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D Gradmann

Publications and source records attributed to D Gradmann.

47 records · Page 3Linked to original sources
PubMed1978

Current-voltage relationships for the plasma membrane and its principal electrogenic pump in Neurospora crassa: I. Steady-state conditions.

The nonlinear membrane current-voltage relationship (I-V curve) for intact hyphae of Neurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus "quasi-linear" cable theory. Under normal conditions of growth and respiration, the membrane I-V curve is best described as a parabolic segment convex in the direction of depolarizing current. At the average resting potential of - 174 mV, the membrane conductance is approximately 190 micronhos/cm2; conductance increase to approximately 240 micronhos/cm2 at -300 mV, and decreases to approximately 130 micronhos/cm2 at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range. Inhibition of the primary electrogenic pump in Neurospora by ATP withdrawal (with 1 mM KCN) depolarizes the membrane to the range of -40 to -70 mV and reduces the slope of the I-V curve by a fixed scaling factor of approximately 0.8. For wild-type Neurospora, compared under control conditions and during steady-state inhibition by cyanide, the I-V difference curve--presumed to define the current-voltage curve for the electrogenic pump--is a saturation function with maximal current of approximately 20 muA/cm2, a half saturation potential near -300 mV, and a projected reversal potential of ca. -400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H+ extruded:1 ATP split. The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-state I-V curves, under the assumption the cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects. The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 muF/cm/2); a second of 50-75 msec; and a third of 20-30 sec, perhaps representing changes of intracellular composition.

Biological Transport, Active↗
PubMed1976

"Metabolic" action potentials in Acetabularia.

The transient depolarizations in Acetabularia which fulfill the essential criteria of an action potential (all-or-none characteristics, triggering by depolarization, propagation, etc.) are investigated. These action potentials are analyzed by conductance measurements and voltage clamp experiments on the basis of the analog circuit of the membrane (Gradmann, D. 1975, J. Membrane Biol. 25:183). It is concluded that these action potentials do not arise by permeability changes of the passive diffusion channels, but by the active pathway of the electrogenic pump, which consists of a voltage source EP of about --20 mV in series with two nonlinear conducting elements P1 and P2, the latter and EP being shunted by a large quasi capacity CP of some mF cm-2. The nonlinear current-voltage relationship of the carrier system (P1) is not changed during the action potential but has an effect on its time course. However, the elements P2 and CP, which probably reflect metabolic entities, are suggested to control the action potentials.

Acetabularia↗
PubMed1976

Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia.

Membrane potential, Vm, and K+ (86Rb+) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. -170 mV) the K+ influx amounts to 0.24-0.6 pmol-cm-2-s-1 and the K+ efflux to 0.2-1.5 pmol-cm-2s-1. According to the K+ concentrations inside and outside the cell (40:1) the voltage dependent K+ flux (zero at Vm = EK = -90 mV) is stimulated approx. 40-fold for Vm more positive than EK. It is calculated that during one action potential (temporary depolarization to Vm more positive than EK) a cell looses the same amount of K+, which leaks in during 10-20 min in the resting state (Vm = -170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K+ balance of this alga.

Acetabularia↗
PubMed1976

"Action potentials" in Neurospora crassa, a mycelial fungus.

Occasional spontaneous "action potentials" are found in mature hyphae of the fungus Neurospora crassa. They can arise either from low-level sinusoidal oscillations of the membrane potential or from a linear slow depolarization which accelerates into a rapid upstroke at a voltage 5-20 mV depolarized from the normal resting potential (near-180 mV). The "action potentials" are long-lasting, 1-2 min and at the peak reach a membrane potential near-40 mV. A 2-to 8-fold increase of membrane conductance accompanies the main depolarization, but a slight decrease of membrane conductance occurs during the slow depolarization. Two plausible mechanisms for the phenomenon are (a) periodic increases of membrane permeability to inorganic ions, particularly H+ or Cl- and (b) periodic decreases in activity of the major electrogenic pump (H+) or the Neurospora membrane, coupled with a nonlinear (inverse signoid) current-boltage relationship. Identification of action potential-like disturbances in fungi means that such behavior has now been found in all major biologic taxa which have been probed with suitable electrodes. As yet there is no obvious function for the events in fungi.

Action Potentials↗
PubMed1975

Analog circuit of the Acetabularia membrane.

The high membrane potential of Acetabularia (Em=-170mV) is due to an electrogenic pump in parallel with the passive diffusion system (Ed=-80mV) which could be studied separately in the cold, when the pump is blocked. Electrical measurements under normal conditions show that the pump pathway consists of its electromotive force Ep with two elements P1 and P2 in series; P2 is shunted by a large capacitance (Cp=3mF cm-2). The nonlinear current-voltage relationship of P1 (light- and temperature-sensitive) could be determined separately; it reflects the properties of a carrier-mediated electrogenic pump. The value of Ep(-190 mV) indicates a stoichiometry of 2:1 between electrogenically transported charges and ATP. The electrical energy normally stored in Cp, compares well with the metabolic energy, stored in the ATP pool. The nonlinear current-voltage relationship of P2 (attributed to phosphorylating reactions) is also sensitive to light and temperature and is responsible for the region of negative conductance of the overall current-voltage relationship. The power of the pump (1 muW cm-2) amounts to some percent of the total energy turnover. The high Cl- fluxes (1 nmol cm-2 sec-1) and the electrical properties of the plasmalemma are not as closely related as assumed previously. For kinetic reasons, a direct and specific Cl- pathway between the vacuole and outside is postulated to exist.

Acetabularia↗
PubMed1975

Oscillations of an electrogenic pump in the plasma membrane of Neurospora.

The presence of the poky mutation in Neurospora crassa produces mitochondria which are defective in cytochromes b and aa3 but which compensate by means of an alternate, cyanide-insensitive oxidase. As previously reported (Slayman, Rees, Orchard & Slayman, J. Biol. Chem., 250:396, 1975) cyanide blockade of the poky strain carrying the partial suppressor f results in a metabolic downshift of only 56%, compared with a downshift of 98% in wild-type Neurospora; the downshift is accompanied by exponential decay of ATP in the wild type, but by an undershoot and monotonic recovery of ATP in poky f. Whereas the membrane potential declines with ATP in wild-type Neurospora, it oscillates near the resting level (ca. -- 185 mV) in poky f. Oscillations begin with a depolarizing swing of 30--100 mV, followed by slight hyperpolarization, then by 2--4 damped cycles having a frequency near 1/min. Similar oscillations arise with antimycin, salicyl hydroxamic acid, and several uncoupling agents, and depend on partial maintenance of respiration through either the defective cytochrome chain or the alternate oxidase. Small oscillations (maximally +/- 30% of the control value) in membrane conductance also occur, roughly in phase with the oscillations of membrane potential. The amplitude of these, in comparison with the nonlinearity of the normal current-voltage relationship for the membrane, strongly suggests that they arise as a secondary consequence of the voltage changes. Therefore, since it has previously been argued (Slayman, Long & Lu, J. Membrane Biol. 14:305, 1973) that most of the resting membrane potential in the organism arises from active extrusion of H+ ions, the simolest interpretation of the cyanide-induced voltage oscillations is that current through the H+ pump is modulated cyclically. The ultimate mechanism for this modulation is unresolved, but could plausible involve a metabolic feedback system, oscillations of intracellular pH, or both. In many respects the observed voltage oscillations resemble the well-known oscillations of mitochondrial H+ flux which are produced by sudden metabolic shifts.

Adenosine Triphosphate↗