NS510, a High Affinity Fluorescent Catecholamine Sensor for Monitoring Norepinephrine Exocytosis

Abstract

Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations in the dystrophin gene, is characterized by progressive muscle degeneration/ weakness. Enhanced store-operated Ca 2þ entry (SOCE), a Ca 2þ influx mechanism coordinated by STIM1 and Orai1, is proposed to potentiate Ca 2þ -mediated damage in DMD. To determine the role of SOCE in promoting muscular dystrophy, we crossed mdx mice with tamoxifen-inducible, musclespecific Orai1 knockout mice (mdx-Orai1 KO). Analysis of transcript levels revealed that Orai1 expression was $1.5x higher in muscle from mdx mice compared to WT mice, while nearly undetectable in muscle from mdx-Orai1 KO mice. Levels of Orai1 transcript correlated with SOCE activity measured using Mn 2þ quench of fura-2 fluorescence in single flexor digitorum brevis muscle fibers. Specifically, SOCE was increased in mdx fibers and absent in fibers from mdx-Orai1 KO mice. Moreover, compared to WT fibers, mdx fibers exhibited: i) increased myoplasmic Ca 2þ levels; ii) reduced Ca 2þ store content; and iii) a significant prolongation in the rate of electrically-evoked Ca 2þ transient decay. All of these effects were normalized in fibers from mdx-Orai1 KO mice. Ex vivo contractility experiments revealed that extensor digitorum longus muscles from mdx mice displayed a reduction in maximal tetanic specific force (150.353.5 mN/mm 2 ) compared to WT muscles (199.6511.1 mN/mm 2 ), a parameter ameliorated in muscles from mdx-Orai1 KO mice (188.1510.9 mN/mm 2 ). When exposed to 10 consecutive eccentric contractions, mdx muscles exhibited a higher damage-induced force reduction than WT muscles (residual force after the 10 th eccentric contraction was 35.553.8% and 61.851.9%, respectively). Interestingly, Orai1 ablation partially protected muscles from damage in mdx-Orai1 KO mice (residual force was 46.152.6% after 10 contractions). Our results indicate that enhanced Orai1dependent SOCE exacerbates the dystrophic phenotype and Orai1 deficiency promotes sarcolemmal integrity/stability during eccentric contraction. Adrenal chromaffin cells (ACCs) comprise the neuroendocrine arm of the sympathetic nervous system and secrete catecholamines to coordinate the physiological response to stress. Although SERT is prominently expressed in ACCs, its role there has remained unclear. Our data show that SERT interacts with 5-HT 1A recep-