Na⁺-alanine cotransport in frog single proximal tubule cells causes cell swelling followed by volume regulation (Mounfield & Robson, 1998). Regulation occurs as a consequence of solute efflux. A previous study identified a quinidine-sensitive conductance that may provide a K⁺ efflux pathway (Robson & Hunter, 1997). The aim of the following study was to determine whether this conductance plays a role in volume regulation.

Frogs were humanely killed and single frog proximal tubule cells were isolated by enzyme digestion (Hunter, 1989). Cell length was measured using an optical technique. Cells were superfused with high Na⁺ amphibian Ringer solution and 5 mM L-alanine (substitution of mannitol) added to the bath, in the absence and presence of 1 mM quinidine. Na⁺-alanine cotransport currents were examined in whole-cell patches with high Na⁺ and high Cs⁺ amphibian Ringer solutions in the bath and pipette, respectively. To activate cotransport currents 5 mM L-alanine was added to the bath in either the absence or presence of 1 mM quinidine. Whole-cell potential was held at -40 mV and then stepped between +40 and -100 mV in 20 mV steps. Chord conductance was calculated between +40 and -100 mV and reversal potential (V_rev) determined by polynomial regression. All values are expressed as means ± S.E.M. Statistical significance was tested using Student’s t test and assumed at the 5 % level.

Control cell length was 20.5 ± 0.54 µm (n = 25). This increased by 0.33 ± 0.05 µm (n = 25) on the addition of L-alanine. In the presence of quinidine L-alanine did not change length, 18.3 ± 0.73 versus 18.3 ± 0.67 µm (n = 11). L-Alanine increased whole-cell conductance and depolarised V_rev: 18.6 ± 2.84 to 33.9 ± 3.51 µS cm^-2 and -5.70 ± 1.85 to 25.4 ± 3.6 mV (n = 5), respectively. In the presence of quinidine, L-alanine also increased conductance and shifted V_rev: 14.7 ± 2.9 to 22.0 ± 3.45 µS cm^-2 and 0.16 ± 1.19 to 13.5 ± 2.10 mV (n = 5), respectively. However, the magnitude of the response to L-alanine was reduced.

In summary, Na⁺-alanine-induced cell swelling in single proximal tubule cells is prevented by the K⁺ channel inhibitor quinidine. Quinidine decreased Na⁺-alanine cotransport currents, suggesting that the effect of quinidine may be via a direct inhibitory action on the Na⁺-cotransporter itself.

This work was supported by The Wellcome Trust. K. Khandel was a Wellcome Vacation student.

Hunter, M. (1989). J. Physiol. 416, 13P.

Mounfield, P.R. & Robson, L. (1998). J. Physiol. 510, 145-153. abstract

Robson, L. & Hunter, M. (1997). J. Physiol. 500, 605-616. abstract