Irreversible effects of calcium ions on the plasma membrane calcium pump

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mm Ca²⁺, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca²⁺ affinity at 37°C and is unaffected by serine-protease inhibitors. The activation caused by 1 mm Ca²⁺ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 m Ca²⁺ is not inhibited. Preincubations at 0°C also lead to activation, to a level up to half that seen at 37°C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca²⁺-containing solutions at 37°C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 m Ca²⁺. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca²⁺ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.We thank The Wellcome Trust for a research grant, the Medical Research Council for an equipment grant and the Regional Transfusion Service (Sheffield) for bank blood supplies.     

Effect of calcium ions on outward currents in the somatic membrane of mollusk giant neurons

Potassium currents through the somatic membrane of giant neurons ofHelix pomatia in normal (10 mM Ca) Ringer's solution and low-calcium (1 mM Ca) solution were studied by the voltage clamp method. With a decrease in the Ca concentration to 1 mM peak potassium conductance versus membrane, potential curves and inactivation curves were shifted along the voltage axis in the negative direction by about 10 mV. Inactivation of the delayed potassium current was slowed in low Ca solution. The effect of a decrease in external calcium concentration on volt-ampere and inactivation characteristics increased with a rise in external pH. These effects of a low Ca concentration on potassium mechanisms of the giant neuron somatic membrane can be attributed to changes in the negative surface potential in the region of the potassium channels.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Institute of Biology, Hungarian Academy of Sciences, Tihany. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 400–409, July–August, 1976.     

Primary structure of the cAMP-dependent phosphorylation site of the plasma membrane calcium pump

The primary structure of a region of the erythrocyte plasma membrane calcium pump which is phosphorylated by the cAMP-dependent protein kinase has been determined. The sequence is A-P-T-K-R-N-S-S(P)-P-P-P-S-P-D. The site is located between the calmodulin binding domain and the C-terminus of the ATPase. The ATPase is phosphorylated only at this site by the cAMP-dependent protein kinase, and the phosphorylation is inhibited by calmodulin. The effect of the phosphorylation is to decrease the Km for Ca2+ of the purified ATPase from about 10 microM to about 1.4 microM and to increase the Vmax of ATP hydrolysis about 2-fold.     

Cadmium inhibits plasma membrane calcium transport

Summary The interaction of Cd²⁺ with the plasma membrane Ca²⁺-transporting ATPase of fish gills was studied. ATP-driven Ca²⁺-transport in basolateral membrane (BLM) vesicles was inhibited by Cd²⁺ with anI ₅₀ value of 3.0nm at 0.25 m free Ca²⁺ using EGTA, HEEDTA and NTA to buffer Ca²⁺ and Cd²⁺ concentrations. The inhibition was competitive in nature since theK _0.5 value for Ca²⁺ increased linearly with increasing Cd²⁺ concentrations while theV _max remained unchanged. The Ca²⁺ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd²⁺ occurs directly on the Ca²⁺ binding site of the Ca²⁺-transporting ATPase and not via the Ca²⁺-binding sites of calmodulin. It is suggested that Cd²⁺-induced inhibition of Ca²⁺-transporting enzymes is the primary effect in the Cd²⁺ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca²⁺ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd²⁺ on Ca²⁺-dependent enzymes may derive from increased free-Ca²⁺ levels when Cd²⁺ supersedes Ca²⁺ on the ligands.     

Effects of ATP on calcium binding to synaptic plasma membrane

The release of labeled norepinephrine from preloaded synaptosomes requires the presence of potassium and calcium. ATP-dependent binding of calcium to synaptic plasma membranes (SPM) may provide a means of maintaining the cation in a readily available pool for the triggering of transmitter release. A high Ca-binding capacity was demonstrated in SPM. The Km for calcium is 5.5 X 10(-5) M. The dependence of the system on the gamma phosphate of ATP was demonstrated by an increase in Ca-binding with increasing ATP concentration and by competitive inhibition of binding by ADP and AMP. Magnesium is also required for ATP-dependent Ca-binding. The optimum pH for the Ca binding was 7.0. Pretreatment of SPM with phospholipase A2 lowered the binding capacity. Sulfhydryl groups are also critical for ATP-dependent Ca binding to occur. A model for ATP-dependent Ca-binding was proposed.     

Giant plasma membrane vesicles to study plasma membrane structure and dynamics

The plasma membrane (PM) is a highly heterogenous structure intertwined with the cortical actin cytoskeleton and extracellular matrix. This complex architecture makes it difficult to study the processes taking place at the PM. Model membrane systems that are simple mimics of the PM overcome this bottleneck and allow us to study the biophysical principles underlying the processes at the PM. Among them, cell-derived giant plasma membrane vesicles (GPMVs) are considered the most physiologically relevant system, retaining the compositional complexity of the PM to a large extent. GPMVs have become a key tool in membrane research in the last few years. In this review, I will provide a brief overview of this system, summarize recent applications and discuss the limitations.     

Effect of miconazole on the structure and function of plasma membrane of Candida albicans

Abstract Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae , the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.     

Activation of a pea membrane protein kinase by calcium ions

Membranes from the buds of Pisum sativum L. contain a protein kinase which is activated 5- to 15-fold by micromolar levels of calcium. Best calcium activations were found with light-membrane fractions, and on density gradients these band at a similar position to the plasma membrane. Other heavier membranes, however, also contain a calcium-dependent protein kinase. The activity of the calcium-dependent protein kinase is inhibited by added phospholipids and phospholipase, in contrast to protein-kinase C. Calcium-dependent protein-kinase activity can be inhibited by 40% by low concentrations of the calmodulin inhibitor, trifluoperazine, but inhibitions are detected only after prior incubation of the membranes for some hours in ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid. Substantial calcium-dependent protein-kinase activity remains uninhibited by trifluoperazine indicating that there may be calmodulin-dependent and calmodulin-independent, but calcium-activated, protein kinases in pea membranes. The calcium-activated protein kinase seems to be intrinsically bound to membranes and only slight or partial solubilisation is obtained by the detergents nonidet P-40, (3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate or octyl glucose. Better solubilisation is obtained by acetone treatment. There is some retention of calcium activation after partial solubilisation. A calcium-independent protein kinase has also been detected in membrane preparations; it has a substrate specificity different from that the calcium-dependent enzyme. Our results indicate, therefore, that there may be at least three protein kinases attached to pea shoot membranes.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - TFP trifluoperazine     

Regulation of plasma membrane calcium fluxes by mitochondria

The role of mitochondria in cell signaling is becoming increasingly apparent, to an extent that the signaling role of mitochondria appears to have stolen the spotlight from their primary function as energy producers. In this chapter, we will review the ionic basis of calcium handling by mitochondria and discuss the mechanisms that these organelles use to regulate the activity of plasma membrane calcium channels and transporters.     

Effect of magnesium ions on red cell membrane properties

Summary Spectrin forms aggregates in solution when incubated at relatively high concentrations (several millimolar) of divalent cations. According to the evidence of electron microscopy, aggregates of globular appearance and, rather uniform size are cooperatively formed from spectrin dimers, no intermediate structures being seen. Inter-dimer chemical cross-linking of spectrin in intact red cell membranes is enhanced if magnesium ions at a concentration of 0.5mm or more are present. On the other hand, the elimination of magnesium from the interior of intact cells causes no significant change in shear elastic modulus, measured by micropipette assays, nor is there any dependence of membrane filtration rate on intracellular free magnesium concentration in the range 0–1mm. Magnesium-depleted cells are, however, converted into echinocytes within a short period, in which, control cells, exposed to ionophore and external magnesium ions, remain completely discoid. Magnesium-depleted cells also undergo structural, changes on heating below the temperature at which vesiculation sets in. These reveal themselves by the transformation of the cells to a unique characteristic shape, by grossly reduced filtrability, and by extensive agglutination of the cells when treated with a bifunctional reagent. Magnesium ions thus regulate the stability, but not to any measurable extent the gross elasticity, of the red cell membrane.     

Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane

The effects of various divalent cations in the external solution upon the Ca spike of the barnacle muscle fiber membrane were studied using intracellular recording and polarizing techniques. Analysis of the maximum rate of rise of the spike potential indicates that different species of divalent cations bind the same membrane sites competitively with different dissociation constants. The overshoot of the spike potential is determined by the density of Ca (Sr) ions in the membrane sites while the threshold membrane potential for spike initiation depends on the total density of divalent cations. The order of binding among different divalent and trivalent cations is the following: La+++, UO2++ > Zn++, Co++, Fe++ > Mn++ > Ni++ > Ca++ > Mg++, Sr++.     

The permeability of thymocyte plasma membranes for calcium ions]

The passive calcium transport through the plasmatic membranes of thymocytes of cattle has been investigated. It has been found that this process is a reaction of the first order. The kinetic values of the reaction have been calculated.     

Effect of calcium ions on potassium conductivity of latrotoxin channels

It has been shown that inhibition of potassium current through latrotoxin channels by calcium ions is followed by electrostatic interaction of these ions with a total charge on the mouth of the channel.