Isolation and patch clamp measurements of xylem contact cells for the study of their role in the exchange between apoplast and symplast of leaves

Because of their importance for nutrition, a method was developed to patch xylem contact cells in leaves of Vicia Saba and maize. Since the lignification of older cells was a major obstacle for isolating protoplasts which could be patched, only young leaves (fourth fully developed leaf) were used. An important step in the isolation of these cells was the infiltration of the leaves and their exposure to enzymes for several hours, allowing mesophyll cells to be removed whilst having most of the xylem contact cells attached to the xylem. Channel activity in cell-attached mode or in excised patches could only be observed if an internal coating of sigmacote was used to block diffusion ions out of the pipette glass. Two different types of K+ channels were identified by measuring the reversal potential at different concentrations of KCl. One channel (SC) had a symmetric IV curve with a high probability of remaining open, irrespective of membrane potential; the other channel was an inward rectifier. The symmetical channel could be blocked weakly by Na+ but it was permeable to NH4(+).