Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons.

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [(3)H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca(2+) oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca(2+) dynamics the in neurons.

The action of selective CRAC channel blockers is affected by the Orai pore geometry.

As the molecular composition of calcium-release activated calcium (CRAC) channels has been unknown for two decades, elucidation of selective inhibitors has been considerably hampered. By the identification of the two key components of CRAC channels, STIM1 and Orai1 have emerged as promising targets for CRAC blockers. The aim of this study was to thoroughly characterize the effects of two selective CRAC channel blockers on currents derived from STIM1/Orai heterologoulsy expressed in HEK293 cells. The novel compounds GSK-7975A and GSK-5503A were tested for effects on STIM1 mediated Orai1 or Orai3 currents by whole-cell patch-clamp recordings and for the effects on STIM1 oligomerisation or STIM1/Orai coupling by FRET microscopy. To investigate their site of action, inhibitory effects of these molecules were explored using Orai pore mutants. The GSK blockers inhibited Orai1 and Orai3 currents with an IC(50) of approximately 4µM and exhibited a substantially slower rate of onset than the typical pore blocker La(3+), together with almost no current recovery upon wash-out over 4min. For the less Ca(2+)-selective Orai1 E106D pore mutant, I(CRAC) inhibition was significantly reduced. FRET experiments indicated that neither STIM1-STIM1 oligomerization nor STIM1-Orai1 coupling was affected by these compounds. These CRAC channel blockers are acting downstream of STIM1 oligomerization and STIM1/Orai1 interaction, potentially via an allosteric effect on the selectivity filter of Orai. The elucidation of these CRAC current blockers represents a significant step toward the identification of CRAC channel-selective drug compounds.

Canonical transient receptor potential (TRPC) 1 acts as a negative regulator for vanilloid TRPV6-mediated Ca2+ influx.

TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca(2+) selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca(2+) and Na(+) currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca(2+) influx in HEK293 cells.