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First published online October 7, 2005
Journal of Experimental Biology 208, 3957-3969 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01858

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Electrical properties and fusion dynamics of in vitro membrane vesicles derived from separate parts of the contractile vacuole complex of Paramecium multimicronucleatum

Kazuyuki Sugino^*, Takashi Tominaga, Richard D. Allen and Yutaka Naitoh

Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, HI 96822, USA

Author for correspondence (e-mail: naitoh{at}pbrc.hawaii.edu)

Accepted 24 August 2005

The contractile vacuole complex of Paramecium multimicronucleatum transforms into membrane-bound vesicles on excision from the cell. The I–V relationship was linear in a voltage range of –80 to +80 mV in all vesicles, despite being derived from different parts of the contractile vacuole complex. No voltage-gated unit currents were observed in membrane patches from the vesicles. Vesicles derived from the radial arm showed a membrane potential of >10 mV, positive with reference to the cytosol, while those derived from the contractile vacuole showed a residual (<5 mV) membrane potential. The electrogenic V-ATPases in the decorated spongiome are responsible for the positive potential, and Cl^– leakage channels are responsible for the residual potential. The specific resistance of the vesicle membrane (6 k cm²) increased, while the membrane potential shifted in a negative direction when the vesicle rounded. An increase in the membrane tension (to 5x10^–3 N m^–1) is assumed to reduce the Cl^– leakage conductance. It is concluded that neither voltage- nor mechano-sensitive ion channels are involved in the control of the fluid segregation and membrane dynamics that govern fluid discharge cycles in the contractile vacuole complex.

The membrane vesicles shrank when the external osmolarity was increased, and swelled when the osmolarity was decreased, implying that the contractile vacuole complex membrane is water permeable. The water permeability of the membrane was 4–20x10^–7 µm s^–1 Pa^–1. The vesicles containing radial arm membrane swelled after initially shrinking when exposed to higher external osmolarity, implying that the V-ATPases energize osmolyte transport mechanisms that remain functional in the vesicle membrane. The vesicles showed an abrupt (<30 ms), slight, slackening after rounding to the maximum extent. Similar slackening was also observed in the contractile vacuoles in situ before the opening of the contractile vacuole pore. A slight membrane slackening seems to be an indispensable requirement for the contractile vacuole membrane to fuse with the plasma membrane at the pore. The contractile vacuole complex-derived membrane vesicle is a useful tool for understanding not only the biological significance of the contractile vacuole complex but also the molecular mechanisms of V-ATPase activity.

Key words: contractile vacuole complex, membrane vesicle, membrane potential, membrane resistance, membrane dynamics, membrane tension, patch clamp, water permeability, V-ATPase, Paramecium multimicronucleatum