The zebrafish is a model organism of increasing importance in the study of human congenital and inherited disease (Briggs, 2002). However, little information regarding cellular and molecular function in this organism is available. The nephron of the teleost kidney lacks a loop of Henle, but does posses a distal tubule which appears to act in an analogous fashion to the mammalian diluting segment (Nishimura et al. 1983). Here we report the successful expression of a ROMK-related channel from this species.

A search of the NCBI GENBANK database identified a partial sequence of high similarity to rat ROMK2 for a clone from an adult Danio rario cDNA library (Image Consortium ID: 3815817). To verify the identity of this clone, a PCR reaction was performed using primers which were designed against the original partial sequence. The full-length clone (1.8 kb) was sequenced (Lark Technologies) and is predicted to encode a 370 amino acid protein (zfROMK2), 54 % identical to rat ROMK2. The full-length cDNA was sub-cloned into pcDNA3 and used to transcribe cRNA (mMessage mMachine^TM -Ambion) which was expressed in oocytes isolated by standard methods from humanely killed Xenopus laevis (n = 3). Stage V and VI oocytes were injected with 50 nl of H₂O (control) or 50 nl H₂O containing 50 ng (1 ng nl^-1) of cRNA encoding zfROMK2 or 1 ng (0.02 ng nl^-1) of cRNA encoding rat ROMK2. The oocytes were incubated at 18 °C, and functional expression was assessed by two microelectrode voltage clamp 3-4 days following injection.

At an external [K⁺] of 2.00 mM, oocytes injected with rat ROMK2 displayed a resting membrane potential (V_m) of -96.00 ± 1.36 mV (mean ± S.E.M., n = 16), and at a clamp potential of 0 mV an outward current (I₀) of 3.15 ± 0.16 µA that was sensitive to 5 mM Ba²⁺ (1.47 ± 0.07 µA: P < 0.001, Student’s paired t test). Similarly, in oocytes expressing zfROMK2 V_m was -92 ± 1.52 mV and I₀ was 1.93 ± 0.72 µA (n = 16) which was also sensitive to Ba²⁺ (0.72 ± 0.09 µA: P < 0.001). Control oocytes displayed a V_m of -26.20 ± 0.76 mV and I₀ of 0.13 ± 0.76 µA (n = 12) which was unaffected by Ba²⁺ (0.09 ± 0.01 µA).

These results suggest that adult Danio rario express a ROMK2- like potassium selective channel. A consideration of the predicted zfROMK2 amino acid sequence indicates that there are likely to be differences in the regulatory mechanisms controlling the activity of this channel. This, along with the sites of expression and physiological role of this protein require further study.

We thank the National Kidney Research Fund for financial support. S.H. is a graduate student supported by the Iranian ministry of Health and Medical education and Arak medical university.