Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, PC252
Principal cells from the mouse cortical collecting duct possess an apamin sensitive K+ conductance
M. Fisher1, H. Taylor1, A. Neal1, G. Cooper1, L. Robson1
1. Biomedical Science, University of Sheffield, Sheffield, United Kingdom.
K+ secretion by principal cells of the cortical collecting duct plays a pivotal role in the maintenance of whole body potassium homeostasis . The majority of this secretion is known to take place via the apically expressed ROMK channel . However, recent studies have indicated an important contribution of calcium activated K+ channels such as the large conductance BK channel . The aim of the current study was to investigate the existence of small conductance calcium activated (SK) K+ channels in mouse cortical collecting duct principal cells. Intact collecting duct tubules were isolated using enzyme digestion of the renal cortex and tubules were mounted between glass holding pipettes. Whole cell recordings were achieved via the basolateral aspect of cells, with high Na+ in the bath and high K+ in the pipette. Principal cells were identified by their negative reversal potential (Vrev) as described previously . Once the whole cell configuration was achieved, currents were allowed to reach steady-state and then 10μM apamin was added to the extracellular solution. Clamp potential was stepped from a holding value of -40 mV, to between +100 and -100 mV in 20 mV steps. Statistical analysis was tested using Student’s t test and assumed at the 5% level. In a second set of experiments mRNA expression for SK family members in whole mouse cortex and isolated collecting ducts was examined. Total whole cell currents in principal cells showed a characteristic inwardly rectifying profile. Mean conductance at +100mV was 0.76 ± 0.12 vs 2.29 ± 0.56 nS at -100mV (n=6, p<0.01). Addition of 10μM apamin reduced both the outward and inward conductances by around 50%. The mean conductance at +100mV was 0.76 ± 0.12 vs 0.36 ± 0.10 nS, in the absence and presence of apamin, respectively, n=6, p<0.05. While mean conductance at -100mV was 2.29 ± 0.56 vs 1.09 ± 0.47 nS, in the absence and presence of apamin, respectively, n=6 p<0.05. The apamin sensitive currents had a Vrev of -60.95 ± 4.54 (n=6) and also demonstrated inward rectification (mean conductance at +100 mV was 0.40 ± 0.05 vs 1.20 ± 0.44 nS at -100 mV, p<0.05). The SK mRNA expression in mouse kidney cortex and collecting ducts indicated the presence of SK2 mRNA but an absence of SK3 or SK1 mRNA. These data suggest that mouse collecting duct principal cells possess an apamin-sensitive K+ conductance that may be attributable to SK2. Future work will need to investigate the importance of this channel to K+ homeostasis.
Where applicable, experiments conform with Society ethical requirements