scholarly journals Golgi membranes contain an electrogenic H+ pump in parallel to a chloride conductance.

1983 ◽  
Vol 97 (4) ◽  
pp. 1303-1308 ◽  
Author(s):  
J Glickman ◽  
K Croen ◽  
S Kelly ◽  
Q Al-Awqati
Keyword(s):  
Membrane Potential ◽  
Proton Pump ◽  
Gradient Centrifugation ◽  
Chloride Conductance ◽  
F1 Atpase ◽  
Chloride Uptake ◽  
Golgi Vesicles ◽  
Galactosyl Transferase ◽  
Golgi Membranes ◽  
Electrogenic Proton Pump

Rat liver Golgi vesicles were isolated by differential and density gradient centrifugation. A fraction enriched in galactosyl transferase and depleted in plasma membrane, mitochondrial, endoplasmic reticulum, and lysosomal markers was found to contain an ATP-dependent H+ pump. This proton pump was not inhibited by oligomycin but was sensitive to N-ethyl maleimide, which distinguishes it from the F0-F1 ATPase of mitochondria. GTP did not induce transport, unlike the lysosomal H+ pump. The pump was not dependent on the presence of potassium nor was it inhibited by vanadate, two of the characteristics of the gastric H+ ATPase. Addition of ATP generated a membrane potential that drove chloride uptake into the vesicles, suggesting that Golgi membranes contain a chloride conductance in parallel to an electrogenic proton pump. These results demonstrate that Golgi vesicles can form a pH difference and a membrane potential through the action of an electrogenic proton translocating ATPase.

Acidification of rat liver lysosomes: quantitation and comparison with endosomes

1993 ◽  
Vol 265 (4) ◽  
pp. C901-C917 ◽  
Author(s):  
R. W. Van Dyke
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Rat Liver ◽  
Proton Pumps ◽  
Intact Cells ◽  
Early Endosomes ◽  
Medium Permeability ◽  
Electrogenic Proton Pump ◽  
Secondary Lysosomes ◽  
Permeability Studies

Both lysosomes and endosomes are acidified by an electrogenic proton pump, although studies in intact cells indicate that the steady-state internal pH (pHi) of lysosomes is more acid than that of endosomes. We undertook the present study to examine in detail the acidification mechanism of purified rat liver secondary lysosomes and to compare it with that of a population of early endosomes. Both endosomes and lysosomes exhibited ATP-dependent acidification, but proton influx rates were 2.4- to 2.7-fold greater for endosomes than for lysosomes because of differences in both buffering capacity and acidification rates, suggesting that endosomes exhibited greater numbers or rates of proton pumps. Lysosomes, however, exhibited a more acidic steady-state pHi due in part to a slower proton leak rate. Changes in medium Cl- increased acidification rates of endosomes more than lysosomes, and the lysosome ATP-dependent interior-positive membrane potential was only partially eliminated by high-Cl- medium. Permeability studies suggested that lysosomes were less permeable to Na+, Li+, and Cl- and more permeable to K+ and PO4(2-) than endosomes. Na-K-adenosine-triphosphatase did not appear to regulate acidification of either vesicle type. Endosome and lysosome acidification displayed similar inhibition profiles to N-ethylmaleimide, dicyclohexyl-carbodiimide, and vanadate, although lysosomes were somewhat more sensitive [concentration producing 50% maximal inhibition (IC50) 1 nM] to bafilomycin A1 than endosomes (IC50 7.6 nM). Oligomycin (1.5-3 microM) stimulated lysosome acidification due to shunting of membrane potential. Overall, acidification of endosomes and lysosomes was qualitatively similar but quantitatively somewhat different, possibly related to differences in the density or rate of proton pumps as well as vesicle permeability to protons, anions, and other cations.

Voltage dependence of chloride current through Xenopus muscle membrane in alkaline solutions

1980 ◽  
Vol 58 (9) ◽  
pp. 999-1010 ◽  
Author(s):  
Peter C. Vaughan ◽  
James G. McLarnon ◽  
Donald D. F. Loo
Keyword(s):  
Steady State ◽  
Membrane Potential ◽  
Chloride Conductance ◽  
Resting Potential ◽  
Muscle Membrane ◽  
Alkaline Solutions ◽  
Constant Field ◽  
Confined Space ◽  
Initial Current ◽  
Test Current

Three-microelectrode voltage-clamp experiments have been conducted on surface fibres of Xenopus laevis sartorius muscles. When potassium and chloride were substituted by rubidium and sulphate, negligibly small currents were observed. In solutions containing rubidium and chloride at pH 8.4–8.8 normally polarized fibres exhibited instantaneous current–voltage relations that were linear over a wide voltage range. Chloride conductance varied widely from fibre to fibre; the mean resting conductance at −80 mV was 7.4 × 10−4 ± 2.6 × 10−4 S/cm2 (mean ± SE). When hyperpolarizing voltage steps were made, conductance declined from the initial to the steady state; inward currents saturated near 14 μA/cm2. In experiments performed on fibres depolarized by immersion in K+-and Rb+-rich solutions it was found that resting conductance did not increase by as much as would be expected from constant field – constant permeability precepts, by comparison with normally polarized fibres. Despite the low chloride transmembrane concentration ratio, rectification in the steady state was similar in depolarized and normally polarized fibres.When a two-pulse protocol was employed to test the availability of chloride conductance after conditioning of the system at some voltage, it was found that the test current, the initial current at the onset of the test voltage step, depended sigmoidally on the conditioning voltage. The sigmoid relationships had asymptotic limits: after hyperpolarizing conditioning the test current was minimal, after depolarizing conditioning, maximal. Normalized sigmoid relations were superimposable, whether from normally polarized or chronically depolarized cells.When the protocol was repeated using different test potentials and initial currents following a particular conditioning voltage were plotted against the test potential, families of straight lines were obtained. The slopes of the members of these families were dependent on the conditioning voltage: the more negative the conditioning step the lower the slope. The lines projected through a mutual intersection at a voltage slightly more positive than the resting potential. This is interpreted as indicating that there is some voltage, slightly positive with respect to the membrane potential, at which the initial current is independent of the conditioning voltage.It is concluded that the state of the chloride conductance mechanism is a function of the deviation of the membrane from the resting potential rather than of the absolute membrane potential and that relaxations from initial to steady states reflect properties of the permeation mechanism rather than accumulation or depletion of chloride in a confined space, although some contribution by a mechanism such as the latter cannot be completely ruled out.

Voltage-dependent modulation of GABAA receptor channel desensitization in rat hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2151-2160 ◽  
Author(s):  
K. W. Yoon
Keyword(s):  
Membrane Potential ◽  
Hippocampal Neurons ◽  
Membrane Conductance ◽  
Cell Voltage ◽  
Chloride Conductance ◽  
Membrane Voltage ◽  
Hippocampal Cell Culture ◽  
Voltage Dependent ◽  
Agonist Concentration ◽  
Rate Of Recovery

1. The mechanism of the time-dependent decline in gamma-amino-butyric acid (GABA)-induced chloride conductance, referred to as desensitization, was studied in dissociated rat hippocampal cell culture with the use of a whole-cell voltage-clamp recording. 2. In most cells the gradual decline of membrane conductance was dependent simultaneously on the agonist concentration and membrane voltage. Even in the continued presence of GABA, desensitization could be prevented by holding the membrane potential > 0 mV in a near symmetrical chloride gradient across the cell membrane. 3. The “recovery” from desensitization occurred after removal of the agonist with a time constant of approximately 35 s. The rate of recovery from desensitization was independent of membrane voltage. 4. When the membrane potential was jumped from a negative to a positive membrane potential during steady state of desensitization, the GABA-induced chloride conductance gradually “relaxed” to the undesensitized state. This phenomenon of gradual increase in chloride conductance or “reactivation” from desensitization was both voltage and agonist dependent. 5. The process of recovery of the GABA ionophore from the desensitized state is distinct from the process of reactivation, which is dependent both on the voltage and agonist. 6. These observations suggest that the ligand-bound GABA receptor has two alternate states, i.e., permissive (activated) and desensitized. The rates of transition between these two states are voltage dependent.

scholarly journals Proton pump-driven cutaneous chloride uptake in anuran amphibia

2003 ◽  
Vol 1618 (2) ◽  
pp. 120-132 ◽  
Author(s):  
Lars Jørn Jensen ◽  
Niels Johannes Willumsen ◽  
Jan Amstrup ◽  
Erik Hviid Larsen
Keyword(s):  
Proton Pump ◽  
Chloride Uptake

scholarly journals The transverse distribution of phospholipids in the membranes of Golgi subfractions of rat hepatocytes

1984 ◽  
Vol 219 (1) ◽  
pp. 261-272 ◽  
Author(s):  
J A Higgins
Keyword(s):  
Phospholipase C ◽  
Morphological Changes ◽  
Residual Activity ◽  
Membrane Vesicles ◽  
Rat Hepatocytes ◽  
Membrane Bilayer ◽  
Transverse Distribution ◽  
Golgi Vesicles ◽  
Golgi Membranes ◽  
In Trans

The transverse distribution of phospholipids in the membranes of subfractions of the Golgi complex was investigated by using phospholipase C and 2,4,6-trinitrobenzenesulphonic acid as probes. In trans-enriched Golgi membranes, 26% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate or for hydrolysis by phospholipase C, and 72% of the phosphatidylcholine is hydrolysed by phospholipase C. In cis-enriched Golgi membranes, 45% of the phosphatidylethanolamine is available for reaction with trinitrobenzenesulphonate and for hydrolysis by phospholipase C, and 95% of the phosphatidylcholine is hydrolysed by phospholipase C. Under the conditions used with either probe the contents of the Golgi vesicles labelled with either [3H]palmitic acid or [14C]leucine were retained. Galactosyltransferase activity of the membrane vesicles was partially inhibited by the experimental procedures used to investigate the transverse distribution of phospholipids. However, the residual activity was latent, suggesting that the vesicles remained closed. Trinitrobenzenesulphonic acid caused no detectable morphological change in either Golgi fraction. Phospholipase C treatment caused morphological changes, including fusion of vesicles and the appearance of ‘signet-ring’ profiles in some vesicles; however, the vesicles remained closed and the bilayer was retained. It appears, therefore, that neither probe causes major disruption of the Golgi vesicles nor gains access to the inner surface of the membrane bilayer. These observations suggest that phospholipids have a transverse asymmetry in Golgi membranes, that this distribution differs in trans and cis membranes, and that the phospholipid structure of Golgi membranes is inconsistent with a simple flow of membrane bilayer from endoplasmic reticulum to Golgi membranes to plasma membrane.

scholarly journals Membrane potential of Plasmodium-infected erythrocytes.

1982 ◽  
Vol 93 (3) ◽  
pp. 685-689 ◽  
Author(s):  
R B Mikkelsen ◽  
K Tanabe ◽  
D F Wallach
Keyword(s):  
Membrane Potential ◽  
Plasma Membranes ◽  
Metabolic Inhibitors ◽  
Erythrocyte Membranes ◽  
Weak Acids ◽  
Atpase Inhibitor ◽  
Lipophilic Anion ◽  
Parasite Stage ◽  
Infected Cells ◽  
Electrogenic Proton Pump

The membrane potential (Em) of normal and Plasmodium chabaudi-infected rat erythrocytes was determined from the transmembrane distributions of the lipophilic anion, thiocyanate (SCN), and cation, triphenylmethylphosphonium (TPMP). The SCN- and TPMP-measured Em of normal erythrocytes are -6.5 +/- 3 mV and -10 +/- 4 mV, respectively. The TPMP-measured Em of infected cells depended on parasite developmental stage; "late" stages (schizonts and gametocytes) were characterized by a Em = -35 mV "early stages (ring and copurifying noninfected) by a low Em (-16 mV). The SCN-determined Em of infected cells was -7 mV regardless of parasite stage. Studies with different metabolic inhibitors including antimycin A, a proton ionophore (carbonylcyanide m-chlorophenylhydrazone [CCCP] ), and a H+ -ATPase inhibitor (N,N'-dicyclohexylcarbodiimide, [DCCD] ) indicate that SCN monitors the Em across the erythrocyte membrane of infected and normal cells whereas TPMP accumulation reflects the Em across the plasma membranes of both erythrocyte and parasite. These inhibitor studies also implicated proton fluxes in Em-generation of parasitized cells. Experiments with weak acids and bases to measure intracellular pH further support this proposal. Methylamine distribution and direct pH measurement after saponin lysis of erythrocyte membranes demonstrated an acidic pH for the erythrocyte matrix of infected cells. The transmembrane distributions of weak acids (acetate and 5,5-dimethyloxazolidine-2,4-dione) indicated a DCCD-sensitive alkaline compartment. The combined results suggest that the intraerythrocyte parasite Em and delta pH are in part the consequence of an electrogenic proton pump localized to the parasite plasma membrane.

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