1 endocytosis, which decreases Cl - secretion. However, the PKC-dependent posttranslational modification causing NKCC1 endocytosis is not known. Ubiquitin has been reported to be a signal causing endocytosis of the Na + -Cl - cotransporter during PKC activation. Thus, we hypothesize that PKC-dependent ubiquitination of NKCC1 may be a signal for its endocytosis. We used Madin-Darby Canine Kidney (MDCK) cells stably expressing eGFP-NKCC1 to monitor NKCC1 endocytosis by fluorescence microscopy. Phorbol 12-myristate 13-acetate (PMA) was used to activate PKC and PYR-41 to inhibit the ubiquitin ligase E1. We combined PYR + PMA to test whether inhibiting the ubiquitin ligase blocks the effect of PKC on NKCC1 endocytosis. To quantify our results, we used ImageJ to count the number of endocytosed vesicles containing eGFP-NKCC1 and the number of nuclei. A one-way ANOVA found a significant difference within our conditions (P < 0.001). A Tukey’s post-hoc test shows that the number of vesicles/cell in control (1.2 ± 0.3, n = 51) is not significantly different from DMSO (1.7 ± 0.5, n = 24, P = 0.9), the solvent for PMA and PYR. PMA (14.8 ± 2.3, n = 27), our positive control for inducing NKCC1 endocytosis, significantly increases the number of vesicles/cell compared to control (P < 0.001). Surprisingly, PYR alone (8.0 ± 0.9, n = 47) significantly increases the number of vesicles/cell compared to control (P < 0.01). PYR alone has significantly less vesicles/cell compared to PMA (P < 0.03). Finally, PYR + PMA (11.3 ± 2.1, n = 54) is not significantly different from PYR nor PMA (P = 0.4 and 0.5 respectively). Our results suggest that blocking the ubiquitin ligase E1 with PYR unmasked a novel pathway for NKCC1 internalization that may be SUMOylation dependent. PKC-activation induces NKCC1 internalization may be ubiquitin-independent, or one of the previously identified PKC isoforms (PKCε or PKCδ) acts via ubiquitination and the other via SUMOylation. Our results provide new insight on the complex regulation of fluid secretion by NKCC1. A better understanding of this mechanism may help in the future to develop therapeutic approaches for secretory diarrhea or cystic fibrosis. Indiana Space Grant Consortium, NIH 1R15DK139519-01 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.