SEAL-PROMOTING SOLUTIONS AND PIPETTE PERFUSION FOR PATCH CLAMPING PLANT-CELLS

Patch-clamp technology has greatly increased our knowledge of plant me mbrane transport. However, the success of patch clamping crucially rel ies on establishing a high resistance (G Omega) seal between the membr ane and the patch-clamp pipette. This can prove problematic in many pl ant-cell preparations. It is therefore of great importance to develop protocols for protoplast isolation, maintenance and seal formation tha t improve seal rate. This study investigated whether the pH and the Kf and the Cl- concentration of the pipette solution had an effect on th e seal formation. High pH and absence of K+ significantly promoted mem brane sealing, whereas the concentration of Cl- had no effect. To reap the benefit of seal-promoting pipette solutions and yet retain the op tion to adjust this solution to experimental requirements, a pipette p erfusion apparatus was implemented. The perfusion system was successfu lly applied in cell-attached patch, excised-patch and whole-cell confi gurations, using plasma membrane and tonoplast of three different spec ies. The system enables complete solution exchange within minutes and is potentially of great benefit in the study of channel selectivity, t he application of (cytoplasmic) channel blockers and the study of prim ary and secondary transport.