Identification and lateral membrane localization of cyclin M3, likely to be involved in renal Mg²⁺ handling in seawater fish

The kidney of marine teleosts isthe major site of Mg² excretion and produces urine with a highMg²⁺ concentration. However, the transporters involved in Mg²⁺ excretion are poorly understood. The cyclin M (Cnnm; also known as ancient conserved domain protein) family comprises membrane pro-teins homologous to the bacterial Mg²⁺ and Co²⁺ efflux protein, CorC. To understand the molecular mechanism of Mg²⁺ homeostasis in marine teleosts, we analyzed the expression of the Cnnm family genes in the seawater (SW) pufferfish, torafugu (Takifugu rubripes), and the closely related euryhaline species, mefugu (Takifugu obscurus). Database mining and phylogenetic analysis indicated that the Takifugu genome contains six members of the Cnnm family: two orthologs of Cnnm1, one of Cnnm2, one of Cnnm3, and two of Cnnm4. RT-PCR analyses indicated that Cnnm2, Cnnm3, and Cnnm4a are expressed in the kidney, whereas other members are mainly expressed in the brain. Renal expression of Cnnm3 was upregulated in SW mefugu, whereas renal expression of Cnnm2 was upregulated in freshwater (FW) mefugu. No significant difference was observed in renal expression of Cnnm4a between SW and FW mefugu. In situ hybridization and immunohistochemical analyses of the SW mefugu kidney revealed that Cnnm3 is expressed in the proximal tubule, and its product localizes to the lateral membrane. When Cnnm3 wasexpressed in Xenopus laevis oocytes, whole cellular Mg²⁺ contentand free intracellular Mg²⁺ activity significantly decreased. Theseresults suggest that Cnnm3 is involved in body fluid Mg²⁺ homeostasis in marine teleosts.