Transport of Na-22(+) and Ca-45(2+) by Xenopus laevis oocytes expressing mRNA from lobster hepatopancreas

This paper describes the development of a functional assay system to expres s crustacean epithelial electrogenic 2Na(+)/1H(+) antiporters in Xenopus la evis oocytes. Subsequent publications will use this assay method to establi sh nucleotide and amino acid sequence information about this transporter by functionally screening an hepatopancreatic cDNA library. In this method, o ocytes were injected with hepatopancreatic mRNA (50 ng) isolated from Homar us americanus, while control oocytes received injections of an equivalent v olume of distilled water. Three to five days post-injection, oocytes were i ncubated in media containing either Na-22(+) or Ca-45(2+) for specific time intervals and the rates of ion transfer into the oocytes were monitored un der a variety of experimental conditions. Uptakes of both radiolabelled cat ions were stimulated by mRNA injection. mRNA-stimulated Na-22(+) uptake was significantly (P < 0.05) inhibited by addition of calcium, amiloride, or b y an antiporter-specific monoclonal antibody to the external medium. mRNA-s timulated Ca-45(2+) uptake was significantly (P < 0.05) inhibited by additi on of sodium, amiloride, cadmium, zinc, or by the antiporter-specific monoc lonal antibody (also inhibitory for Na-22(+) transport) to the external med ium. The kinetics of Na-22(+) influx in mRNA-injected oocytes were sigmoida l functions of external sodium concentration, exhibiting a Hill Coefficient (n) of approximately 3.0. Both calcium and amiloride significantly (P < 0. 05) reduced sigmoidal sodium influx kinetics by alterations in the J(max) ( amiloride) or K-Na (calcium) of the transporter. Size fractionation of hepa topancreatic mRNA resulted in a single fraction that was most stimulatory f or sodium and calcium transport and which likely contains the antiporter tr anscript. The results of this study provide the basis for using Na-22(+) an d Ca-45(2+) transport assays of lobster mRNA-injected oocytes to functional ly screen an hepatopancreatic cDNA library for clones that will provide ful l length nucleotide and amino acid sequences of the invertebrate electrogen ic 2Na(+)/1H(+) antiporter protein. (C) 2001 Wiley-Liss, Inc.