Roger Eckert

Calcium‐mediated inactivation of calcium current in Paramecium

1. The Ca current seen in response to depolarization was investigated in Paramecium caudatum under voltage clamp. Inactivation of the current was measured with the double pulse method; a fixed test pulse of an amplitude sufficient to evoke maximal inward current was preceded by a conditioning pulse of variable amplitude (0‐120 mV).

Kinetics of calcium‐dependent inactivation of calcium current in voltage‐clamped neurones of Aplysia californica.

Ca currents flowing during voltage‐clamp depolarizations were examined in axotomized Aplysia neurones under conditions that virtually eliminated other currents. Moderate to large currents exhibited a two‐component time course of relaxation that can be approximated reasonably well by the sum of two exponentials. The rapid phase (tau 1 approximately equal to 70 ms at 0 mV) plus the slower phase (tau 2 approximately equal to 300 ms at 0 mV) ride upon a steady, non‐inactivating current, I infinity. Conditions that diminish the peak current amplitude, such as reduced stimulus depolarization, inactivation remaining from a prior depolarization, or partial blockade of the Ca conductance by Cd, slowed both phases of inactivation, and all selectively eliminated the tau 1 phase, such that weak currents exhibited only the slower phase of decline. Injection of EGTA slowed both phases of inactivation, decreased the extent of the tau 1 phase, and increased the intensity of I infinity and of the current during the tau 2 phase. For a given voltage, the rate of inactivation increased as the peak current strength was increased, and decreased as the peak current strength was decreased. For a given peak current the rate of inactivation decreased as depolarization was increased. The relation of inactivation to prior Ca2+ entry was essentially linear for small currents, but decreased in slope with time during strong currents. The relation also became shallower with increasing depolarization, suggesting an apparent decrease in the efficacy of Ca in causing inactivation at more positive potentials. The basic kinetics of Ca current inactivation along with experimentally induced changes in those kinetics were simulated with a binding‐site model in which inactivation develops during current flow as a function of the entry and accumulation of free Ca2+. This demonstrated that a single Ca‐mediated process can account for the two‐component time course of inactivation, and that the nearly bi‐exponential shape need not arise from two separate processes. The two‐component time course emerges as a consequence of a postulated hyperbolic reaction between diminishing probability of channels remaining open and the accumulation of intracellular free Ca2+. The occurrence of a single‐ or a two‐component time course of inactivation thus appears to depend on the levels of internal free Ca2+ traversed during current flow.

Calcium dependence of ciliary activity in the oviduct of the salamander Necturus

1. Ciliary activity in the oviduct of the mud puppy Necturus maculosus was monitored by a photometric technique in normal, decalcified, and Triton X‐extracted preparations to investigate the regulatory role of calcium ions.

Calcium‐dependent repolarization in Paramecium

1. Intracellular injection, recording and current‐passing methods were used to investigate the role of intracellular Ca in the modulation of electrical behaviour in the ciliate Paramecium caudatum.

An electrophysiological study of the regulation of ciliary beating frequency in Paramecium.

1. The role of the surface membrane in the control of ciliary beat frequency in Paramecium was examined by intracellular electrophysiological techniques and pressure injection of Ca2+ and EGTA. Experiments were done on wild type P. caudatum and on both the wild type and a pawn mutant of P. tetraurelia. 2. The increased frequency of beating that accompanies reversal of power stroke orientation in response to depolarization in the wild type fails to occur during depolarization in the mutant pawn, which also fails to exhibit ciliary reversal upon depolarization. 3. Injection of moderate amounts of EGTA blocked the frequency increase without interfering with reversal of the beat in response to depolarization of the wild type. Larger injection of EGTA also prevented reversed beating. 4. The beat frequency in the normal (forward‐swimming) direction increased during hyperpolarization in pawn. The hyperpolarizing frequency‐voltage relations were quantitatively similar to those of the wild type. 5. Injection of EGTA to a final concentration of 10 mM into wild type cells neither modified the resting frequency nor blocked the frequency increase which normally accompanies hyperpolarization. 6. Transient ciliary reversal in both pawn and wild type produced by injection of Ca2+ could be terminated by the passage of inward current. The power stroke returned to the normal forward‐swimming direction and the ciliary beating frequency increased. Upon termination of the inward current the cilia of Ca2+‐injected cells again beat in reverse for many seconds. 7. The results support previous reports that increased frequency of beating and ciliary reversal seen in response to depolarization both require the entry of Ca2+ through the surface membrane. On the other hand, the results indicate that frequency increase with hyperpolarization is independent of an altered rate of Ca2+ entry. 8. Increased frequency during hyperpolarization appears to be related more closely to electrotonic membrane current than to membrane potential. It is proposed that inward current might activate high frequency beating by altering the ionic environment of the axoneme within the restricted volume of the cilium by electrophoretic means.

Cyclic AMP enhances calcium-dependent potassium current in Aplysia neurons.

Summary1.The effect on the Ca-dependent potassium current,IK(Ca), of procedures that increase intracellular cAMP levels was studied inAplysia neurons using three different pharmacological approaches.2.Exposure to cAMP analogues which were either resistant to or protected from phosphodiesterase hydrolysis caused an increase inIK(Ca) from 30 to 50% in 10 min. The degree of reversibility of this effect varied from complete with db cAMP to very little with pcpt cAMP.3.Exposure to cholera toxin, which stimulates the synthesis of endogenous cAMP, increasedIK(Ca) 25% in 10 min and the effect was not reversible. Both approaches were effective in all seven neuron types studied.4.Application of serotonin plus phosphodiesterase inhibitor caused an increase inIK(Ca) in neuron R15 but not in the other neuron types. Application of pentylene tetrazole (PTZ) led to a decrease inIK(Ca).5.It is proposed that elevation of cyclic AMP mediates an increased sensitivity of theIK(Ca) channel to Ca ions.

Cilia: Activation Coupled to Mechanical Stimulation by Calcium Influx

Ciliated epithelial cells in the oviduct of Necturus maculosus were stimulated mechanically by brief dimpling with a microstylus. This treatment produlced a transient depolarization of the membrane, and a transient increase in the frequency of ciliary beating. The increase in frequency of ciliary beating was related to the concentration of extracellular calcium ion, decreasing with reductiotn in calcium. Addition of lanthanum was followed by a decrease in spontaneous ciliary aictivity and a hyperpolarization of the membrane. In the presence of lanthanum, the transietnt depolarization in response to mechanical stimulation had a shorter timte course, and the concomitant increase in ciliary frequency was greatly reduced. It is concluded that calciuml ions enter the cell as a result of mechanical stimulationi of the membrane, and that calcium influx leads to an increase in the frequency of ciliary activity.

Two components of Ca-dependent potassium current in identified neurones ofAplysia californica

Outward tail currents measured inAplysia neurones after termination of depolarizing voltage-clamp pulses consist of rapidly decaying voltage-dependent K currents and slow tail currents of much slower time course. The rapidly decaying voltage-dependent tail currents were blocked with aminopyridines, and measurements of the slow tail currents were made following decay of any residual rapid tail currents. The slow tail current exhibited two components of differing sensitivity to externally applied tetraethylammonium (TEA) ions. In some neurones of the abdominal ganglion (L-2, L-4), virtually all of the slow tail current was resistant to blockage by TEA, while in others (L-3, L-6) 80% or more of the slow tail current was blocked by low TEA concentrations (KD<1 mM), the remaining slow tail current being resistant to TEA. This TEA-resistant slow tail current was identified as a K current because it reversed near the K equilibrium potential (EK), the reversal potential was shifted by changes in the external K concentration, and it could be blocked by injection of Cs+. It was abolished by replacement of external Ca2+ by Co2+ or Ba2+, by addition of Cd2+, or by injection of EGTA, and thus determined to be a Ca-dependent current. Intracellular injection of TEA or external application of aminopyridine or apamine had little or no effect on the TEA-resistant slow tail current. Quinidine reduced the TEA-sensitive, but not the TEA-resistant current. Both the TEA-sensitive and the TEA-resistant components of the slow tail current exhibited similar time courses of decay. Thus, neurones ofAplysia appear to contain different proportions of two classes of Ca-dependent K channels that differ in their sensitivity to certain channel blocking agents.

Ionic and pharmacological modification of input resistance and excitability in paramecium

Abstract 1. 1. Pharmacological agents and specific ionic conditions modify membrane properties of Paramecia. 2. 2. Lanthanum, TEA and tetracaine increase input resistance and regenerative response amplitude indicating a blockage of the calcium leakage current normally occurring during regenerative depolarizations. 3. 3. TEA blocks late repolarizing current as in nerve and muscle. 4. 4. Lanthanum does not block activated early inward current as in metazoan calcium spike systems. 5. 5. Alteration of the external ( K )/( Ca ) 1 2 ratio and increasing ionic strength at constant ( K )/( Ca ) 1 2 yield results consistent with the hypothesis that ciliary reversal is determined by electrophysiological factors controlling internal calcium concentration.