(667) EMC is Required for CFTR Biogenesis and Activation in vivo

Background: Cystic Fibrosis Transmembrane-conductance Regulator, CFTR, is a multi-transmembrane protein which functions as a chloride/bicarbonate channel and plays an important role in maintaining water homeostasis on the apical surface of epithelial cells throughout the whole body. Mutations in the cftr gene leading to loss/defective CFTR function causes cystic fibrosis (CF). Among the effort to enhance mutant CFTR expression/function on the plasma membrane, intensive studies have focused on the cellular mechanisms mediating CFTR degradation. However, CFTR biogenesis machineries are not well understood, and improved understanding of these machineries could potentially enhance mutant CFTR expression.

Endoplasmic Reticulum (ER) membrane protein complex (EMC) is a well-conserved ubiquitous protein complex expressed on the ER membrane. It is recognized as a membrane domain insertase and plays a critical role in mediating the biosynthesis of multi-transmembrane proteins[1, 2], especially the ion channels. However, whether EMC regulates CFTR biogenesis is not clear.

Methods: In this study, we generated intestinal epithelial cell specific EMC subunit 3 (EMC3) knockout (KO) mice by crossing EMC3 flox mice with Villin-Cre mice. Intestinal crypts were harvested and analyzed by RT-PCR, Mass-spectrometry (MS) and Western blotting. Intestinal organoids were used to study CFTR function through cAMP- and Calcium-dependent pathways as well as monitor calcium flux between wildtype (WT) and KO mice.

Results: Although EMC3 KO mice were viable after birth, they were smaller compared to their WT littermates, implicating a functional impairment of intestinal epithelium in EMC3 KO mice. Molecular analysis of the protein profile collected by MS of intestinal crypts from these mice revealed the downregulation of the EMC complex, CFTR and many other ion transporters. Western blot confirmed a significant reduction of CFTR protein in EMC3 KO villi while CFTR transcription was not altered. Consistently, forskolin- and cpt-cAMP-stimulated intestinal organoid fluid secretion was greatly reduced and delayed (Figure 1A). Interestingly, we found that EMC3 deficiency completely inhibited carbachol-mediated intestinal organoid fluid secretion (Figure 1B) while muscarinic receptors (carbachol receptors) were upregulated at both transcriptional and translational levels in EMC3 KO villi. These data indicated that downstream signaling of muscarinic receptors were compromised in EMC3 KO epithelial cells. Indeed, muscarinic receptor specific agonist, Oxotremorine M, failed to induce intracellular calcium flux as well as fluid secretion in EMC3 KO organoids. Pathway analysis of the above MS data supported these observations.

Conclusions: We conclude that EMC plays a critical role in vivo in CFTR biogenesis and activation, especially the Ca²⁺-mediated CFTR activation. Targeting the EMC complex could potential enhance mutant CFTR expression/function.

Acknowledgements: The authors would like to thank Dr. Jim Chmiel for editing. This study was supported by CFF postdoc-to-faculty transition award (HUANG20F5).

References: [1] O'Donnell JP, Phillips BP, Yagita Y, Juszkiewicz S, Wagner A, Malinverni D, et al. The architecture of EMC reveals a path for membrane protein insertion. Elife. 2020;9.

[2] Bai L, You Q, Feng X, Kovach A, Li H. Structure of the ER membrane complex, a transmembrane-domain insertase. Nature. 2020;584(7821):475-8.