Metabolic characterization and GAPDH dependent regulation of ENaC in hPheo1 wild-type and SDHB knockdown cells

by Selleckchem | Mar 08 2023

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells providing an important area of investigation in understanding SDHB-related disease.

Comments：

The study you described investigates the expression and activity of epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) in a progenitor cell line derived from a human pheochromocytoma (PCC). The researchers compared hPheo1 wild-type (WT) cells to cells in which succinate dehydrogenase subunit B (SDHB) had been knocked down (KD). SDHB mutations are associated with an increased risk of malignant and aggressive disease in patients with PCC and paraganglioma (PGL).

The researchers first performed targeted metabolomic studies to quantify changes in glycolysis pathway intermediates and citric acid cycle intermediates in the two types of cells. Next, they performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity of ENaC and ASIC ion channels in the two cell types.

The results of the study showed that both ENaC alpha and ASIC1/2 were expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborated these results and further indicated that channel activity was decreased in SDHB KD cells. Additionally, experiments showed a more significant decrease in membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells.

Overall, the study provides evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, which could have implications for understanding SDHB-related disease and potential targets for future treatment options.