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SUMMARYThe contractile vacuole complex of Paramecium multimicronucleatumtransforms 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 (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 4–20×10–7 μm s–1Pa–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 (

The precise relationship between the rate of contractile vacuole fluid discharge and the vacuole diameter at the start of systole was determined in cells of Paramecium multimicronucleatum subjected to various external conditions. The rate of discharge was higher when the diameter was larger. When the rate of discharge was plotted against the diameter, the points fell around a single parabolic line passing through the origin and were independent of the external conditions employed. This implies that the rate of discharge is proportional to the square of the vacuole diameter. We have previously proposed a hypothesis in which membrane tension in the contractile vacuole is altered as its planar membrane becomes tubular or as tubules become planar membrane (termed the membrane area-proportional tension hypothesis). We propose here that it is this change in membrane tension which determines the vacuole pore shape and sets the subsequent rate of fluid discharge.

To examine the forces needed for discharge of the fluid contents from the contractile vacuole of Paramecium multimicronucleatum, the time course of the decrease in vacuole diameter during systole (the fluid-discharging period) was compared with that of various vacuole discharge models. The observed time course did not fit that predicted by a model in which contraction of an actin–myosin network surrounding the vacuole caused discharge nor that predicted by a model in which the surface tension of the lipid bilayer of the vacuole caused discharge. Rather, it fitted that predicted by a model in which the cell’s cytosolic pressure was responsible for discharge. Cytochalasin B, an effective inhibitor of actin polymerization, had no effect on the in vivo time course of systole. An injection of a monoclonal antibody raised against the proton pumps of the decorated spongiomes (now known to be the locus of fluid segregation in P. multimicronucleatum) disrupted the decorated spongiomes and reduced the rate of fluid segregation, whereas it did not alter the time course of systole. We conclude that in P. multimicronucleatum the internal pressure of the contractile vacuole is caused predominantly by the cytosolic pressure and that the fluid-segregation mechanism does not directly affect the fluid-discharge mechanism. Elimination of this cytosolic pressure by rupturing the cell revealed the presence of a novel fluid-discharge mechanism, apparently centered in the vacuole membrane. The involvement of tubulation of the vacuole membrane as the force-generating mechanism for fluid discharge in disrupted cells is discussed.

A voltage-clamped Paramecium produced an inward membrane current upon thermal or mechanical stimulation of its anterior region, whereas it produced an outward membrane current upon similar stimulation of its posterior region. Anterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a positive direction, showing sign reversal at a positive membrane potential, whereas posterior thermo- and mechanoreceptor currents decreased when the membrane potential was shifted in a negative direction, showing sign reversal at a membrane potential more negative than the resting potential. The reversal potential for both anterior receptor currents shifted in a positive direction when external [Ca2+] was increased, whereas those for both posterior receptor currents shifted in a positive direction when external [K+] was increased. External [Mg2+], [Mn2+], [Na+], [Rb+] and [TEA+] had similar effects on the thermo- and mechanoreceptor currents. Thermoreceptor currents decreased whereas mechanoreceptor currents increased as the ambient temperature was raised. When a mechanical stimulus was applied to the membrane where a thermoreceptor current was being produced, an algebraic summation of these receptor currents occurred. It is concluded that thermoreceptor currents are dependent on ion channels different from those responsible for the mechanoreceptor currents, although the ionic pores for the channels are similar to each other in various respects. A possibility that a thermoreceptor mechanism exclusively shares a Ca2+ pore in the anterior membrane, or a K+ pore in the posterior membrane, with a mechanoreceptor mechanism is discussed.

The overall membrane potential response of the ciliate Paramecium caudatum to a rise in the temperature of its environment was depolarizing when the ambient temperature before stimulation (Te) was equal to or higher than the culture temperature (Tc), but hyperpolarizing when Te was lower than Tc. The anterior region of the cell responded to a rise in temperature with a localized membrane depolarization. The posterior region was depolarized when Te was equal to or higher than Tc, but hyperpolarized when Te was lower than Tc. The Te-dependent polarity reversal of the posterior response was responsible for the comparable reversal of the overall response. The temperature at which the polarity reversal of the posterior response took place shifted according to Tc. This shift caused a comparable shift in the temperature at which polarity reversal occurred for the overall response. The Tc -dependent polarity reversal of the posterior response and its Tc-dependence are major causes of thermoaccumulation mediated by ciliary activity of Paramecium in regions with temperatures close to Te.