The channel regulator IsK plays a role in swelling stimulated K⁺ efflux in mouse renal proximal tubule cells (Millar et al. 2001). The aim of the following study was to determine whether the basolateral K⁺ channel plays a role in IsK-regulated K⁺ efflux by comparing proximal convoluted tubules isolated from wild-type (WT) and IsK knockout (KO) mice.

WT and KO mice were humanely killed by cervical dislocation and tubules isolated used a modified method (Schafer et al. 1997). Tangential cortical slices (< 0.5 mm) were taken and incubated in MEM medium plus type II collagenase (0.5 mg ml^-1), protease (0.1 mg ml^-1), glycine (0.375 mg ml^-1) and soyabean trypsin inhibitor (0.05 mg ml^-1) at 37 °C without agitation. The incubation medium was poured off every 10 min and replaced with fresh enzyme mix. Once the liberated tubules had settled, the enzyme mix was replaced with ice-cold medium plus 1 % BSA. Proximal convoluted tubules were identified by the presence of Bowman’s capsule and/or by size and the presence of convolutions. Cell-attached patches of the basolateral membrane were taken under the control condition and in the presence of a hypotonic shock. The bath contained high Na⁺ Ringer solution, while the pipette contained high K⁺ Ringer solution. Both Ringer solutions were made hypotonic by omitting 40 mM mannitol. Command potential was stepped between +100 and 0 mV. Currents were analysed by pCLAMP 6 and reversal potentials calculated by linear regression. In hypotonic Ringer solution currents were investigated using a macroscopic analysis. Data are expressed as means ± S.E.M. and statistical significance was tested using Student’s t test and assumed at the 5 % level.

In 6/9 patches, inward currents were observed. The single channel conductance, measured between +100 and +20 mV, was 26.3 ± 2.5 pS (n = 6). The reversal potential of the channels was +41.7 ± 14.11 mV, close to the expected V_rev for E_K. At +100 mV, the macroscopic control current was -12.4 ± 2.3 pA (n = 8). This was unchanged in hypotonic Ringer solution, -7.0 ± 2.8 pA (n = 6). There was also no significant difference between WT and KO macroscopic currents, -7.0 ± 2.8 pA (n = 6) versus -11.9 ± 4.4 pA (n = 7), respectively.

In summary, the channel identified on the basolateral membrane of mouse convoluted proximal tubule exhibits a V_rev that is consistent with a K⁺-selective conductance. This channel is neither regulated by hypotonic shock nor IsK.

This work was funded by The Wellcome Trust and BBSRC.

Millar, I.D., White, S.J., Kibble, J.D. & Robson, L. (2001). J. Physiol. 531.P, 123P.

Schafer, J.A., Watkins, M.L., Li, L., Herter, P., Haxelmans, S. & Schlatter, E. (1997). Am. J. Physiol. 273, F650-657.