In several tissues, the proteins KCNQ1 and KCNE1 (IsK) combine to form voltage-gated K⁺ channels. In the proximal tubule (PT) of murine kidney, KCNE1 mediates a cell to lumen K⁺ flux which may serve to counteract membrane depolarization due to electrogenic Na⁺-coupled uptake of glucose or amino acids (Vallon et al. 2001). The aim of the present study was to demonstrate expression of KCNE1 and KCNQ1 mRNA in isolated mouse PTs and also to determine KCNE1 protein expression in the kidney.

129/SV mice were killed by cervical dislocation and the kidneys rapidly removed for extraction of total RNA or for protein extraction. RT-PCR was performed on RNA extracted from total mouse kidney (TMK: n = 5 preparations) and from S1 and S2 segments of proximal tubules (n = 5 preparations) isolated by enzymatic digestion of cortical slices. For PCR, we used oligonucleotide primers designed against murine KCNE1 and KCNQ1 (Grahammer et al. 2001). The predicted size of PCR products was 355 and 421 base pairs (bp), respectively. PCR products were separated by electrophoresis on 2 % agarose gels and visualized by ethidium bromide fluorescence (302 nm).

Western blotting was performed on lysates (n = 3) extracted from TMK centrifuged at 15 000 g representing a crude membrane fraction, and then centrifuged at 50 000 g to obtain a cytosolic fraction. Extracted proteins were separated by 4-12 % gradient PAGE, and then electroblotted on to a nitrocellulose membrane. The membrane was incubated with an antibody against KCNE1 developed ‘in house’ against the C-terminal peptide of KCNE1 and visualized by standard chemiluminescent methods.

PCR products of 355 and 421 bp corresponding to KCNE1 and KCNQ1, respectively, were obtained from PT. Western blotting for KCNE1 revealed a major band at 15 kDa predominantly in the membrane fraction of the mouse kidney samples.

These experiments indicate that mRNAs for KCNQ1 along with KCNE1 are expressed in mouse proximal tubules and the protein for KCNE1 is also localized in mouse kidney membrane fractions. These results are consistent with the hypothesis that KCNQ1 and KCNE1 have a role in setting the membrane potential in the proximal tubule.

The Wellcome Trust financed this work.