A novel calcium channel Cavß2 splice variant with unique properties predominates in the retina.

Cavß subunits are essential for surface expression of voltage-gated calcium channel complexes and crucially modulate biophysical properties like voltage-dependent inactivation. Here, we describe the discovery and characterization of a novel Cavß2 variant with distinct features that predominates in the retina. We determined spliced exons in retinal transcripts of the Cacnb2 gene, coding for Cavß2, by RNA-Seq data analysis and quantitative PCR. We cloned a novel Cavß2 splice variant from mouse retina, which we are calling ß2i, and investigated biophysical properties of calcium currents with this variant in a heterologous expression system as well as its intrinsic membrane interaction when expressed alone. Our data showed that ß2i predominated in the retina with expression in photoreceptors and bipolar cells. Furthermore, we observed that the ß2i N-terminus exhibited an extraordinary concentration of hydrophobic residues, a distinct feature not seen in canonical variants. The biophysical properties resembled known membrane-associated variants, and ß2i exhibited both a strong membrane association and a propensity for clustering, which depended on hydrophobic residues in its N-terminus. We considered available Cavß structure data to elucidate potential mechanisms underlying the observed characteristics but resolved N-terminus structures were lacking and thus, precluded clear conclusions. With this description of a novel N-terminus variant of Cavß2, we expand the scope of functional variation through N-terminal splicing with a distinct form of membrane attachment. Further investigation of the molecular mechanisms underlying the features of ß2i could provide new angles on the way Cavß subunits modulate Ca2+ channels at the plasma membrane.

Effects of alpha-calcitonin gene-related peptide on osteoprotegerin and receptor activator of nuclear factor-κB ligand expression in MG-63 osteoblast-like cells exposed to polyethylene particles.

BACKGROUND: Recent studies demonstrated an impact of the nervous system on particle-induced osteolysis, the major cause of aseptic loosening of joint replacements. METHODS: In this study of MG-63 osteoblast-like cells we analyzed the influence of ultra-high molecular weight polyethylene (UHMWPE) particles and the neurotransmitter alpha-calcitonin gene-related peptide (CGRP) on the osteoprotegerin/receptor activator of nuclear factor-κB ligand/receptor activator of nuclear factorκB (OPG/RANKL/RANK) system. MG-63 cells were stimulated by different UHMWPE particle concentrations (1:100, 1:500) and different doses of alpha-CGRP (10-7 M, 10-9 M, 10-11 M). RANKL and OPG mRNA expression and protein levels were measured by RT-PCR and Western blot. RESULTS: Increasing particle concentrations caused an up-regulation of RANKL after 72 hours. Alpha-CGRP showed a dose-independent depressive effect on particle-induced expression of RANKL mRNA in both cell-particle ratios. RANKL gene transcripts were significantly (P < 0.05) decreased by alpha-CGRP treatment after 48 and 72 hours. OPG mRNA was significantly down-regulated in a cell-particle ratio of 1:500 after 72 hours. Alpha-CGRP concentrations of 10-7 M lead to an up-regulation of OPG protein. CONCLUSION: In conclusion, a possible osteoprotective influence of the neurotransmitter alpha-CGRP on particle stimulated osteoblast-like cells could be shown. Alpha-CGRP might be important for bone metabolism under conditions of particle-induced osteolysis.