Two Different Conductances Contribute to the Anion Currents in Coffea arabica Protoplasts

The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two components: a voltage-gated conductance (G _s ) and a voltage-independent one (G _l ). G _s is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials. G _l and G _s also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with G _s shows strong outward rectification and its permeability sequence is F⁻ > NO₃ ⁻ > Cl⁻. In the same conditions the current associated with G _l does not rectify and its permeability sequence is F⁻≫ NO₃ ⁻= Cl⁻. Furthermore, at potentials over +50 mV G _s , but not G _l , increases with a time constant of several minutes. Finally the gating of G _s is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances similar to the ones described here have been found in many plant preparations but G _l -type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding to G _s ). We show that in coffee protoplasts G _l and G _s are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities. Received: 25 November 1996/Revised: 9 April 1997     

Vacuolar chloride regulation of an anion-selective tonoplast channel

Fluctuations in intravacuolar chloride concentrations affected the tonoplast inward (anion flux into the vacuole) currents of sugar beets (Beta vulgaris). Rising vacuolar chloride concentrations induced increases in the levels of nitrate, acetate and phosphate inward currents. These currents, evoked at physiological vacuolar potentials, showed a linear relationship with the concentration of vacuolar chloride between 6 and 100 mm. Single channel currents revealed that rises in vacuolar chloride increased the frequency and probability of channel openings at a given tonoplast potential by reducing the mean closed time of the anion channel. In addition, there was an increase in the gating charge for the channel and a decrease in the free-energy favoring the transition of the channel from the closed to the open state. Vacuolar chloride had a very different effect on malate currents. Increasing chloride concentrations resulted in decreased frequency and open probability of the channel openings, a decrease in the gating charge and an increase in the mean closed time of the channel. Our results support the role for vacuolar chloride concentrations regulating the influx of anions into the vacuole, in addition to osmoregulation. The activation of channel activity by chloride will provide a pathway for the storage of nutrients, such as nitrate and phosphate into the vacuole, while the reduction of the malate currents will allow the use of malate for mitochondrial oxidation and cytoplasmic pH control.This work was supported by the National Science and Engineering Research Council of Canada.     

Permeant anions are not required for norepinephrine secretion from pheochromocytoma cells

The ‘chemiosmotic’ model for secretion proposed by Pollard and his colleagues (Int. Rev. Cytol. 58, 159–197, 1979) was tested with pheochromocytoma cells. Contrary to the prediction of this model, norepinephrine secretion did not require the presence of a permeant anion in the medium. Secretion was not blocked by replacing much of the Cl^? of the medium with isethionate or by replacing all of the Cl^? salts of the medium with isotonic sucrose. Biochemical evidence is presented to indicate that the cells secreted by the normal exocytotic mechanism in the sucrose medium. Making the normal bathing medium hypertonic with 300 mM sucrose increased the basal level of norepinephrine release, but alos suppressed secretion in response to a strong secretagogue (1 mM Ba²⁺). The data indicate that the Pollard model does not apply to pheochromocytoma cells, but suggest the possible involvement of osmotic pressure in exocytosis.     

Influence of enzymatic phospholipid cleavage on the permeability of the erythrocyte membrane. II. Protein-mediated transfer of monosaccharides and anions

In order to investigate the influence of membrane lipids on transport via the protein domain of the erythrocyte membrane, a number of facilitated diffusion processes was studied by tracer flux techniques in whole cells after cleavage of up to 65% of the phosphatidylcholine or the sphingomyelin by phospholipase A₂ from Naja naja or bee venom, or by sphingomyelinase, respectively.The mediated fluxes of l-arabinose, which is transported by the glucose carrier, and of l-lactate, which uses a specific monocarboxylate carrier, were markedly inhibited by cleavage of either phosphatidylcholine or sphingomyelin. These phospholipid dependencies are in line with earlier data on cholesterol dependencies (Deuticke, B. (1977) Rev. Physiol. Biochem. Pharmacol. 78, 1–97). They can only in part be explained by changes of membrane fluidity. More specific interactions of the degradation products with the carrier proteins seem also to play a role.Sulfate and oxalate transfer, which proceed via the inorganic anion-exchange system, are essentially unaffected by cleavage of phosphatidylcholine and less sensitive to sphingomyelin cleavage than the two other processes. This also agrees with earlier data on cholesterol independency of sulfate transfer. The inorganic anion-exchange protein thus seems to be less dependent on the surrounding lipids in its conformation and its mode of action than the two other carriers.     

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S1₃ → S₀ is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.     

Difficulties of molecular dissociation of Clostridium botulinum type G progenitor toxin

Clostridium botulinum type G progenitor toxin was chromatographed on DEAE-Sephadex and Q-Sepharose equilibrated with 0.05 M Tris-HCl buffer, pH 8.0, containing 0.2 M urea. The toxin was eluted in a single protein peak from DEAE-Sephadex, but it was eluted in four protein peaks from Q-Sepharose; the third peak was toxic and the others were nontoxic. The third peak, appearing to be the toxic component, had a molecular mass of 150,000. In SDS-polyacrylamide gel electrophoresis, purified type G progenitor toxin migrated in six bands, with molecular masses of 150,000, 140,000, 58,000, 10,800, 10,600, and 10,400. Type G progenitor toxin may be composed of a toxin component with a molecular mass of 150,000 and a nontoxic component in a manner similar to progenitor toxins of other types. Type G toxic component, whether it was reduced or not, migrated in a single band to the same relative positions in SDS-PAGE; type A toxic component reduced with 2-mercaptoethanol migrated in two bands.