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Research Article

Developmental and functional studies of the SLC12 gene family members from Drosophila melanogaster

¹National Institutes of Health ²National Institute of Dental and Craniofacial Research

Submitted 25 August 2009 ; revised 5 October 2009 ; accepted in final form 6 October 2009

The electroneutral cation-chloride cotransporter gene family, SLC12, contains nine members in vertebrates. These include seven sodium and/or potassium-coupled chloride transporters and two membrane proteins of unknown function. Although SLC12 family members have been identified in a number of lower species, the functional properties of these proteins are unknown. There are five SLC12 homologues in Drosophila melanogaster including at least one member on each of the four main branches of the vertebrate phylogenetic tree. We have employed in situ hybridization to study the expression patterns of the Drosophila SLC12 proteins during embryonic development. Our studies indicate that all five members of this family are expressed during early embryogenesis (stages 1-6) but that spatial and temporal expression patterns become more refined as development proceeds. Expression during late embryogenesis was seen predominantly in the ventral nerve cord, salivary gland, gut and anal pad. In parallel studies we have carried out transport assays on each of the five Drosophila homologues expressed as recombinant proteins in the cultured insect cell line High Five. Under our experimental conditions we found that only one of these proteins, CG4357, transported the potassium congener 86Rb. Additional experiments established that rubidium transport via CG4357 was saturable (K_m = 0.29+0.05 mM), sodium-dependent (K_1/2 = 53+11 mM), chloride-dependent (K_1/2 = 48+5 mM) and potently inhibited by bumetanide (K_I = 1.17+0.08 µM), a specific inhibitor of Na⁺-K⁺-2Cl^- cotransporters. Taken together our results provide strong evidence that CG4357 is an insect orthologue of the vertebrate Na⁺-K⁺-2Cl^- cotransporters.

Drosophila embryogenesis; Na+-K+-2Cl- cotransport; cation-chloride cotransport; in situ hybridization