Characterization of two novel ammonia transporters, HIAT1α and HIAT1β, in the American Horseshoe Crab, Limulus polyphemus

Abstract

The American horseshoe crab, Limulus polyphemus, excretes nitrogenous waste in the form of toxic ammonia across their book gills. The mechanism of this branchial excretion is yet unknown. In the current study, two isoforms of a novel ammonia transporter, LpHIAT1α and LpHIAT1β, have been identified in L. polyphemus. Both isoforms have 12 predicted transmembrane regions and share 82.7% of amino acid identity to each other, and 77–86% amino acid homology to HIAT1 found in fish and crustaceans. In L. polyphemus, both isoforms were expressed in the gills, coxal glands, and brain. Slightly higher mRNA expression levels of LpHIAT1α were observed in the peripheral mitochondria-poor region of the gill (PMPA), central mitochondria-rich region of the gill (CMRA), and brain compared to the LpHIAT1β isoform. A functional expression analysis of LpHIAT1α and LpHIAT1β in Xenopus laevis oocytes resulted in a significantly lower uptake of the radiolabeled ammonia analogue 3H-methylamine when compared to controls, indicating an ammonia excretory function of the proteins. Exposure to elevated environmental ammonia (HEA, 1 mmol l−1 NH4Cl) caused an increase in mRNA expression of LpHIAT1β in the ion-conductive ventral gill half. High mRNA expression of both isoforms in the brain, and the observation that LpHIAT1α and LpHIAT1β likely mediate cellular ammonia excretion, suggests that these highly conserved ammonia transporters have an important housekeeping function in cellular ammonia elimination.