Bombyx mori Homologs of STIM1 and Orai1 Are Essential Components of the Signal Transduction Cascade That Regulates Sex Pheromone Production

Abstract

Sex pheromone production in the pheromone gland (PG) of the silkmoth, Bombyx mori, is mediated by store-operated channels (SOCs) acting downstream of pheromone biosynthesis activating neuropeptide (PBAN) binding. Although recent studies have implicated STIM1 and Orai1 as essential components of SOCs, little is known about the molecular nature of the SOCs involved in sex pheromone production. In this study we cloned silkmoth homologs of STIM1 and Orai1 and sought to determine whether they comprise the PG SOC pathway. BmSTIM1 is expressed in multiple tissues and, in the PG, is encoded by two transcripts of differing size. BmOrai1A and BmOrai1B, which are identical except for a 37-residue N-terminal truncation in BmOrai1B, arise from alternative splicing of the bmorai1 locus and are expressed as independent transcripts in various tissues. In the PG, only BmOrai1B is actively transcribed. Fluorescent chimeras demonstrated that BmSTIM1 expression is restricted to the endoplasmic reticulum, whereas both BmOrai1A and BmOrai1B localize to the cell surface. In Ca(2+)-free medium, thapsigargin-mediated depletion of endoplasmic reticulum Ca(2+) stores resulted in redistribution of BmSTIM1 to the plasma membrane, but only when the BmOrai1 homologs were also overexpressed. Translocation was dependent on the BmSTIM1 C terminus "CRAC activation domain." Ala mutation of Lys(380), Lys(383), Lys(384), Arg(382), and Arg(385) suggests that translocation involves electrostatic interactions. Translocation was also seen following PBAN stimulation in cells co-expressing BmSTIM1, BmOrai1B, and the PBAN receptor. In vivo RNA interference-mediated knockdown of BmSTIM1 and BmOrai1 significantly reduced sex pheromone production without affecting cell viability.