Low Divalent Cation Research Articles

Ca2+ transport properties and determinants of anomalous mole fraction effects of single voltage-gated Ca2+ channels in hair cells from bullfrog saccule.

We studied the permeation properties of two distinct single voltage-gated Ca2+ channels in bullfrog saccular hair cells to assess the roles of the channels as physiological Ca2+ transporters and multi-ion pores. By varying the permeant ions (Ba2+, Ca2+) and concentrations (2-70 mM), we estimated the affinity constant (K(D)) of the two channels as follows (mM): L-type channel, K(D,Ba) = 7.4 +/- 1.0, K(D,Ca) = 7.1 +/- 2.2 (n = 7); non-L-type channel, K(D,Ba) = 5.3 +/- 3.2, K(D,Ca) = 2.0 +/- 1.0 (n = 8). Using ionic concentrations close to physiological conditions (2 mM Ca2+ and 1.0 mM Mg2+), the conductance of the L-type channel was approximately 2 pS. We determined the mechanisms by which ions traverse the pore of these single Ca2+ channels, using mixtures of Ba2+ and Ca2+ at total concentrations above (70 mM) or close to (5 mM) the K(D) of the channels. We found evidence for an anomalous mole fraction effect (AMFE) only when the total divalent ion concentration was 5 mM, consistent with a multi-ion pore. We show that AMFE arises from the boundaries between the pore and bulk solution in the atria of the channel, which is derived from the presence of depletion zones that become apparent at low divalent cation concentrations. The present findings provide an explanation as to why previous whole-cell Ca2+ currents that were recorded in quasi-physiological Ca2+ concentrations (approximately 2-5 mM) showed clear AMFE, whereas single Ca2+ channel currents that were recorded routinely at high Ca2+ concentrations (20-110 mM) did not.

P2X7 receptors in rat parotid acinar cells: formation of large pores

1. Permeabilization of cells mediated by P2X(7) receptors occurs to varied degrees in native and heterologous expression systems. Previous studies on P2X(7) receptors in parotid acinar cells suggested that ATP does not permeabilize these cells. 2. Modification of the assay conditions showed that ATP permeabilizes freshly dissociated rat parotid acinar cells to the fluorescent dye YOPRO-1. 3. The pharmacological and physiological properties of this effect indicate that permeabilization is mediated by the P2X(7) receptor. Adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzBzATP) were effective agonists with EC(50) values of 49.3 and 0.6 microM, respectively. 4. Permeabilization was best observed in low divalent cation concentrations and at physiological temperatures. Previous studies failed to detect permeabilization because of the sensitivity of this effect to temperature and divalent cations. 5. An important consideration in understanding the effect of divalent cations is that the fluorescence of YOPRO-1/nucleic acid complexes is directly quenched by addition of divalent cations. This must be considered if quantitative study of the interaction of divalent cations with P2X(7) receptors is carried out using fluorescent DNA-binding dyes. 6. In summary, our data show that P2X(7) receptors in parotid acinar cells can form large pores in the plasma membrane. This property likely contributes to signalling and may be cytotoxic and have particular significance in damaged or inflamed salivary glands.

Identification of a β-like DNA Polymerase Activity in Bovine Heart Mitochondria

A new DNA polymerase activity, distinct from DNA polymerase γ, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase γ on several chromatographic columns and appears to copurify with a 38 ± 2-kDa polypeptide. Unlike DNA polymerase γ, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent Km values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3′ to 5′ exonuclease activity. Many of these characteristics resemble a β-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.

Non-genomic inhibition of human P2X7 purinoceptor by 17beta-oestradiol.

1. Effects of oestrogen on the current evoked by ATP and benzoylbenzoyl ATP (BzATP) in CV-1 monkey kidney cells transformed by SV 40 (COS cells) expressing the human P2X7 (hP2X7) purinoceptor were studied using standard patch-clamp techniques. 2. 17beta-Oestradiol rapidly and reversibly inhibited the whole-cell hP2X7 receptor cation current. This inhibitory action resulted in a rightward shift of the dose-response curve to ATP and BzATP in the presence of physiological as well as low divalent cation concentrations. 3. The inhibitory effect of 17beta-oestradiol on the BzATP- or ATP-induced cation current was concentration dependent. The half-maximal inhibition was obtained with 3 microM 17beta-oestradiol. Progesterone and 17alpha-oestradiol had almost no effect on the hP2X7 receptor cation current. 4. The inhibition of the hP2X7 receptor cation current by 17beta-oestradiol did not depend on the membrane potential. 17beta-Oestradiol added to the extracellular side of outside-out patches inhibited BzATP-activated single-channel currents. 5. Activation of the hP2X7 receptor in both COS and U937 (human macrophage) cells did not induce the formation of large non-specific pores. 6. Since COS cells do not express endogenous nuclear oestrogen receptor, this study shows that, at pharmacological concentrations, 17beta-oestradiol inhibited the hP2X7 receptor cation channel in a non-genomic manner.

Physicochemical Studies of Hard-to-Cook Beans (Phaseolus vulgaris)

Model aqueous systems were used for the study of the cooking quality of beans (Phaseolus vulgaris). These systems were created using graded contents of calcium and magnesium ions, in a ratio of 4:1, to prepare aqueous media with cation content corresponding to 0-400 mg litre -1 of calcium carbonate. The model systems were used to measure the quality of boiled beans in water of low and high divalent cation content. A boiling time of 60 min was used and the hardness of the boiling beans was measured by an Instron Universal Testing Machine. Cooking of beans in water of low divalent cation content equivalent to 0-50 mg litre -1 CaCO 3 resulted in well-boiled beans for both types (easy-to-cook and hard-to-cook) of beans. Boiling in water of high divalent cation content led to the formation of very hard beans for both types of bean, while boiling in water of moderate divalent cation content (normal drinking water: 150-250 mg litre -1 CaCO 3 ) differentiated between the two categories of beans. These results were further tested by adsorption experiments with divalent cations, on beans boiled (a) in different proportions of boiling water, and (b) in model aqueous systems. The divalent cation content of boiling water and the phytic acid content of the beans proved to be the crucial factors in the hard-to-cook phenomenon of beans.

The effect of cations on the settling and dewatering of activated sludges: Laboratory results

The effect of cations on the settling and dewatering of activated sludge was investigated using laboratory scale activated sludge reactors. Three parameters were investigated: the effect of the calcium to magnesium ratio; the effect of increasing divalent cation concentrations; and the effect of increasing monovalent cation concentrations. The study showed that the settling and dewatering properties of the activated sludge were dependent on both the concentrations and ratios of cations in the feed. Some activated sludge systems required both calcium and magnesium in the feed while others only required one of these cations for good settling and dewatering properties. A minimum of 0.7–2.0 meq/L each of calcium and magnesium was necessary for acceptable settling and dewatering. Increasing the feed concentrations of calcium and magnesium above this level improved floc strength, settling and dewatering properties and increased the bound protein concentration. The poor settling and dewatering of suspensions receiving low divalent cation concentrations in the feed could also be improved by batch addition of calcium and/or magnesium. However, improvements were greater when an equal concentration of the cation was added to the feed. Addition of the monovalent cation, sodium, to the feed resulted in a deterioration in settling and dewatering properties when the monovalent to divalent cation ratio exceeded approximately 2 to 1, expressed on an equivalent basis. However, the deterioration could be reversed by increasing the calcium and magnesium concentration in the feed which reduced the monovalent to divalent cation ratio below 2 to 1.

An ATP-gated cation channel with some P2Z-like characteristics in gastric smooth muscle cells of toad.

1. Whole-cell and single-channel currents elicited by extracellular ATP were studied in freshly dissociated smooth muscle cells from the stomach of the toad Bufo marinus using standard patch clamp and microfluorimetric techniques. 2. This ATP-gated cation channel shares a number of pharmacological and functional properties with native rat myometrium receptors, certain native P2Z purinoceptors and the recently cloned P2X7 purinoceptor. But, unlike the last two, the ATP-gated channel does not mediate the formation of large non-specific pores. Thus, it may represent a novel member of the P2X or P2Z class. 3. Extracellular application of ATP (> or = 150 microM) elicited an inward whole-cell current at negative holding potentials that was inwardly rectifying and showed no sign of desensitization. Na+, Cs+ and, to a lesser degree, the organic cation choline served as charge carriers, but Cl- did not. Ratiometric fura-2 measurements indicated that the current is carried in part by Ca2+. The EC50 for ATP was 700 microM in solutions with a low divalent cation concentration. 4. ATP (> or = 100 microM) at the extracellular surface of cell-attached or excised patches elicited inwardly rectifying single-channel currents with a 22 pS conductance. Cl- did not serve as a charge carrier but both Na+ and Cs+ did, as did choline to a lesser extent. The mean open time of the channel was quite long, with a range in hundreds of milliseconds at a holding potential of -70 mV. 5. Mg2+ and Ca2+ decreased the magnitude of the ATP-induced whole-cell currents. Mg2+ decreased both the amplitude and the activity of ATP-activated single-channel currents. 6. ADP, UTP, P1, P5-di-adenosine pentaphosphate (AP5A), adenosine and alpha, beta-methylene ATP (alpha, beta-Me-ATP) did not induce significant whole-cell current. ATP-gamma-S and 2-methylthio ATP (2-Me-S-ATP) were significantly less effective than ATP in inducing whole-cell currents, whereas benzoylbenzoyl ATP (BzATP) was more effective. BzATP, alpha, beta-Me-ATP, ATP-gamma-S and 2-Me-S-ATP induced single-channel currents, but a higher concentration of alpha, beta-Me-ATP was required. 7. BzATP did not induce the formation of large non-specific pores, as assayed using mag-fura-2 as a high molecular mass probe.

Structural Changes in Base-paired Region 28 in 16 S rRNA Close to the Decoding Region of the 30 S Ribosomal Subunit are Correlated to Changes in tRNA Binding

Escherichia coli30 S ribosomal subunits undergo a reversible change under low monovalent or divalent cation concentration and become inactive in tRNA binding and 50 S subunit association. In the inactive form, 16 S rRNA base-pairs (921–922)·(1395–1396) and (923–925)·(1391–1393), which are part of region 28, are unstable and an alternate arrangement, (921–923)·(1532–1534), is detected by psoralen photochemical crosslinking. Site-directed mutagenesis has been used to investigate whether changes in base-paired region 28 or the alternate secondary structure is responsible for the inactivity of the subunit. 30 S subunits with the substitution C1533A or with deletion of nucleotides 1534 to 1542 can still be inactivated like the wild-type 30 S subunit. On the other hand, 30 S subunits that contain sequence changes in the 920 to 926 region show moderate to severe decreases in tRNA binding even under activating conditions. When 30 S subunits containing these mutations were subjected to chemical probing, they failed to show the normal hyper-reactivity of nucleotide G926 and, instead, reactivity was shifted to G925 or to G928, and G929. Two mutations in the 920 region result in structures in which A1394 is base-paired rather than being unpaired as normal; deletion but not substitution of A1394 resulted in loss of tRNA binding activity and depression of the reactivity of G926. Mutations were made to insert or delete a nucleotide at position 920. The deletion mutant but not the insertion mutant has decreased tRNA binding activity and also low reactivity of G926. We conclude that structural changes in region 28 account for the active/inactive difference in tRNA binding. Molecular models of region 28 were made using the program MC-SYM. Models that include a hydrogen bond interaction between A1394 and G1392 account for the G926 reactivity in the wild-type sequence and account for the effects of most of the mutations in changing the G926 reactivity.

Actin-bound nucleotide/divalent cation interactions.

At this point, it may be worthwhile to list, in summary form, the important aspects of divalent cation and nucleotide binding to actin that have been reviewed here: 1) High affinity divalent cation binding to actin is very tight, with equilibrium dissociation constant KCa approximately 1 nM and KMg approximately 5 nM at pH 7.0. 2) The binding kinetics of Ca++ are diffusion limited. Dissociation is slow, with k-Ca approximately 0.015 sec at pH 7.0 (and low ionic strength). 3) The binding kinetics of Mg++ are limited by the characteristics of the Mg++ aquo-ion and are much slower than for Ca++; k-Mg approximately 0.0012 at pH 7.0. 4) Increase in pH or ionic strength weakens divalent cation binding at the high affinity site, primarily by increasing k-Ca and k-Mg. 5) Exchange of Mg++ for Ca++ (or vice versa) at the high affinity site is by a competitive pseudo-first order process with an apparent rate constant (kapp) intermediate between k-Ca and k-Mg and dependent upon the cation concentration ratio [Ca]/[Mg] present. 6) High affinity ATP binding is modulated by the high affinity divalent cation. The cation concentration range over which this modulation occurs is about 100-fold higher for Mg++ than for Ca++, again because of the different characteristics of the Mg++ and Ca++ aquo-ions. 7) At low divalent cation concentrations, ATP dissociation from actin is limited by dissociation of the tightly-bound divalent cation. 8) At high divalent cation concentrations, ATP dissociation probably occurs via dissociation of the divalent cation-nucleotide complex and is quite slow, with dissociation rate constant approximately 0.0005 sec-1. 9) Competitive nucleotide exchange on actin may be described by a pseudo-first order model analogous to that for divalent cation exchange. The pseudo-first order rate constants depend upon the divalent cation concentration. The overall nucleotide exchange rate constant kex depends upon these constants and the solution nucleotide concentration ratio, e.g. [ATP]/[ADP]. The following circumstances develop from the characteristics of the high affinity binding of divalent cation and nucleotide to actin: 1) The standard methods for actin preparation convert in vivo Mg-actin into Ca-actin. 2) Converting Ca-actin back to Mg-actin is not easy. A very low ratio of [Ca]/[Mg] is necessary, which usually requires the use of Ca-cheltors, and a long time (5-10 min) must be allowed for complete exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic factors regulating the interaction of Gardnerella vaginalis hemolysin with red blood cells

We have studied the hemolytic properties of an exotoxin released by Gardnerella vaginalis (Gvh). We found that hemolysis induced by Gvh is modulated by the composition of the isotonic buffer in which the red cells are suspended. In particular, low pH enhances its lytic activity, whereas low ionic strength and divalent cations diminish it. The inhibitory effects of reduced salt concentration and divalent cations occur despite normal binding of the toxin to the cells. This suggests that some post-binding step is impared. The toxin is also able to damage cholesterol-containing lipid vesicles, and even on these model membranes it is more active at low pH. From this point of view, Gvh has some similarity to Clostridium perfringens θ-toxin, a membrane-damaging toxin belonging to the family of ‘thiol-activated’ cytolysins produced by Gram-positive bacteria.

Lipid surface charge does not influence conductance or calcium block of single sodium channels in planar bilayers

We have studied the effects of membrane surface charge on Na+ ion permeation and Ca2+ block in single, batrachotoxin-activated Na channels from rat brain, incorporated into planar lipid bilayers. In phospholipid membranes with no net charge (phosphatidylethanolamine, PE), at low divalent cation concentrations (approximately 100 microM Mg2+), the single channel current-voltage relation was linear and the single channel conductance saturated with increasing [Na+] and ionic strength, reaching a maximum (gamma max) of 31.8 pS, with an apparent dissociation constant (K0.5) of 40.5 mM. The data could be approximated by a rectangular hyperbola. In negatively charged bilayers (70% phosphatidylserine, PS; 30% PE) slightly larger conductances were observed at each concentration, but the hyperbolic form of the conductance-concentration relation was retained (gamma max = 32.9 pS and K0.5 = 31.5 mM) without any preferential increase in conductance at lower ionic strengths. Symmetrical application of Ca2+ caused a voltage-dependent block of the single channel current, with the block being greater at negative potentials. For any given voltage and [Na+] this block was identical in neutral and negatively charged membranes. These observations suggest that both the conduction pathway and the site(s) of Ca2+ block of the rat brain Na channel protein are electrostatically isolated from the negatively charged headgroups on the membrane lipids.

Monitoring of the effects of dysprosium shift reagents on cell suspensions.

The effects of two widely used paramagnetic shift reagents for cationic NMR, dysprosium tripolyphosphate [Dy(PPP)2(7-)] and dysprosium triethylenetetramine hexaacetate [Dy(TTHA)3-], on the cell structure of dog and human erythrocytes, dog kidney cortical tubules and rat hepatocytes were investigated. The effect of shift reagents on cell integrity was monitored by measuring the hematocrit values for erythrocytes, by measuring the lactate dehydrogenase (LDH) release and by electron microscopy for cortical tubules and hepatocytes. The quantitation of the dyprosium penetration inside cells was accomplished by atomic absorption, atomic emission and neutron activation. More severe effects were observed with Dy(PPP)2(7-) than with Dy(TTHA)3-, and were dependent on the divalent cation concentration and on the shift reagent concentration. Very serious cell damage was observed after 60 min incubation in the presence of 10 mumol Dy(PPP)2(7-)/mL suspension at low or high divalent cation concentration. The situation was improved at 5 mumol Dy(PPP)2(7-)/mL suspension especially at high divalent cation concentration (2.5 mM). Incubation with Dy3+, PPP5- or Dy(TTHA)3- caused little or no structural effects but dysprosium was found to penetrate slowly inside tubules with Dy(TTHA)3-. Both Dy3+ and Dy(PPP)2(7-) penetrated rapidly inside cells. Dysprosium was found to bind to the isolated cytosol but not to isolated membranes, eliminating the possibility of extracellular membrane binding.

Similar properties of cGMP-activated channels between cones and rods in the carp retina

Using patch-clamp techniques, properties of cGMP-activated channel were studied at a single-channel level in order to examine (1) whether any differences are recognized between the cGMP-activated channels of rods and cones in the same animal species, and (2) whether the channel properties of the same photoreceptor class differ in different animal species. Experiments were performed on inside-out membrane patches excised from outer segments of rods and morphological subtypes of cones in the carp retina. Single-channel activities could be recorded when the patches were perfused with low concentrations of cGMP (less than 10 microM). Throughout five morphological subtypes of cones and rod, single-channel currents showed no significant rectification at membrane hyperpolarization in a low divalent cation solution, and single-channel conductances were almost the same: 13.8 +/- 0.2 pS (mean +/- S.E.M., n = 23) in cones and 12.7 +/- 0.8 pS (n = 3) in rods. These values were significantly smaller than that reported in catfish cones (about 50 pS), and that in rods of the toad and the tiger salamander (about 25 pS). In rods and all subtypes of cones of the carp, open durations of cGMP-activated channels were brief. In addition, kinetic parameters of channel openings and closings showed no differences throughout all subtypes of cones and rod.

Effects of Low Molecular Weight Peptides and Divalent Cations on Degradation and Binding of Angiotensin II

The effects of peptide inhibitors (bestatin and amastatin) and divalent cations (Ca2+ and Co2+) on the velocity of Asp1 liberation from angiotensin II (A-II) by human placental membrane fractions and binding of 125I A-II to human placental membranes were tested at 22 degrees C and 4 degrees C. Asp1 liberation was measured by high performance liquid chromatography. As expected, the degradation and binding of A-II were temperature sensitive, with both being at 4 degrees C than at 22 degrees C. While amastatin (10(-4) M) and bestatin 10(-6) M) significantly reduced the velocity of Asp1 liberation from A-II to about 45%, amastatin (10(-4) M) and bestatin (10(-4) M) increased 125I A-II binding to 125% and 130%, respectively. Ca2+ (10 mM) and Co2+ (10 mM) activated the velocity of Asp1 liberation from A-II to 140% and 120%, respectively at 22 degrees C. Ca2+ (10(-1) M) and Co2+ (10 mM) also enhanced 125I A-II binding about 130%. Previously we showed that the A-II degrading activity found in human placental membrane fractions is mainly due to aminopeptidases A and M. Since amastatin and bestatin are the specific inhibitors for aminopeptidases A and M, and since Ca2+ and Co2+ are the activators for aminopeptidase A and aminopeptidase M, respectively, it is conceivable that the enzymes regulate the levels of A-II and, therefore, that they may play an important role in the binding of A-II to human placental membrane fractions.

Investigations about various Possible Functions of the L -Amino Acid Oxidase in the Cyanobacterium Anacystis nidulans

Abstract The cyanobacterium A nacystis nidulans was grown on nitrate or L-arginine as sole nitrogen source and in the presence of different divalent cation concentrations (1 mᴍ MgSO4 and 0.1 mᴍ CaCl2 or 0.1 mᴍ MgSO4 and 0.05 mᴍ CaCl2). The L-amino acid oxidase previously reported to be present in Anacystis nidulans (E . K. Pistorius and A. E. Gau, Biochim. Biophys. Acta 849, 203, 1986) was shown to be involved in L-arginine catabolism in cells grown with the lower divalentcation concentration. Under these conditions L-arginine was partly degraded via 2-ketoarginine and 4-guanidinobutyrate. On the other hand, at higher cation concentrations the ʟ-amino acid oxidase activity seem ed to be not sufficient to provide enough NH4 + from ʟ-arginine for cell growth. Under those conditions photosystem II activity was initially reduced, and growth on ʟ-arginine could only start after photosystem II activity increased again and after arginase was induced. The arginase pathway was functional in A . nidulans grown on ʟ-arginine independently of the divalent cation concentration in the medium. A tentative scheme of the various functiona roles of the ʟ-amino acid oxidase protein in A . nidulans is given. This model combines the here presented and the previous results and suggests that the ʟ-amino acid oxidase is functional in photosynthetic and respiratory activities as well as in ʟ-arginine degradation in A . nidulans. All these activities of the ʟ-amino acid oxidase protein are greatly influenced by the divalent cation concentration in the growth medium.

CGMP-dependent channel protein from photoreceptor membranes: single-channel activity of the purified and reconstituted protein.

The cGMP-dependent channel protein has been purified from bovine rod photoreceptor membranes and incorporated into planar lipid membranes. At low divalent cation concentrations, cGMP stimulated single-channel current fluctuations. The probability Po of the channel being open strongly depended on the cGMP concentration (EC50 = 31 microM; Hill coefficient, n = 2.3); whereas the single-channel conductance (lambda = 26 pS) was independent of the agonist concentration. The agonist-stimulated increase in the probability of an open channel was largely due to shorter closed times and, to a lesser extent, due to the channel staying open for a longer time. The current-voltage relationship of the single open channel deviated from ohmic behavior, and the open probability decreased at more negative membrane potentials. The rectification of the macroscopic cGMP-induced current in artificial bilayers that contained many channel copies can be accounted for by the voltage dependence of channel gating together with the nonlinearity of the current-voltage curve of an open channel. Current fluctuations exhibited a variety of sublevels, indicating that the channel may exist in more than one conductive state.

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation.

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.

Location of a δ-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.

Electrokinetic and hydrodynamic properties of sarcoplasmic reticulum vesicles: A study by laser doppler electrophoresis and quasi-elastic light scattering

The electrical properties of sarcoplasmic reticulum vesicles were studied using electrophoretic mobility measurements by laser Doppler velocimetry. Combined with quasielastic light scattering analysis, this method enabled us to monitor changes in membrane surface charge density upon addition of NaCl, CaCl 2, and MgCl 2 solutions. At low ionic strength (4.7 m m), and in the absence of divalent salts, sarcoplasmic reticulum vesicles displayed a zeta potential value of −32.6 mV and a surface charge density of 5.4 10 −3 C/m 2, corresponding to one elementary negative charge per 30 nm 2 or about 2900 negative charges per vesicle. Upon addition of NaCl (up to 100 mM) vesicles became more negative (from 2900 to 6200 negative charges per vesicle), suggesting chloride binding or adsorption on the membrane. A noticeable increase of the vesicular size was observed upon addition of calcium or magnesium (25 to 30% increase of the hydrodynamic radius for 2.5 m m CaCl 2 or MgCl 2). This effect was prevented or suppressed by addition of relatively low concentrations of NaCl (50 m m). At low divalent cation concentrations one-third of the total net charge of the vesicles was neutralized suggesting a specific binding of cation on the membrane. At higher salt concentrations no modification of this parameter was observed. These results support the Gouy and Chapman theory about the electrical properties of sarcoplasmic reticulum vesicles.

Divalent cation binding to phospholipids: An EPR study

Divalent cation association to sonicated phospholipid liposomes has been examined with electron paramagnetic spectroscopy. Spectra were obtained suggesting that, in some cases, divalent cations associated with acidic phospholipid head groups are highly mobile. Using the amplitude of its characteristic sextet signal as a measure of free Mn(H2O)+6+, the apparent affinities of cardiolipin and phosphatidylserine for Mn2+ were measured as a function of monovalent electrolyte. Monovalent cations having smaller nonhydrated radii were more effective in displacing Mn from the phospholipids. Under conditions of low divalent cation concentrations, it is shown that the Gouy-Chapman diffuse double layer theory predicts a Mn-affinity (KA) inversely proportional to the square of monovalent salt concentration. Although this relationship was closely obeyed for Mn binding to cardiolipin, the fall-off in KA with added sodium chloride was slower in the cases of Mn binding to phosphatidylserin or phosphatidic acid. When phosphatidylcholine or cholesterol was incorporated into mixed vesicles along with a fixed amount of charged phospholipid, the Mn-binding strength was roughly proportional to the weight fraction of the latter. This result is consistent with: (1) a random dispersal of lipids in the bilayer, and (2) a 1:2 divalent cation-phospholipid interaction.