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Membrane potential responses of Paramecium caudatum to an application of K+-rich solution were examined to understand the mechanisms underlying K+-induced backward swimming. A wild-type cell impaled by a microelectrode produced action potentials followed by a sustained depolarization in response to an application of a K+-rich test solution. After termination of the application, a prolongation of the depolarization (depolarizing after-potential) took place. Behavioral mutants incapable of exhibiting K+-induced backward swimming did not show depolarizing afterpotentials. Upon short application of K+-rich solution, the timing and duration of the ciliary reversal of the wild-type cell coincided well with the K+-induced depolarization. The duration of the depolarizing afterpotential decreased as the duration of the application increased. The depolarizing afterpotential recovered slowly after it had been suppressed by a preceding application of the K+-rich solution. By injection of an outward current into the wild-type cell, the action potentials were evoked normally during the period when the K+-induced depolarizing afterpotential was suppressed. We concluded that the prolongation of the depolarizing membrane potential response following the application of the K+-rich solution represents the Ca2+ conductance responsible for the K+-induced backward swimming in P. caudatum and that the characteristics of the K+-induced Ca2+ conductance are distinct from those of the Ca2+ conductance responsible for the action potentials. [ABSTRACT FROM AUTHOR]

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation. [ABSTRACT FROM AUTHOR]

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca2+] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca2+ signals also occur when AED is applied in presence of the fast Ca2+ chelator, BAPTA. (ii) Extracellular La3+ application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca2+]i transient (while injected La3+ causes a sustained fluorescence signal). (iii) Simply increasing [Ca2+]o causes a similar rapid, short-lived [Ca2+]i transient. All these phenomena, (i–iii), are compatible with activation of an extracellular ``Ca2+/(polyvalent cation)-sensing receptor'' known from some higher eukaryotic systems, where this sensor (responding to Ca2+, La3+ and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores (``alveolar sacs'') are physically linked to the cell membrane and they can also be activated by the Ca2+ releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca2+ signal also in absence of Ca2+o we largely exclude a ``Ca2+-induced Ca2+ release'' (CICR) mechanism. Our finding of increased cortical Ca2+ signals after store depletion and re-addition of extracellular Ca2+ can be explained by a ``store-operated Ca2+ influx'' (SOC), i.e., a Ca2+ influx superimposing store activation. AED stimulation in presence of Mn2+o causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca2+ influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca2+/polyamine-sensing receptor, (ii) release of Ca2+ from subplasmalemmal stores, (iii) and Ca2+ influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca2+] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca2+] microdomains (≤0.5 μm, ≤33 msec) upon stimulation. [ABSTRACT FROM AUTHOR]

A stopped-flow spectrophotometric technique was used to study the kinetics of Ca flux into ciliary membrane vesicles from Paramecium tetraurelia wild-type and several 'pawn' mutants with defective Ca conductances. 15 mm Arsenazo III was used as metallochromic indicator and as intravesicular Ca trap. The absolute amount of Ca-permeable vesicles was significantly reduced in preparations from the 'pawn' mutants compared to wild-type. However, influx kinetics were identical for vesicles from wild-type and 'pawn' mutant Paramecia when the fraction of Ca-permeable vesicles was taken into account. Ca influx was rapid with a time constant of about 1.5 sec and an initial saturation rate of arsenazo III of about 50%/single vesicle ×sec. Ca influx rates were half-maximal at approximately 20 μ m Ca. Comparisons of Ba toxicity tested with a behavioral assay, Ca inward conductances under voltage-clamp conditions and Ca influx kinetics between wild-type and the 'pawn' mutants pwA (d4-94), leaky pwB (d4-96) and the double mutant pwA/pwB indicated that Ca transport in all types of ciliary membrane vesicles occurred through similar Ca gates. [ABSTRACT FROM AUTHOR]

We had previously shown that an influx of extracellular Ca2+ (Ca2+), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca2+ e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts'' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca2+] e ≥ 5 × 10−6 m, while normally a [Ca2+] e = 0.5 m m is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca2+] e even beyond levels usually available to cells. Quenching of [Ca2+] e by EGTA application to levels of resting [Ca2+] i or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca2+ e . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca2+-mobilization from subplasmalemmal pools (``alveolar sacs'') and, as a superimposed step, a Ca2+-influx, since exocytotic membrane fusion can occur at [Ca2+] e even slightly below resting [Ca2+] i . The other important conclusion is that increasing [Ca2+] e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+] e also drives detachment of ``ghosts'' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. [ABSTRACT FROM AUTHOR]

The concentration of intracellular calcium, [Ca2+]i, in Paramecium was imaged during cold-sensitive response by monitoring fluorescence of two calcium-sensitive dyes, Fluo-3 and Fura-Red. Cooling of a deciliated Paramecium caused a transient increase in [Ca2+]i at the anterior region of the cell. Increase in [Ca2+]i was not observed at any region in Ca(2+)-free solution. Under the electrophysiological recording, a transient depolarization of the cell was observed in response to cooling. On the voltage-clamped cell, cooling induced a transient inward current under conditions where K+ currents were suppressed. These membrane depolarizations and inward currents in response to cooling were lost upon removing extracellular Ca2+. The cold-induced inward current was lost upon replacing extracellular Ca2+ with equimolar concentration of Co2+, Mg2+ or Mn2+, but it was not affected significantly by replacing with equimolar concentration of Ba2+ or Sr2+. These results indicate that Paramecium cells have Ca2+ channels that are permeable to Ca2+, Ba2+ and Sr2+ in the anterior soma membrane and the channels are opened by cooling. [ABSTRACT FROM AUTHOR]

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 n m to 1.0 μ m) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca based depolarizations. Addition of either Na or Mg improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 μ m neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 μ m), lysozyme is a secretagogue. A transient inward current, recorded in Ca alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 m m) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca conductance. This receptor-operated Ca conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca influx at high concentrations to cause exocytosis. [ABSTRACT FROM AUTHOR]

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless dior trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca release, and the prospects of their purification are discussed. [ABSTRACT FROM AUTHOR]

The effects of proteolysis on a hyperpolarization- and Ca-dependent K channel from the surface membrane of Paramecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca concentrations between 10 and 10 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca-dependent enzymes it is suggested that this Paramecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca-dependent K channels (BK channels) and the hyperpolarization-, Ca-dependent K channels in Paramecium. [ABSTRACT FROM AUTHOR]

The Ca-inward current of Paramecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec, or about 1.2 ·10 molecules/cell·sec. Because no other factors than the Ca-inward current were found to affect cGMP formation in Paramecium, we used it as a quantitative measure of Ca channel activity. After a transient stimulation of cGMP formation by 1 mm Ba, an additional increase of Ba to 5 mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1 mm Ba revealed a time-dependent inactivation of the Ca channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization of Paramecium reduced the maximal response, but did not shift the dose-response curve for Ba. Veratridine, which activates the Ca channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response of Paramecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol. The extremely high rate of cGMP turnover in Paramecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel of Paramecium is proposed. [ABSTRACT FROM AUTHOR]

The membrane currents of wild type Paramecium tetraurelia and the behavioral mutant teaA were analyzed under voltage clamp. The teaA mutant was shown to have a greatly increased outward current which was blocked completely by the combined use of internally delivered Cs+ and external TEA+. This, along with previous work (Satow, Y., Kung, C., 1976, J. Exp. Biol. 65:51-63) identified this as a K+ current. It was further found to be a calcium-activated K+ current since this increased outward K+ current cannot be elicited when the internal calcium is buffered with injected EGTA. The mutation pwB, which blocks the inward calcium current, also blocks this increased outward K+ current in teaA. This shows that this mutant current is activated by calcium through the normal depolarization-sensitive calcium channel. While tail current decay kinetic analysis showed that the apparent inactivation rates for this calcium-dependent K+ current are the same for mutant and wild type, the teaA current activates extremely rapidly. It is fully activated within 2 msec. This early activation of such a large outward current causes a characteristic reduction in the amplitude of the action potential of the teaA mutant. The teaA mutation had no effect on any of the other electrophysiological parameters examined. The phenotype of the teaA mutant is therefore a general decrease in responsiveness to depolarizing stimuli because of a rapidly activating calcium-dependent K+ current which prematurely repolarizes the action potential. [ABSTRACT FROM AUTHOR]

The membrane of Paramecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation, fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that the Paramecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca concentration. The function of the Ca-dependent Na conductance is discussed. [ABSTRACT FROM AUTHOR]