Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta

Abstract

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP ₃R-mediated Ca ²⁺ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca ²⁺, associated with deficient store-operated calcium entry, although SERCA and IP ₃R have normal stability. Notably, steady state ER Ca ²⁺ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca ²⁺ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca ²⁺-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca ²⁺ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

Author Summary

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissues characterized by fracture susceptibility and growth deficiency. Most OI cases are caused by autosomal dominant mutations in the genes encoding type I collagen, COL1A1 and COL1A2. Delineation of novel gene defects causing dominant and recessive forms of OI has led to the understanding that the bone pathology results not only from abnormalities in type I collagen quantity and primary structure, but also from defects in post-translational modification, folding, intracellular transport and extracellular matrix incorporation. Recently, mutations in TMEM38B, which encodes the integral ER membrane K ⁺ channel TRIC-B, have been identified as causative for the OI phenotype. However, the mechanism by which absence of TRIC-B causes OI has not been reported. Using cell lines established from three independent probands, we have demonstrated that absence of TRIC-B leads to abnormal ER Ca ²⁺ flux and store-operated calcium entry (SOCE), although ER steady state Ca ²⁺ is normal. Disruption of intracellular calcium dynamics alters the expression and activity of multiple collagen interacting chaperones and modifying enzymes within the ER. Thus TRIC-B deficiency causes OI by dysregulation of collagen synthesis, through the impairment of calcium-dependent gene expression and protein-protein interactions within the ER.