Taka-aki Ohkawa

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: I. Effects of La3+, verapamil, EGTA, W-7, and TFP on the action potential

SummaryThe cytoplasmic streaming of the normal internodal cell of giant algaChara stops transiently at about the peak of action potential. Application of La3+ or verapamil (a calcium channel blocker) or removal of external Ca2+ by EGTA caused a partial depolarization of the resting potential, partial decrease of the membrane conductance and a marked decrease of the amplitude of action potential. Under these conditions, the conductance at the peak of action potential reduced markedly and the streaming of cytoplasm did not cease during action potential (excitation-cessation (EC) uncoupling). The effects of Ca2+ channel blockers could not be removed by addition of CaCl2 to the external medium. In contrast, the effect of EGTA on the excitability could be removed to a greater extent and the cytoplasmic streaming ceased at about the peak of action potential by the addition of Ca2+ externally. Application of calmodulin antagonists W-7 or TFP caused similar effects on the action potential and on the cytoplasmic streaming.

A kinetic analysis of the electrogenic pump of Chara corallina. I: Inhibition of the pump by DCCD

SummaryThe current-voltage curve of theChara membrane was obtained by applying a slow ramp depo- and hyperpolarization by use of voltage clamp. With the progress of poisoning by DCCD (dicyclohexylcarbodiimide) theI–V curve moved by about 50 mV (depolarization) along the voltage axis, reducing its slope, and finally converged to theid-V curve of the passive diffusion channel. Changes ofip-V curve of the electrogenic pump channel could be obtained by subtracting the latter from the former.The sigmoidalip-V curve could be simulated satisfactorily by adopting a simple reaction kinetic model. Kinetic parameters of the successive changes of state of the H+ ATPase could be evaluated. Changes of these kinetic parameters during inhibition gave useful information about the molecular mechanism of the electrogenic pump.Depolarization of the membrane potential, decrease of membrane conductance, and decrease of pump current during inhibition of the pump with DCCD are caused mainly by the decrease of conductance of the pump channel. The decrease of this pump conductance is caused principally by a marked decrease of the rate constant for releasing H+ to the outside.

Role of calcium ion in the excitability and electrogenic pump activity of theChara corallina membrane: II. Effects of La3+, EGTA, and calmodulin antagonists on the current-voltage relation

SummaryThe steady N shapeI/V curves were obtained by applying slow ramp hyper- and depolarization pulses toChara cells under the voltage-clamp condition. Application of calcium channel blocker, 20 μm La3+, to theChara membrane caused, in about 30 min, a marked reduction of the transient inward current and later almost complete blocking of the pump current, while the steady outward current remained almost unaffected. Removal of external Ca2+ with 0.5mm EGTA caused similar results. Application of calmodulin antagonists, 10 μm TFP or 20 μm W-7, also gave very similar results, i.e., the decrease of the transient inward current and of H+-pump activity. These results suggest that not only the excitatory mechanisms but also the H+-pump activity ofChara membrane are regulated by calmodulin within a comparatively narrow range of internal Ca2+ level.

A kinetic analysis of the electrogenic pump ofChara corallina: III. Pump activity during action potential

SummaryThe current-voltage curve (I–V curve) of theChara membrane was obtained by applying a slow ramp hyper- and depolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50μm DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steadyI–V curve within two hours, which gave theid-V curve of the passive diffusion channel. Theip-V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. The sigmoidalip-V curve could be simulated satisfactorily with a simple reaction kinetic model which assumes a stoichiometric ratio of 2. The emf of the pump (Ep) is given as the voltage at which the pump current changes its sign. The conductance of the pump (gp) can be calculated as the chord conductance from theip-V curve, which is highly voltage dependent having a peak at a definite voltage. The changes of emf and conductance during excitation were determined by use of the current clamp (I=0). Since theEp andgp(V) are known, the changes, during excitation, of emf (Ed) and conductance (gd) of the passive diffusion channel can be calculated. The marked increase of the membrane conductance and the large depolarization during the action potential are caused by the marked increase of the conductance of the passive diffusion channel and the large depolarization of its emf. The conductance of the electrogenic pump decreases to about half at the peak of action potential, while the pump current increases almost to a saturated level.

A kinetic analysis of the electrogenic pump ofChara corallina: II. Dependence of the pump activity on external pH

SummaryThe current-voltage curve of theChara membrane was obtained by applying a slow ramp de- and hyperpolarization by use of voltage clamp. By inhibiting the electrogenic pump with 50μm DCCD (dicyclohexylcarbodiimide), theI–V curve approached a steady state within 100 min, which gave theid-V curve of the passive diffusion channel. Theip-V curve of the electrogenic pump channel was obtained by subtracting the latter from the former. With the increase of external pH, theid-V curve showed only a slight change, while theip-V curve of the pump channel showed almost a parallel shift, in the hyperpolarizing direction, along the voltage axis in the pH range between 6.5 and 7.5. The sigmoidalip-V curve in this pH range could be simulated satisfactorily with the five-state model reported previously (U. Kishimoto, N. Kami-ike, Y. Takeuchi & T. Ohkawa,J. Membrane Biol.80:175–183, 1984) as well as with a lumped two-state model presented in this report. The analysis based on these models suggests that the electrogenic pump of theChara membrane is mainly a 2H+/1ATP pump. The forward rate constant in the voltage-dependent step increased with the increase of external pH, while the backward one decreased. On the other hand, the forward rate constant in the voltage-independent step remained almost unchanged with the increase of external pH, while the backward one increased markedly. The pump conductance at the resting membrane potential showed either a slight increase or a decrease with the increase of external pH, depending on the sample. Nevertheless, the pump current showed generally a slight increase with the increase of external pH.

A kinetic analysis of the electrogenic pump ofChara corallina: IV. Temperature dependence of the pump activity

SummaryThe sigmoidal current-voltage curve (ip-V curve) of the electrogenic H+-pump of theChara membrane was simulated satisfactorily with a simple reaction kinetic model which assumed consecutive changes in state of H+-ATPase. Four rate constants, i.e., forward and backward ones in voltage-dependent and-independent steps could be evaluated from the data. The emf of the pump (Ep), the voltage at which the pump current changes its sign, varies only slightly with temperature. However, the pump current (ip) is highly temperature dependent, and there-fore the conductance (gp) of the pump, calculated as the chord conductance from theip-V curve, is also highly voltage dependent having a peak at a level somewhat less negative than the resting potential. In contrast togp, the conductance (ip) of the passive channel does not change appreciably with temperature. Arrhenius plots ofgp and also of the rate constants showed a clear bend at about 19°C. Great temperature dependence of the kinetic parameters offers useful information on the pumping mechanism of theChara membrane.

Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

SummaryThe relationship between the external Ca2+ concentrations [Ca2+]0 and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond −350∼−400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called “punch-through” (H.G.L. Coster,Biophys. J.5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca2+]0. Increasing [Ca2+]0 caused a more negative and decreasing it caused a more positive shift of BP. BP was at about −700 mV in 200 μM La3+ solution. [Mg2+]0 depressed the membrane electrical tolerance which was supposed to be due to competition with Ca2+ at the Ca2+ binding site of the membrane. Such a depressive effect of Mg2+ was almost masked when the [Ca2+]0/[Mg2+]0 ratio was roughly beyond 2.

Effects of local anesthetics on the Chara plasmalemma

The effects of lidocaine, tetracaine, procaine and bupivacaine (less than 1000 microM) on the Chara corallina internodal cell were studied. These local anesthetics depolarized the membrane at rest, while they affected the rising phase and the peak level of action potential not appreciably. Instead, they prolonged the time course of the falling phase of action potential as slowly as the repolarization was imperfect, even after enough lapse beyond the refractory period. Consequently, an action potential appeared to enhance the degree of depolarization at rest. Such a depolarization with stimulus/excitation was named use-dependent depolarization, while the depolarization without excitation, the resting one. The order of the potency of the use-dependent depolarization almost coincided with that of the nerve-blocking potency. During depolarization the change in membrane conductance was not simple. However, the conductance-voltage (Gm-Vm) relationship curve in the presence of local anesthetic suggested that depolarization was due to, not only the decrease in the electrogenic H(+)-pump, but also the increase in the diffusion conductance.

Electrical tolerance (breakdown) of the Chara corallina plasmalemma: II. Inductive property of membrane and effects of pHo and impermeable monovalent cations on breakdown phenomenon.

SummaryChanges in the chord conductanceG and the membrane electromotive forceEm in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–Vm relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pHo in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pHo 7.In some cellsEm shifted to a positive level as large as +50∼+70 mV during the breakdown phenomenon. Such a large positive shift ofEm is caused mainly by the increase in conductance of Cl− and partly Ca2+ and K+.