Effects of cell differentiation on ion conductances and membrane voltage in LLC-PK1 cells

1432-2013

Renal proximal tubule ; Slow-whole cell ; Patch-clamp ; K+ conductance ; Non-selective cation conductance ; Cell culture

Springer Online Journal Archives 1860-2000

Medicine

Abstract LLC-PK1 cells serve as a widely used model for the renal proximal tubule. Until now, little has been found out about their membrane voltage (V m) and ionic conductances (g). Several studies have shown changes in cell properties during differentiation and ageing. The aim of this study was to examine the relationship between V m or g and the age of these cells. Therefore, we investigated single cells, subconfluent and confluent monolayers of LLC-PK1 cells aged 1–8 days with the slow-whole-cell patch-clamp technique. The V m of all cells was-34±2 mV (n=75) and the membrane conductance (g m) was 2.3±0.3 nS (n=30). V m in cells aged up to 2 days was-24±3 mV (n=22) whereas V m in cells aged 5–8 days was -50±3 mV (n=15). An increase of extracellular K+ from 3.6 to 18.6 mmol/l led to a depolarization in all cells of 4±1 mV (n=31) and an increase of g m by 17±13% (n=15). Complete replacement of extracellular Na+ by N-methyl-D-glucamine (NMDG) led to a hyperpolarization of 19±2 mV (n=38) and gm was lowered by 27±14% (n=17). A reduction in extracellular Cl− from 147 to 32 mmol/l showed no significant effect on V m (n=16) or g m (n=11). Amiloride (10 μmol/l) had no significant effect on V m (n=13) or g m (n=7). The reduction of the extracellular osmolarity from 290 to 160 mosmol/l led to a hyperpolarization of 11±1 mV (n=18) and an increase in g m by 326±117% (n=12). There was no significant correlation between g m and cell age. LLC-PK1 cells used in this study have a K+ conductance and a non-selective cation conductance in parallel. With increasing age, LLC-PK1 cells became more and more conductive for K+ and lost their nonselective cation conductance. There is no evidence for a significant amiloride-sensitive Na+ or Cl− conductance in these cells. The K+ conductance could be activated by osmotically induced cell swelling.

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