Dot1a‐AF9‐AF17‐mediated transcriptional changes in the epithelial sodium channel (ENaC) are translated into changes in ENaC‐mediated Na+ transport

Abstract

We previously reported that Dot1a‐AF9 complex inhibits the transcription of α‐ENaC by hypermethylating histone H3 K79 at its promoter. Aldosterone downregulates this complex through various mechanisms. AF17 upregulates transcription of all ENaC genes and their key regulators by competitively binding to and promoting nuclear export of Dot1a. However, the significance of these transcriptional mechanisms in regulating Na+ transport has not been fully defined. To address this question, mouse inner medullary collecting duct cell line‐3 (mIMCD3) and mouse cortical collecting duct M1 cells were used to determine ENaC activity as measured by benzamil‐sensitive intracellular Na+ concentration ([Na+]i) using fluorescence imaging or benzamil‐sensitive equivalent short circuit current (Isc) using an EVOM ohm/volt meter. In both cell lines, addition of benzamil significantly decreased the basal [Na+]i and Isc values. Benzamil‐sensitive [Na+]i or Isc values were significantly decreased by overexpression of Dot1a or AF9, but increased by AF17 overexpression. The effect of aldosterone on benzamil‐sensitive [Na+]i and Isc was impaired by blocking either transcription or translation. Our data suggest that ENaC is largely responsible for Na+ uptake in these cells, and that transcriptional changes in ENaC genes mediated by Dot1a, AF9, AF17 or aldosterone treatment are translated into changes in ENaC‐mediated Na+ transport.