Calcium activates an electrogenic proton pump in neurospora plasma membrane

Calcium ionophoresis into coenocytic cells of Neurospora crassa activates the plasma membrane proton pump as measured by current-voltage analysis. This is direct evidence that intracellular calcium regulates the activity of a key transport enzyme found in higher plants and fungi.     

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.     

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

Summary The nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora 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 membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of –174 mV, the membrane conductance is 190 mhos/cm²; conductance increases to 240 mhos/cm² at –300 mV, and decreases to 130 mhos/cm² at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range.Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of –40 to –70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of 20 A/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-stateI–V curves, under the assumption that 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 F/cm²) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.     

The plasma membrane ATPase ofNeurospora: A proton-pumping electroenzyme

Probably the best marker enzyme for plasma membranes of eukaryotic cells is a magnesium-dependent, vanadate-inhibited ATPase whose primary function is the transmembrane transport of cations. In animal cells, different species of the enzyme transport different cations: sodium ions released in unequal exchange for potassium ions, calcium ions extruded alone (perhaps), or protons secreted in equal exchange for potassium ions. But in plants and fungi only proton secretion has been clearly demonstrated. A useful model cell for studying the proton-secreting ATPase has been the ascomycete fungusNeurospora, in which the enzyme drives an outward current of protons that can exceed 50 µA/cm² and can support membrane potentials greater than 300 mV. Both thermodynamic and kinetic studies have shown that the proton-pumping ATPase ofNeurospora normally transports only a single proton for each ATP molecule split; and kinetic modelling studies have suggested (contrary to conventional assumptions) that the fast steps in the overall reaction are transmembrane transit of the proton and its dissociation following transport, while the slow steps are the binding of protons and/or ATP. The primary structure of theNeurospora enzyme, recently deduced by gene sequencing, is very close to that of the yeast (Saccharomyces) enzyme, and the hydropathic patterns for both closely resemble those for the animal-cell plasma-membrane ATPases. All of these enzymes appear to have 6–10 membrane-spanning -helices, plus a large cytoplasmic headgroup which bears the catalytic nucleotide-binding site. Structural data, taken together with the electrical-kinetic behavior, suggest that the catalytic headgroup functions as an energized gate for protons. From a geometric point of view, action of such a gate would transfer the membrane field across the transported ion, rather than vice versa.     

Calcium effects on electrogenic pump and passive permeability of the plasma membrane ofChara corallina

Summary Removal of Ca²⁺ from the medium results in depolarization of theChara internodal cell and an increase in membrane conductance (G _m). The increase in conductance is associated with an increase in K⁺ conductance, as judged by Ca²⁺ effects on the K⁺ dependence of clamp current. The voltage dependence ofG _m is also affected by Ca²⁺, as is the time course of the response of clamp current to a step change in voltage. Mg²⁺ restores the low conductance and the fast response to a voltage change, but not hyperpolarization at neutral pH, suggesting that there is an additional, independent effect on the electrogenic pump. The membrane does not show the normal ability to increase proton conductance at high pH in the absence of Ca²⁺; this is also restored by Mg²⁺ as well as by Ca²⁺.     

Characterization of an electrogenic ATP and chloride-dependent proton translocating pump from rat renal medulla

To study acidification mechanisms in the distal nephron, microsomes were prepared from rat renal medulla by differential centrifugation. Microsomes were enriched in the enzyme marker gamma-glutamyl transferase and contained an ATP-dependent proton pump, as evidenced by ATP-dependent, 3,3',4',5-tetrachlorosalicylanilide-reversible quenching of acridine orange fluorescence. Acidification was vanadate-insensitive, but was completely inhibited by micromolar N-ethylmaleimide. Maximal acidification was achieved in the presence of halide (Cl-, Br-) only and was not attainable with potassium-valinomycin diffusion potentials without halide ion. Microsomal ATPase activity was neither chloride- nor N-ethylmaleimide-sensitive. A chloride conductance was observed only with vesicles which had undergone ATP-dependent acidification. An ATP-dependent, N-ethylmaleimide-inhibitable, 3,3',4',5-tetrachlorosalicylanilide-reversible, and chloride-attenuated quench of bis(1,3-dibutylbarbituric acid-(5] pentamethinoxonol fluorescence was seen, consistent with net transfer of positive charge into the vesicles. Nonetheless, positive intravesicular potentials increased the ATP-dependent initial acidification rate, perhaps by increasing availability of chloride ion to the transport site. Our results are consistent with an electrogenic, ATP-dependent proton pump regulated by a voltage-sensitive chloride site.     

A chemoreceptive bilayer lipid membrane based on an auxin-receptor ATPase electrogenic pump

Auxin-binding proteins have been extracted from coleoptiles and primary leaves of maize and diffusion--reconstituted in phosphatidyl choline/partially-oxidized cholesterol membranes. Measurement of membrane ion flux at 25 mV external potential with buffered KCl electrolyte was performed for the receptor support matrix and various combinations of ATP, receptor and naphthalene-1-acetic acid. Addition of the three components in any order results in a substantial increase in current with a limit-of-detection for auxin of about 10(-7)M. The pH-dependence of the response is consistent with previous suggestions that an ATPase pump acts to translocate protons in the presence of K+ and Mg2+ and that the pump can be activated by auxin. This work provides the first direct link between the binding of a plant hormone to a putative receptor and the evocation of a biochemical response.     

Contribution of an electrogenic pump to the resting membrane polarization in a crustacean heart.

1. In the neurogenic heart of the isopod crustacean Porcellio dilatatus, external K+ removal depolarized the membrane (K0 effect) whereas subsequent restoration of K+ resulted in a rapid hyperpolarization (K1 effect). 2. The amplitude of the K1 effect depended on the duration of the prior K+ deprivation and on the subsequent K+ concentration. 3. The membrane resistance slightly increased during the K0 effect; during the K1 effect, it only returned to its control value. 4. Ouabain, cooling and replacement of external Na+ by Li+ also produced depolarization. 5. The K1 effect was suppressed by ouabain and markedly depressed by lowering the temperature to 4-6 degrees C. It was abolished if Li+ replaced Na+ during the prior privation of K+; moreover Li+ was unable to act as a substitute for external K+ in generating the K1 effect if used at equivalent concentration, but enhanced the effect at high concentration. 6. The findings are consistent with the presence of an electrogenic sodium pump in the myocardium of Porcellio contributing to the resting membrane potential. 7. Changes in the spontaneous rhythm observed during K0 and K1 are further suggestive of the presence of an electrogenic Na+ pump in the pacemaker neurons of the cardiac ganglion. Another explanation is also proposed. 8. The magnitude of the spontaneous contractions of the heart was increased during the K0 effect and markedly decreased during the K1 effect. An indirect effect of the changes in internal Na+ concentration on the contractile processes is suggested.     

A contribution of an electrogenic Na+ pump to membrane potential in Aplysia neurons

The resting membrane potential (RMP) of Aplysia neurons is very temperature-dependent, and in some cells increases with increasing temperature by as much as 2 mv/°C. RMP at room temperature may significantly exceed the potassium equilibrium potential, which can be determined by measurement of the equilibrium point of the spike after potential. The hyperpolarization on warming is completely abolished by ouabain, replacement of external Na⁺ by Li⁺, removal of external K⁺, and by prolonged exposure to high Ca⁺⁺, while it is independent of external chloride but is increased by cocaine (3 x 10^-3 M). In an identified cell that shows a marked temperature dependence of RMP, both the potassium equilibrium potential and the membrane resistance were found to be relatively independent of temperature. The hyperpolarization on warming, which may increase RMP by as much as 50%, can most reasonably be ascribed to the activity of an electrogenic Na⁺ pump.     

Crossing-over across the centromere in the mating-type chromosome ofNeurospora crassa

The frequency of crossing-over in the two short regions on opposite arms, adjacent to the centromere of the mating-type chromosome ofNeurospora crassa is controlled, independently in each arm, by at least two genes with equal and additive effect. These genes segregate on inbreeding and cause great variability in both the frequency of recombination and the frequency of second-division segregation of loci situated within these regions. Recombination values between loci situated beyond these sensitive regions is not affected; the relative increase or decrease in their centromere distances may be attributed to change in the recombination frequency about the centromere only.     

Relationship between the composition of phospholipids and respiratory activity of choline-deficient mutants ofNeurospora crassa

Phosphatidylcholine is one of the most frequent phospholipid components of the inner mitochondrial membrane ofNeurospora crassa. Quantitative analysis of phospholipids of the wild strain ofNeurospora crassa and of its twocho mutants showed that these strains did not significantly differ in the content of phosphatidylcholine. Mutants cultivated in a medium without choline contained, as compared with the wild strain, an increased amount of phosphatidylserine and a decreased quantity of phosphatidic acid. Respiratory activity increased and sensitivity to inhibitors of respiration changed. It is likely that the presence of choline in the growth medium exerts a regulatory effect on the cell metabolism of these mutants.     

Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis

The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25 degrees C. We have measured the Ca2+-ATPase activity (v) under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component, but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM Mg2+, it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.     

Cell physiological aspects of the plasma membrane electrogenic H<Superscript>+</Superscript> pump

This article deals with cell physiological aspects of the plasma membrane electrogenic proton (H⁺) pump and emphasizes the contribution of the giant algal cells of the Characeae in elucidating the mechanism of the pump. First, a history of the development of intracellular perfusion techniques in characean internodal cells is described, including preparation of tonoplast-free cells. Then, an outline of the hypothesis of the electrogenic H⁺ pump proposed by Kitasato is introduced, who prophesied the existence of an electric potential generated by an active H⁺ efflux. Subsequently, a history of finding ATP as the direct energy source of the electrogenic ion pump is presented. Quantitative agreement between the pump current and the ATP-dependent H⁺ efflux supports the notion that the ion carried by the electrogenic ion pump is H⁺. The role of the H⁺ pump in regulation of the cytosolic pH is discussed. Mechanisms of light-induced potential change through photosynthesis-controlled activation of the H⁺ pump are discussed in terms of changes in the levels of adenine nucleotides and in modulation of the K_m value for the ATP of H⁺-ATPase. Recent progress in the molecular mechanism of the blue-light-induced activation of the H⁺-ATPase in guard cells is presented. However, there are cases where H⁺-ATPase activity is inhibited by blue light, indicating the flexibility of the control mechanisms of H⁺-ATPase activity. Finally, modulation of H⁺-pumping or H⁺-ATPase activities in response to environmental factors, such as anoxia, membrane excitation, osmotic and salt stresses, nutrient deficiencies and aluminum toxicity are described. Discussions are presented on the regulation of the electrogenic H⁺ pump.     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.