Whole cell potassium currents in fetal rat alveolar type II cells cultured on Matrigel matrix

Abstract

Patch-clamp studies were performed on fetal rat alveolar type II cells isolated at 19 days of gestation and cultured on either plastic for 7 days or Matrigel matrix (40-50 microliters/cm2) for 10 days. Before study, cells cultured on Matrigel matrix were dissociated from alveolar-like structures with enzymes, replated, and washed with cold buffer at a constant flow rate to remove residual gel. This wash significantly improved obtaining of successful seals. Potassium current-voltage relationships and maximum whole cell K+ conductance (normalized to membrane capacitance) were significantly changed with time in cells cultured on plastic, but no significant change occurred in cells cultured on Matrigel matrix. Application of 20 mM tetraet hyl ammonium, 2mM 4-aminopyridine, and 5mM BaCl2 significantly inhibited K+ currents, showing differences in channel sensitivity to these agents and a voltage-dependent blockage between culture groups or with time in culture. To conclude, we have developed a new method by which epithelial cells cultured on Matrigel matrix can be successfully studied with the use of patch-clamp techniques. Furthermore, these studies show that fetal type II cells have voltage-activated K+ channels and that channel density and their sensitivity to channel blockers are modulated by the substratum on which the cells are cultured.