ELECTROPORATION OF THE VACUOLE-ATTACHED PATCH-CLAMP CONFIGURATION ALLOWS ACCESS TO THE TONOPLAST RESISTANCE AND ESTIMATION OF ITS SPECIFIC CONDUCTANCE

Electroporation involves the formation of pores in the lipid phase of a membrane. These pores often last for dozens of minutes resulting in long-term electroporation, or they may last for only a few seconds yie lding short-term electroporation. Electroporating pulses (1 V for 30 m s) were given in the vacuole-attached configuration using vacuoles fro m Beta vulgaris and endoplasmic droplets from Chara corallina resultin g in a lowering of the patch resistance and allowing measurement of th e tonoplast resistance. Estimates of tonoplast specific conductances, adjusted for the influence of the tip and seal resistances, of 0.343 p S/mu m(2) (N = 37, S.E. = 0.039 pS/mu m(2)) for Beta vulgaris and 0.44 2 pS/mu m(2) (N = 3, S.E. = 0.040) for Chara corallina were obtained. Electroporation of the patch membrane in the vacuole-attached configur ation was obtained in 77 out of 180 experiments while patch rupture wa s only seen in three trials. Seal resistance stability was confirmed w hen rip withdrawal from this electroporated vacuole-attached configura tion resulted in a return to a resistance in the gigaohm range. The ph ysical persistence of the patch membrane during electroporation was co nfirmed by direct observation and by the retention of betacyanins. Imp lications of this work for studies using the whole-vacuole configurati on are discussed. (C) 1998 Elsevier Science Ireland Ltd. All rights re served.