The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.     

Cooperative phenomena in the membrane potential of parathyroid cells induced by divalent cations

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.     

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.     

Control of regeneration and morphogenesis by divalent cations in Acetabularia mediterranea

A study of the effects of altered calcium and magnesium levels on the capacities of Acetabularia plants to elongate and to form whorls or caps revealed sharp transitions of regenerative state over small concentration changes of external calcium at both reduced and elevated levels. Cobalt and lanthanum, which interfere with calcium fluxes across membranes, also affect regeneration and morphogenesis with sharply defined transition ranges. It is shown that these effects are localized at the growing tip. The nature of the mechanisms involved in the transitions from one morphogenetic state to another in the regenerative process is considered.     

The permeability of endplate channels to monovalent and divalent metal cations

The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.     

Stimulation of phosphatase release from cultured tobacco cells by divalent cations

Upon addition of divalent cations to the incubation medium ofcultured tobacco cells, the release of phosphatase into themedium increased and the time course of the release became biphasic.A rapid release (phase I release) occurred instantaneously afterthe addition and then a release at a constant rate (phase IIrelease) followed. Sodium and potassium ions did not affectthe enzyme release. Lanthanum ions caused the biphasic enzymerelease but inhibited the phase II release. The effects of temperature and metabolic inhibitors indicatedthat phase I release was limited by a diffusion process butphase II release was limited by an enzymatic reaction requiringmetabolic energy. From the results it was concluded that divalent cations enhancedthe enzyme release not only by stimulating the transport ofenzyme to the outside of the cell membrane, but also by liberatingthe enzyme retained on the exterior of the cells, e.g., thecell walls. The released phosphatase could be separated into two fractions,F-I and F-II. Only F-I was released by phase I release, whileboth F-I and F-II resulted from phase II release. This indicatedthat F-I was preferentially trapped on the exterior of the cells. ¹ These experiments were carried out at the Department of Botanyin the Faculty of Science of the University of Tokyo. (Received December 15, 1978; )     

The mitochondrial inner membrane anion channel. Regulation by divalent cations and protons

It is now well established that incubation of mitochondria at pH 8 or higher opens up an electrophoretic anion transport pathway in the inner membrane. It is not known, however, whether this transport process has any physiological relevance. In this communication we demonstrate that anion uniport can take place at physiological pH if the mitochondria are depleted of matrix divalent cations with A23187 and EDTA. Using the light-scattering technique we have quantitated the rates of uniport of a wide variety of anions. Inorganic anions such as Cl-, SO4(2-), and Fe(CN)6(4-) as well as physiologically important anions such as HCO3-, Pi-, citrate, and malate are transported. Some anions, however, such as gluconate and glucuronate do not appear to be transported. On the basis of the finding that the rate of anion uniport assayed in ammonium salts exhibits a dramatic decline associated with loss of matrix K+ via K+/H+ antiport, we suggest that anion uniport is inhibited by matrix protons. Direct inhibition of anion uniport by protons in divalent cation-depleted mitochondria is demonstrated, and the apparent pK of the binding site is shown to be about 7.8. From these properties we tentatively conclude that anion uniport induced by divalent cation depletion and that induced by elevated pH are catalyzed by the same transport pathway, which is regulated by both matrix H+ and Mg2+.     

Modulation of Na+/alanine cotransport in liver sinusoidal membrane vesicles by internal divalent cations

Rat liver basolateral plasma membrane (blLPM) vesicles resuspended in 5 mM Mg2(+)-, Ca2(+)-, Mn2(+)- or Co2(+)-containing media exhibited a markedly lower rate of Na(+)-stimulated L-alanine transport. Divalent cation inhibition of L-alanine uptake was dose dependent, and was observed only when the vesicles were pre-loaded with the divalent cations. The presence or absence of the metal ions in the extravesicular incubation media had no effect on L-alanine transport. Conversely, pretreatment of the vesicles with 0.2 mM of either EGTA or EDTA resulted in higher initial rates of L-alanine transport. This stimulation was overcome by addition of excess divalent cation to the vesicle suspension solution. Since these blLPM vesicles are primarily oriented right-side-out, the divalent cation inhibition of L-alanine transport appears to be a result of their interaction with cytosolic components of the cell membrane. Total Na+ flux as measured with 22Na+ was not affected by intravesicular 5 mM Mg2+ or Ca2+, indicating that the inhibition was not due to dissipation of the Na+ gradient. These observations suggest that intracellular divalent cations may serve to modulate L-alanine transport across the liver cell plasma membrane.     

Aggregation of nucleosomes by divalent cations.

Conditions of precipitation of nucleosome core particles (NCP) by divalent cations (Ca(2+) and Mg(2+)) have been explored over a large range of nucleosome and cation concentrations. Precipitation of NCP occurs for a threshold of divalent cation concentration, and redissolution is observed for further addition of salt. The phase diagram looks similar to those obtained with DNA and synthetic polyelectrolytes in the presence of multivalent cations, which supports the idea that NCP/NCP interactions are driven by cation condensation. In the phase separation domain the effective charge of the aggregates was determined by measurements of their electrophoretic mobility. Aggregates formed in the presence of divalent cations (Mg(2+)) remain negatively charged over the whole concentration range. They turn positively charged when aggregation is induced by trivalent (spermidine) or tetravalent (spermine) cations. The higher the valency of the counterions, the more significant is the reversal of the effective charge of the aggregates. The sign of the effective charge has no influence on the aspect of the phase diagram. We discuss the possible reasons for this charge reversal in the light of actual theoretical approaches.     

Electric pulse-induced fusion of mouse lymphoma cells: Roles of divalent cations and membrane lipid domains

Summary Mouse leukemic lymphoblasts (L5178Y) brought into close contact by dielectrophoresis underwent cell fusion following the application of electrical pulses in the presence of electrolytes. The electrically fused cells became spherical after switching off the dielectrophoretic field. Fusion between a cell vitally stained with Janus Green and that with Neutral Red resulted in the homokaryon with a mixed color. Intracellular potentials simultaneously recorded from the two cells located on both sides of the homokaryon were identical. The fusion efficiency was remarkably dependent upon temperature, displaying a discontinuity at about 11°C in the Arrhenius plot. The extracellular application of phospholipase-A₂ or-C suppressed the fusion yield. Thus, it appears that the phospholipid domains play a crucial role in the electric pulse-induced cell fusion. Treatment of the cells with proteolytic enzymes markedly enhanced the fusion yield, presumably due to removing the glycocalix and/or giving rise to fusion-potent, protein-free lipid domains. The presence of millimolar concentrations of divalent cations (irrespective of Mg²⁺ or Ca²⁺) as well as of micromolar concentrations of Ca²⁺ (but not Mg²⁺) was prerequisite to the resealing of membranes suffered from electrical breakdown upon exposure to electric pulses. In addition, extracellular Ca²⁺ (but not Mg²⁺) ions at more than micromolar concentrations were indispensable for the cell fusion.     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Protection of cells against membrane damage by haemolytic agents: divalent cations and protons act at the extracellular side of the plasma membrane

The protective effect of Ca2+, Zn2+ and H+ against membrane damage induced by different haemolytic agents has been studied by measuring monovalent cation leakage and haemolysis of erythrocytes, and phosphoryl[3H]choline and adenine nucleotide leakage from Lettre cells prelabelled with [3H]choline. The protective effect of Ca2+ and Zn2+ on erythrocytes damaged by Staphylococcus aureus alpha-toxin, Sendai virus or melittin is unaffected by the addition of A23187, even though this ionophore greatly increases the uptake of 45Ca2+ or 65Zn2+. The same result has been found for the protective effect of Zn2+ on Lettre cells damaged by S. aureus alpha-toxin, Sendai virus, melittin or Triton X-100. Leakage of phosphoryl[3H]choline from prelabelled Lettre cells is inhibited if extracellular pH is lowered; lowering the intracellular pH without affecting the extracellular pH, affords little protection. It is concluded that Ca2+, Zn2+ and H+ protect cells against membrane damage induced by haemolytic agents by an action at the extracellular side of the plasma membrane.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Effects of membrane depolarization and divalent cations on anaphylactic histamine secretion

The effects of membrane depolarization and divalent cations on histamine release have been studied in sensitized mast cells. Membrane potential of these cells has been measured with intracellular microelectrodes. Our results show that mast cells have a large resting potential (-61 +/- 12 mV) however they do not generate active membrane electrical responses when are depolarized by passing current through the recording microelectrode. High external K+ does not increase histamine release. Histamine secretion is supported by alkali-earth divalent cations (Ca2+ greater than Sr2+ greater than Ba2+) but strongly inhibited by transition metals. Ca2+ concentrations above 1 mM inhibit histamine release, however, this effect is not mimicked by Sr2+ and Ba2+.     

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.