Background: PA is the commonest curable cause of hypertension. Whole exome sequencing (WES) in 2011 and 2013 identified common somatic mutations in genes regulating membrane polarisation in 60-80% of aldosterone-producing adenomas (APA). In search of the missing variants, we undertook further WES. One APA from a 46-year-old gentleman revealed a novel somatic mutation (Val380Asp); which introduced a charged amino-acid into the single transmembrane domain of Cell Adhesion Molecule 1 (CADM1). The search for similar mutations led to a Gly379Asp mutation from a PA patient in Munich.
Method: Wild-type (WT) and mutant CADM1 genes were cloned into lentivirus vectors and transduced into adrenocortical (H295R) cells to assess its effect on aldosterone secretion. Previous studies suggest CADM1 contributes to gap junction (GJ) communication between cells. This was assessed using dye transfer assays. Single H295R cells transfected with WT or mutant CADM1 were microinjected with a calcein dye permeable through GJs, and the effects observed using fluorescent microscopy. H295R cells were also treated with peptide gap27, a specific inhibitor of the adrenocortical GJ protein CX-43. Finally WT or mutant H295R cells were co-transfected with CX-43 tagged by mApple or Venus fluorophores and mixed, allowing confocal visualisation of GJ formation between adjacent cells.
Results: Cells transduced with mutant CADM1 showed 3-6-fold increase in aldosterone secretion (P<0.05) and 10-20-fold increase in CYP11B2 expression (P<0.05) compared to WT. RNA sequencing showed CYP11B2 to be the most upregulated gene (30×) in mutant cells. Dye transfer assays showed paucity of dye transfer between neighbouring mutant CADM1 cells, while calcein passed easily through GJs in WT cells. Inhibition of CX-43 caused 2-fold increase in aldosterone secretion, and 3 to 8-fold (<0.05) increase in CYP11B2 expression in non- and angiotensin-II stimulated cells respectively. Protein modelling suggested that mutations increased the angle of ectodomains to cell membrane, from 49o in WT cells, to 62o and 90o in Gly379Asp and Val380Asp respectively; increasing inter-cell distance from 21.2 nm to 24.7 and 27.9 nm. A key role of CADM1 may be to bring opposing CX-43 hemichannels close enough to form GJ channels. Mixing of Venus and mApple-tagged CX-43 transfected cells showed fewer intact GJ channels in CADM1-mutant cells.
Conclusion: Discovery of the CADM1 mutation has again demonstrated the importance of membrane proteins in aldosterone regulation, although CADM1s impact on cation traffic is indirect. The unsuspected role of cell-adhesion in regulating GJs suggests a role for these in the regulation of aldosterone by oscillating Ca2+ currents.
18 - 21 May 2019
European Society of Endocrinology