Kazunori Oami

Membrane potential responses controlling chemodispersal of Paramecium caudatum from quinine

Membrane potential responses of a ciliate protozoan Paramecium caudatum to the external application of quinine were investigated in relation to its motile activities. Wild-type specimens swimming in the reference solution did not enter into a quinine-containing (0.5 mM) test solution due to avoiding responses exhibited at the border between the two solutions, and therefore stayed in the reference solution (chemodispersal). Squirting of a quinine-containing test solution over a wild-type specimen evoked a train of action potentials superimposed on a depolarizing chemoreceptor potential. Squirting of a quinine-containing test solution over a CNR-mutant specimen defective in voltage-gated Ca2+ channel evoked only chemoreceptor potentials, which consisted of an initial transient depolarization, a following transient hyperpolarization and a sustained depolarization. A current-evoked action potential became larger in its amplitude and longer in its duration with the external application of quinine. Under the voltage-clamp condition, the fast inward current did not change whereas the delayed outward current decreased with the external application of quinine. It is concluded that quinine is a potent repellent for Paramecium because it produces a depolarizing chemoreceptor potential which evokes action potentials and prolongs the duration of the action potential.

Centrin controls the activity of the ciliary reversal-coupled voltage-gated Ca2+ channels Ca2+-dependently

In Paramecium, ciliary reversal is coupled with voltage-gated Ca(2+) channels on the ciliary membrane. We previously isolated a P. caudatum mutant, cnrC, with a malfunction of the Ca(2+) channels and discovered that the channel activity of cnrC was restored by transfection of the P. caudatum centrin (Pccentrin1p) gene, which encodes a member of the Ca(2+)-binding EF-hand protein family. In this study, we injected various mutated Pccentrin1p genes into cnrC and investigated whether these genes restore the Ca(2+) channel activity of cnrC. A Pccentrin1p mutant gene lacking Ca(2+) sensitivity of the third and fourth EF-hands lost the ability to restore the channel function of cnrC, and mutation of the fourth EF-hand caused more serious impairment than mutation of the third EF-hand. Moreover, a Pccentrin1p gene lacking the N-terminal 34-amino acid sequence also lost the ability to restore the channel activity. Native-PAGE analysis demonstrated that the N-terminal sequence is important for the Ca(2+)-dependent structural change of Pccentrin1p. These results demonstrate that Pccentrin1p Ca(2+)-dependently regulates the Ca(2+) channel activity in vivo.

DISTRIBUTION OF CHEMORECEPTORS TO QUININE ON THE CELL SURFACE OF PARAMECIUM CAUDATUM

The topological distribution of the chemoreceptors to quinine in the membrane of a ciliate Paramecium caudatum were examined by conventional electrophysiological techniques. A CNR-mutant specimen defective in voltage-gated Ca channels produced a transient depolarization followed by a transient hyperpolarization and a sustained depolarization when 1 mM quinine-containing solution was applied to its entirety. A Ni2+-paralyzed CNR-mutant specimen produced a simple membrane depolarization in response to a local application of 1 mM quinine-containing solution to its anterior end, whereas it produced a transient membrane hyperpolarization in response to an application to its posterior end. An anterior half fragment of a CNR specimen produced a membrane depolarization whereas a posterior half fragment of the specimen produced a transient hyperpolarization upon application of 1 mM quinine-containing solution. Both anterior depolarization and posterior hyperpolarization took place prior to the contraction of the cell body. It is concluded that Paramecium caudatum possesses two kinds of chemoreceptors or two kinds of coupling of the same receptor to different signal transduction pathways to quinine which are distributed in different locations on the cell surface. Activation of the anterior receptor produces a sustained depolarizing receptor potential while activation of the posterior receptor produces a transient hyperpolarizing receptor potential.

Voltage-gated ion conductances corresponding to regenerative positive and negative spikes in the dinoflagellate Noctiluca miliaris

Depolarization-activated and hyperpolarization-activated ion conductances in the membrane of a marine dinoflagellate Noctiluca miliaris were examined under voltage-clamp conditions. Noctiluca exhibited a transient inward current in response to a step depolarization from a holding potential level of −80 mV to a potential level more positive than −50 mV. The I–V relationship for the current exhibited typical N-shaped characteristics similar to those of most excitable membranes. The current was inactivated by a membrane depolarization. The reversal potential of the current shifted in hyperpolarizing direction when the external Na+ concentration was lowered. The transient inward current is assumed to be responsible for the Na+-dependent positive spike in non-clamped specimens of Noctiluca. Noctiluca exhibited a transient outward current in response to a step hyperpolarization from a holding potential level of −20 mV to a potential level more negative than −30 mV. The I–V relationship for the current was a typical N-shape as if it was turned 180° around its origin. The outward current showed a two-step exponential time-decay. The outward current was inactivated by a membrane hyperpolarization. The reversal potential shifted in the depolarizing direction when the external Cl− concentration was lowered. The transient outward current is responsible for the Cl−-dependent negative spike in non-clamped specimens of Noctiluca.

Correlation between Membrane Potential Responses and Tentacle Movement in the Dinoflagellate Noctiluca miliaris

Membrane potential responses and tentacle movement of the marine dinoflagellate Noctiluca miliaris were recorded simultaneously and their time relationships were examined. The food-gathering tentacle of Noctiluca exhibited slow extension-flexion movements in association with the spontaneously recurring membrane potential responses termed the tentacle regulating potentials (TRPs). The flexion of the tentacle began during the slow depolarization of the TRPs. The rate of the flexion increased after the hyperpolarizing (negative) spike following the slow depolarization. The tentacle then extended slowly during the hyperpolarized level of the TRPs. A TRPs-associated flexion did not occur when the external Ca2+ ions were removed. On the contrary, the tentacle showed conspicuous flexion (coiling) when the external Ca2+ concentration was raised. In association with the stimulus-evoked action potential, which triggers bioluminescent flash (flash-triggering action potential; FTP), the tentacle coiled quickly. The FTP-associated coiling took place even in the Ca2+-deprived condition. The coupling mechanisms of the TRPs-associated and FTP-associated tentacle movements were compared, and their biological significance was discussed.

Ionic mechanisms of depolarizing and hyperpolarizing quinine receptor potentials in Paramecium caudatum

Abstract The ionic mechanisms of the depolarizing and the hyperpolarizing quinine receptor potentials in the ciliate Paramecium caudatum were examined by using a behavioral mutant strain. The depolarizing receptor potential was induced by stimulating the anterior end of the specimen, and the hyperpolarizing receptor potential by stimulating the posterior end. The amplitude of both the depolarizing and the hyperpolarizing receptor potentials increased linearly with logarithmic increase in quinine concentration applied. Threshold concentration for inducing the depolarizing receptor potential was lower than that for the hyperpolarizing one. The peak level of the depolarizing receptor potential shifted towards the depolarizing direction with increasing external Ca2+ concentration while that of the hyperpolarizing receptor potential shifted in the depolarizing direction with increasing external K+ concentration. Under voltage-clamp conditions, the specimen produced an inward current in response to anterior stimulation, and an outward current in response to posterior stimulation. Both the peak inward and the peak outward currents showed a linear relationship with membrane potential. Current-voltage relationships of the receptor currents indicated conductance increase during the application of quinine. The depolarizing quinine receptor potential appears to be produced by an activation of Ca2+ channels, and the hyperpolarizing quinine receptor potential by an activation of K+ channels.

Modification of voltage-sensitive inactivation of Na+ current by external Ca2+ in the marine dinoflagellate Noctiluca miliaris

Effects of the external Ca2+ concentration on the depolarization-induced transient inward Na+ current responsible for the Na+ spike in the dinoflagellate Noctiluca miliaris were examined. The peak value and the duration of the Na+ current increased when lowering the external Ca2+ concentration. The threshold potential level for activation and the reversal potential level of the current were not affected by the external Ca2+ concentration. The inactivation took place even in a solution containing EGTA with very low (<10−9M) Ca2+ concentration. Voltage dependency of the inactivation was scarcely affected by the external Ca2+ concentration. It is concluded that inactivation of Na+ channels responsible for the current is dependent on membrane depolarization and that the external Ca2+ modulates the inactivation kinetics. Appearance of a Na+ spike in a solution with reduced Ca2+ concentration is caused by a lowered rate of inactivation of the Na+ channels.

Membrane Potential Responses of Paramecium caudatum to External Na

Abstract The membrane potential responses of Paramecium caudatum to Na+ ions were examined to understand the mechanisms underlying the sensation of external inorganic ions in the ciliate by comparing the responses of the wild type and the behavioral mutant. Wild-type cells exhibited initial continuous backward swimming followed by repeated transient backward swimming in the Na+-containing test solution. A wild-type cell impaled by a microelectrode produced initial action potentials and a sustained depolarization to an application of the test solution. The prolonged depolarization, the depolarizing afterpotential, took place subsequently after stimulation. The ciliary reversal of the cell was closely associated with the depolarizing responses. When the application of the test solution was prolonged, the wild-type cell produced sustained depolarization overlapped by repeated transient depolarization. A behavioral mutant defective in the Ca2+ channel, CNR (caudatum non reversal), produced a sustained depolarization but no action potential or depolarizing afterpotential. The mutant cell responded to prolonged stimulation with sustained depolarization overlapped by transient depolarization, although it did not show backward swimming. The results suggest that Paramecium shows at least two kinds of membrane potential responses to Na+ ions: a depolarizing afterpotential mediating initial backward swimming and repeated transient depolarization responsible for the repeated transient backward swimming.

MODIFICATION OF THE VOLTAGE DEPENDENT NA+ CONDUCTANCE BY EXTERNAL PH IN THE DINOFLAGELLATE NOCTILUCA MILIARIS

Abstract Effects of the external pH on the voltage-dependent Na conductance in the marine dinoflagellate Noctiluca miliaris were examined under voltage-clamp conditions. The depolarizing Na spike (positive spike) in the tentacle regulating potentials became attenuated when the externa! pH was lowered from 8 (normal value) to less than 5. Under voltage-clamp conditions, a depolarization-activated transient inward current corresponding to the positive spike decreased with lowering the external pH to less than 5, and entirely disappeared at pH 3. Threshold for the inward current shifted towards the depolarizing direction while the reversal potential of the current shifted towards the hyperpolarizing direction with lowering the external pH. Rate of increase in the inward current reduced whereas rate of decrease did not change at low pH. Lowering of the external pH did not affect the steady state inactivation of the inward current. It is concluded that the external pH controls appearance of the positive spike through modification of activation kinetics and selectivity of the voltage-dependent Na conductance responsible for the spike.