Separate determination of the electrical properties of the tonoplast and the plasmalemma of the giant-celled alga Valonia utricularis: vacuolar perfusion of turgescent cells with nystatin and other agents.
In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 microM of the H+-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to -22 mV and in an increase of the area-specific membrane resistance from 6.8 x 10(-2) to 40.6 x 10(-2) Omegam2. The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81-94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 microM of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively form the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 x 10(-2) compared to 0.77 x 10(-2) Fm(-2) of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system.
Wang
J
J
Lehrstuhl für Biotechnologie, Biozentrum der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany.
Spiess
I
I
Ryser
C
C
Zimmermann
U
U
D002462:Cell Membrane / Q000502:physiology*; D003594:Cytoplasmic Granules / Q000502:physiology*; D056890:Eukaryota / Q000502:physiology*; D007476:Ionophores / Q000494:pharmacology*; D008564:Membrane Potentials / Q000187:drug effects; D009761:Nystatin / Q000494:pharmacology*
D007476:Ionophores; D009761:Nystatin