Alternative splicing of the KCNJ1 gene in the kidney results in the expression of three major isoforms of the inwardly rectifying K+ channel ROMK, which differ only in their N-terminal amino acid sequence (Giebisch, 2001). Deletion of amino acids 3-68 of ROMK1 (Kir1.1a) produces non-functional channels that exert a dominant negative effect on wild-type ROMK1 (Koster et al. 1998). Here we report the effects of N-terminal deletion of ROMK2 (Kir1.1b) on channel function and membrane trafficking.
Using PCR-based protocols, amino acids 1-56 of rat ROMK2 were deleted. This was subcloned into the Xenopus expression vector pTLN (Lorenz et al. 1996) and N-terminally tagged with EGFP (EGFP-ROMK2Δ56). Female Xenopus laevis were killed humanely and stage V-VI oocytes isolated. Oocytes were injected with 50 nl of RNAse-free H2O or H2O containing either 5 ng pTLN-EGFP, EGFP-ROMK2Δ56 or wild-type EGFP-ROMK2 cRNA. Channel activity was assessed 3-4 days after injection at room temperature by measuring currents at a holding potential of 0 mV maintained by two-microelectrode voltage clamp. The bath solution contained (mM): NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, Hepes 5; ± 5 mM BaCl2. The pH of solutions was adjusted to 7.5 ± 0.05 using HCl or NaOH. To determine the cellular location of the EGFP-tagged proteins, the oocytes were fixed in 1 % paraformaldehyde and sectioned (10-15 mm thick) using a cryostat at -25 °C. Sections were viewed on a Leica confocal laser-scanning microscope (lEx = 488 nm) within 24 h of sectioning. Data are presented as means ± S.E.M. and statistical significance was determined by one-way ANOVA and Tukey’s test.
Oocytes expressing EGFP-ROMK2 displayed Ba2+-sensitive outward currents of 3.06 ± 0.54 mA (n = 11). In contrast, oocytes expressing EGFP-ROMK2Δ56 exhibited a Ba2+-insensitive outward current of 0.12 ± 0.01 mA (n = 10, P < 0.05 cf. EGFP-ROMK2). This current was not significantly different from that of oocytes injected with H2O (0.13 ± 0.01 mA; n = 8) or pTLN-EGFP (0.14 ± 0.01 mA; n = 8). Confocal microscopy showed that in oocytes expressing either EGFP-ROMK2 (n = 4) or EGFP-ROMK2Δ56 (n = 4), the fluorescently tagged proteins were expressed at the plasma membrane. In contrast, fluorescence of oocytes injected either with H2O or pTLN-EGFP (n = 3-5) was solely intracellular.
These results indicate that the N-terminal amino acid sequence (1-56) of ROMK2 is required for normal channel function but is not essential for membrane trafficking of the protein.
This work is supported by the National Kidney Research Fund. V.M.C. is a White-Rose Research Student.
All procedures accord with current UK legislation.