Western Blot Identification of Dopamine and GABA Receptors in Gill of the Bivalve Crassostrea viginica

Abstract

Gill lateral cells of the bivalve Crassostrea virginica are innervated by serotonin and dopamine (DA) nerves from their ganglia and regulate cilia beating rates. DA slows down cilia beating and serotonin speeds them up. GABA a neurotransmitter in vertebrates and many invertebrates rarely has been reported in bivalves. We showed GABA is present in C. virginica and GABA acts as a ganglionic neurotransmitter modulating gill lateral cell cilia activity. We used immunohistofluorescence to localize GABA receptors in ganglia and gill and identify DA receptors in gill lateral cells as D2‐like (D2DR). We hypothesize Western Blot (WB) would verify D2DR and GABA receptors in gill. For WB gill cell lysate was prepared by in NP‐40 detergent buffer containing protease inhibitor, followed by centrifuging to obtain supernatant with solubilized membrane proteins. Up to 30 µg of solubilized protein was subjected to SDS‐PAGE with 10% acrylamide gels and electroblotted onto nitrocellulose. D2DR and GABA receptor immunoreactivity was revealed by 1̊ antibodies followed by HRP‐conjugated 2̊ antibodies. WB showed 70 ‐ 75 kD bands corresponding to DA D2DR and GABA RA1‐6 receptors in gill. The present project allows us to confirm our previous studies showing the presence of DA and GABA and furthers the understanding of their physiological roles in C. virginica.