Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

Fluxes of Ca2+, Sr2+ and Mg2+ in synaptosomes.

Synaptosomes isolated from sheep brain cortex accumulate Ca²⁺, Sr²⁺ and Mg²⁺ when incubated in isosmotic sucrose media containing 5 mM of either of these cations. The maximal levels of cations retained per mg of protein are 100 nmol of Ca²⁺, 85 nmol of Mg²⁺ and 80 nmol of Sr²⁺. The loss of Ca²⁺ or Sr²⁺ from the preloaded synaptosomes is increased by monovalent cations in the following order: Na⁺> K⁺ > Li⁺> choline, whereas for the loss of Mg²⁺ this order is different: K⁺ > Na⁺ > Li ～ choline. The efflux of Ca²⁺ or Sr²⁺ induced by monovalent cations decreases as the temperature is lowered and it is nearly abolished at 0°C, whereas the efflux of Mg²⁺ is much less influenced by temperature. The results suggest that the mechanism of exchange of Ca²⁺ for Na⁺ in synaptosomes operates similarly for Sr²⁺, but not for Mg²⁺.     

Ionic Selectivity and Permeation Properties of Human PIEZO1 Channels

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K⁺, Na⁺, Cs⁺ and Li⁺; organic cations: TMA and TEA, and divalents: Ba²⁺, Ca²⁺, Mg²⁺ and Mn²⁺. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs⁺, Na⁺ and K⁺ had chord conductances in the range of 35–55 pS with the exception of Li⁺, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K⁺, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba²⁺ and 15 pS for Ca²⁺ at -80 mV and 10 pS for Mg²⁺ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K⁺ currents much like the divalents. As expected, the channel K⁺ conductance increased with K⁺ concentration saturating at ~ 45 pS and the K_D of K⁺ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.     

Barium Influx Mediated by the Cardiac Sodium-Calcium Exchanger in Transfected Chinese Hamster Ovary Cells

We examined Ba²⁺ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na⁺/Ca²⁺ exchanger (CK1.4 cells). Ba²⁺ competitively inhibited exchange-me diated ⁴⁵Ca²⁺ uptake with a K _i ∼ 3 mM. Ba²⁺ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na⁺, as expected for Na⁺/Ba²⁺ exchange activity. The maximal velocity of Ba²⁺ accumulation was estimated to be 50% of that for Ca²⁺. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na⁺, Ba²⁺ influx was negligible in the absence of Na⁺ and increased to a maximum at 20–40 mM Na⁺. At higher Na⁺ concentrations, Ba²⁺ influx declined, presumably due to the competition between Na⁺ and Ba²⁺ for transport sites on the exchanger. Unlike Ca²⁺, Ba²⁺ did not appear to be taken up by intracellular organelles: Thus, ¹³³Ba²⁺ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca²⁺ stores that had been depleted of Ca²⁺ by pretreatment of the cells with ionomycin (a Ca²⁺ ionophore) remained empty during a subsequent period of Ba²⁺ influx. Ca²⁺ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca²⁺]_i. Exchange-mediated Ba²⁺ influx was inhibited when cytosolic [Ca²⁺] was reduced to 20 nM or less and was accelerated at cytosolic Ca²⁺ concentrations of 25–50 nM. We conclude that (a) Ba²⁺ substitutes for Ca²⁺ as a transport substrate for the exchanger, (b) cytosolic Ba²⁺ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba²⁺ influx is accelerated by low concentrations of cytosolic Ca²⁺.     

Effects of cations on the sugar receptor-site of the blowfly, Phormia

1) Ca^{+ +} (1 to 10 mM) lowered the binding affinity of sugarreceptor-site for sucrose in the labellar sugar receptor ofthe blowfly, Phormia regina, without changing the maximum-responseamplitude. It also elevated the values of the Hill coefficient(n_H) in some degrees. 2) Other divalent cations such as Mg^{+ +}, Ba^{+ +} or Cd^{+ +} alsoshowed almost the same property as above. The sequence of theeffect is as follows: Ba^{+ +}, Mg^{+ +} x Ca^{+ +} x Cd^{+ +}. Trivalentcation, La^{+ + +} (1 mM), changed the value of n_H from 1 (La^{++ +}-free) to 2. 3) On the contrary, the action of monovalent cations such asK⁺ or Na⁺, of which ionic strength was made the same as thatof the divalents hardly suppressed the response. 4) The results obtained do not support the hypothesis, at leaston the sugar receptor of the fly, that the receptor potentialis attributable to a change of the surface potential (zeta potential)as is proposed for the frog sugar receptor.     

Effeet of Metal Ions on Activity of Exopectic Acid Transeliminase of Erwinia sp.

Contrary to the exopectic acid transeliminase of Clostridium multifermentans, that of Erwinia sp. was activated strongly by Na⁺ and to a much less extent by Ca²⁺. K⁺ had a small stimulating effect on the enzyme activity. Mn²⁺ and Co²⁺, like Ca²⁺, activated the enzyme weakly. Ba²⁺ and Mg²⁺ showed no and a slight inhibitory effect, respectively, on the activity.

An almost total loss of activity was caused by the addition of EDTA to the reaction mixture. In the presence of Na⁺ the enzyme activity was restored by addition of divalent cations. Individual monovalent cations or each of the divalent cations was ineffective in restoring the activity.     

N-terminus of PutCAX2 from <Emphasis Type="Italic">Puccinellia tenuiflora</Emphasis> affects Ca<Superscript>2+</Superscript> and Ba<Superscript>2+</Superscript> tolerance in yeast

Cation/H⁺ exchangers (CAXs) are membrane proteins that transport Ca²⁺ and other cations using the H⁺ gradient generated by H⁺-ATPase or H⁺-pyrophosphatase. This study reports the characterization of CAX2 from Puccinellia tenuiflora with respect to molecular and functional properties. PutCAX2 was cloned from a cDNA library of P. tenuiflora seedlings. The expression of PutCAX2 in shoots and roots was induced by Ca²⁺ and Ba²⁺ treatments. A green fluorescent protein (GFP) marker revealed that PutCAX2 was located on the endoplasmic reticulum (ER) membrane. Four yeast transformants were created using GFP fusion PutCAX2 and truncated PutCAX2s, and their growth in the presence of various cations (Fe³⁺, Al³⁺, Mn²⁺, Cu²⁺, Co²⁺, Ni²⁺, Mg²⁺, Zn²⁺, Na⁺, Li⁺, Ca²⁺, and Ba²⁺) was analyzed. The N-terminally truncated PutCAX2 (GFP-ΔNPutCAX2) and the N and C-terminally truncated PutCAX2 (GFP-ΔNCPutCAX2) transformants grew well in the presence of 100 and 150 mM Ca²⁺ or 8 and 20 mM Ba²⁺, whereas the GFP-PutCAX2 and C-terminally truncated PutCAX2 (GFP-ΔCPutCAX2) transformants did not show any tolerance to Ca²⁺ or Ba²⁺. The Ba²⁺ content in whole yeast cells expressing GFP-ΔNPutCAX2 or GFP-ΔNCPutCAX2 was lower than that in other yeast transformants. Moreover, the efflux experiment showed that the Ba²⁺ efflux rate of yeast cells expressing GFP-ΔNPutCAX2 and GFP-ΔNCPutCAX2 was higher than that of other yeast cells. To our knowledge, this is the first report on the molecular and functional characterization of a novel ER-localized CAX protein from a wild halophyte plant; the results suggest that the N-terminus of PutCAX2 acts as an auto-inhibitory domain, which affects the Ca²⁺ and Ba²⁺ tolerance of yeast.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

The essentiality of calcium ion in the enzymatic activity of Taiwan cobra phospholipase A2

In order to address the mechanism whereby Ca²⁺ wad crucial for the manifestation of the enzymatic activity of phospholipase A₂ (PLA₂), four divalent cations were used to assess their influences on the catalytic activity and the fine structures ofNaja naja atra PLA₂. It was found that substitution of Mg²⁺ or Sr²⁺ for Ca²⁺ in the substrate solution caused a decrease in the PLA₂ activity to 77.5% or 54.5%, respectively, of that in the presence of Ca²⁺. However, no PLA₂ activity was observed with the addition of Ba²⁺. With the exception of Mg²⁺, the nonpolarity of the 8-anilinonaphthalene-1-sulfonate (ANS)-binding site of PLA₂ markedly increased with the binding of cations to PLA₂. In the meantime, the accessibilities of Lys-6 (65) and Tyr-3 (63) toward trinitrobenzene sulfonate andp-nitrobenzenesulfonyl fluoride were enhanced by the addition of Ca²⁺, Sr²⁺, and Ba²⁺, but not by Mg²⁺. The order of the ability of cations to enhance the ANS fluorescence and the reactivity of Lys and Tyr residues toward modified reagents was Ba²⁺> Sr²⁺> Ca²⁺> Mg²⁺, which was the same order as the increase in their atomic radii. These results, together with the observations that the ANS molecule binds at the active site of PLA₂ and that Tyr-3, Lys-6, and Tyr-63 of PLA₂ are involved in the binding with the substrate, suggest that the binding of Ca²⁺ to PLA₂ induces conformational changes at the active site and substrate-binding site. However, the smaller atomic radius with Mg²⁺ or the bigger atomic radii with Sr²⁺ and Ba²⁺ might render the conformation improperly rearranged after their binding to PLA₂ molecule.     

Enhancing effects of transition metals on the salt taste responses of single fibers of the frog glossopharyngeal nerve: specificity of and similarities among Ca2+, Mg2+ and Na+ taste responses

Fibers of the frog glossopharyngeal nerve (water fibers) thatare sensitive to water also respond to CaCl₂, MgCl₂ and NaCl.In the present study, interaction among cations (Ca²⁺, Mg²⁺and Na⁺) on taste cell membrane in frogs was studied using transitionmetals (NiCl₂, CoCl₂ and MnCl₂), which themselves are barelyeffective in producing neural response at concentrations below5 mM. Unitary discharges from single water fibers were recordedfrom fungiform papillae with suction electrode. Transition metalions (0.05–5.0 mM) had exclusively enhancing effects onthe responses to 50 mM Ca²⁺, 100 mM Mg²⁺ and 500 mM Na⁺. Theeffects of transition metal ions were always reversible. Therank order of effectiveness of transition metals at 1 mM inthe enhancement of the responses to 50 mM CaCl₂, 100 mM MgCl₂and 500 mM NaCl was NiCl₂ > CoCl² > MnCl₂. The concentrationof transition metal ions effective to enhance salt responsewas almost the same among Ca²⁺, Mg²⁺ and Na⁺ responses. Theresults suggest that a common mechanism is involved in the enhancementof Ca²⁺, Mg²⁺ and Na⁺ taste responses. The enhanced Mg²⁺ responseand the enhanced Na⁺ response were greatly inhibited by theaddition of Ca²⁺ ions, and the enhanced Ca²⁺ response was inhibitedby the addition of Mg²⁺ or Na⁺ ions, suggesting that competitiveantagonism occurs between Ca²⁺ and Mg²⁺ ions and between Ca²⁺and Na⁺ ions in the presence of Ni²⁺ ions. Ni²⁺ ions had a dualeffect on the Ca²⁺ response induced by low concentration (0.1mM) of CaCl₂: enhancement at lower concentrations (0.02–0.1mM) of NiCl₂ and inhibition at higher concentrations (0.5–5mM)of NiCl₂. The present results suggest that transition metalions do not affect the receptor-antagonist complex, but affectonly the receptor-agonist complex.     

Effects of Seawater Cations and Temperature on Manganese Dioxide-Reductase Activity in a Marine Bacillus

The seawater cations, Na⁺, K⁺, Mg²⁺, and Ca²⁺, each stimulated MnO₂-reductase activity of whole cells and cell extracts of Bacillus 29. Concentrations of Na⁺ and K⁺ which stimulated whole cells and cell extracts maximally were equivalent to those in two- to fivefold diluted seawater. Cell-extract activity was strongly stimulated by Ca²⁺ and Mg²⁺ up to a concentration of 0.01 M Mg²⁺ and 0.002 M Ca²⁺, with little additional stimulation above these concentrations. Whole-cell activity was stimulated biphasically with increasing concentrations of Ca²⁺ and Mg²⁺. Comparison of the effects of individual cations or mixtures of them at concentrations equivalent to their concentration in fivefold diluted seawater showed that more activity was obtained with 0.01 M Mg²⁺ or 0.002 M Ca²⁺ than with 0.1 M Na⁺, and more with 0.1 M Na⁺ than with 0.0022 M K⁺. Fivefold diluted seawater permitted as much or more activity as solutions of individual or synthetic mixtures of the cations. Pre-exposure experiments showed that the ionic history of whole cells was important to their ultimate activity. The MnO₂-reductase activity of induced whole cells exhibited a temperature optimum near 40 C. Cell extracts had different temperature optima (T_opt), depending on whether induced glucose-linked activity (T_opt = 25 C), uninduced glucose-linked, ferricyanide-dependent activity (T_opt = 30 C), or uninduced ferrocyanide-linked activity (T_opt = 40 C) were being measured. Some of these optima are higher than previously reported.     

Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺, Cu²⁺, Co²⁺, Cd²⁺, Mn²⁺, Ba²⁺, Ni²⁺, Zn²⁺, and Li⁺) were analyzed. AtCCX5 expression was found to affect the response to K⁺ and Na⁺ in yeast. The AtCCX5 transformant also showed a little better growth to Zn²⁺. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K⁺ (0.5 mM), and also suppressed its Na⁺ sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K⁺ uptake and was also involved in Na⁺ transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K⁺ uptake and Na⁺ transport in yeast.     

Enhancement of transport in Micrococcus lysodeikticus by sodium ions

Na⁺ (at a concentration of 10 mM) increased the uptake of succinate, glucose and l-valine by Micrococcus lysodeikucus cells considerably. The effect of Na⁺ could be duplicated by Li⁺ only, which, however, was less active. The other cations tested (K⁺, NH₄⁺, Cs⁺, Mg²⁺, Ca²⁺ and Mn²⁺ were ineffective at concentrations up to 100 mM. Addition of Na⁺ increased the affinities of the uptake system for the substrate studied, while uptake capacity remained unaltered.     

Kinetics of Ca+ adsorption and cationic selectivity with a synaptic membrane protein

A study was conducted on the adsorption of ⁴⁵Ca²⁺ to a surface film of a hydrophobic protein derived from synaptic membranes isolated from bovine cerebellum. A kinetic analysis of Ca²⁺ displacement from the protein by various metal and organic cations could be described by a rate law based on diffusion and displacement. The relative rate constants for the displacement of bound Ca²⁺ were in the order Li⁺ <Na⁺, Rb⁺ <Cs⁺ <K⁺, NH₄⁺. Among the alkaline earth series the sequence was Mg²⁺, Sr²⁺ <Ba²⁺. Ca²⁺ adsorption could be described by a theoretical formulation which takes into account an interfacial energy and potential barrier as well as the diffusional process. An attempt was made to consider the effect of energy of hydration of the cations, surface charge, and the chemical environment at the interface on catonic selectivity. The behavior of the cations in this system significantly resemble their behavior in natural membranes, particularly excitatory ones. The structural and physicochemical environment of the protein at the interface is discussed in relation to Ca²⁺ binding and cationic selectivity.     

Characterization of a PutCAX1 gene from Puccinellia tenuiflora that confers Ca and Ba tolerance in yeast

The gene for a novel cation/H⁺ antiporter from Puccinellia tenuiflora, PutCAX1, was cloned from a cDNA library. The PutCAX protein was localized in the vacuolar membrane using a GFP marker. Several yeast transformants were created using full-length and truncated form of PutCAX1 and their growths in the presence of various cations (Mg²⁺, Ca²⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Se²⁺, and Ba²⁺) were analyzed. PutCAX1 expression was found to affect the response to Ca²⁺ and Ba²⁺ in yeast. The PutCAX1 and C-terminally truncated PutCAX1 (ΔCPutCAX1) transformants grew in the presence of 70 mM Ca²⁺ as well as in the presence of 8 mM Ba²⁺. However, the ΔCPutCAX1 transformant was able to grow in the presence of 20 mM Ba²⁺ while the PutCAX1 transformant could not. On the other hand, expression of the N-terminally truncated form and the N- and C-terminally truncated form failed to suppress the Ca²⁺ or Ba²⁺ sensitivity of yeast. These results suggest that PutCAX1 can complement the active Ca²⁺ transporters at some level and confer yeast Ba²⁺ tolerance, and that the N- and C-terminal regions of PutCAX1 play important roles in increasing the Ca²⁺ or Ba²⁺ tolerance of yeast.     

Time-irreversible Subconductance Gating Associated with Ba2+ Block of Large Conductance Ca2+-activated K+ Channels

Ba²⁺ block of large conductance Ca²⁺-activated K⁺ channels was studied in patches of membrane excised from cultures of rat skeletal muscle using the patch clamp technique. Under conditions in which a blocking Ba²⁺ ion would dissociate to the external solution (150 mM N-methyl-d-glucamine⁺ _o, 500 mM K⁺ _i, 10 μM Ba²⁺ _i, +30 mV, and 100 μM Ca²⁺ _i to fully activate the channel), Ba²⁺ blocks with a mean duration of ∼2 s occurred, on average, once every ∼100 ms of channel open time. Of these Ba²⁺ blocks, 78% terminated with a single step in the current to the fully open level and 22% terminated with a transition to a subconductance level at ∼0.26 of the fully open level (preopening) before stepping to the fully open level. Only one apparent preclosing was observed in ∼10,000 Ba²⁺ blocks. Thus, the preopenings represent Ba²⁺-induced time-irreversible subconductance gating. The fraction of Ba²⁺ blocks terminating with a preopening and the duration of preopenings (exponentially distributed, mean = 0.75 ms) appeared independent of changes in [Ba²⁺]_i or membrane potential. The fractional conductance of the preopenings increased from 0.24 at +10 mV to 0.39 at +90 mV. In contrast, the average subconductance level during normal gating in the absence of Ba²⁺ was independent of membrane potential, suggesting different mechanisms for preopenings and normal subconductance levels. Preopenings were also observed with 10 mM Ba²⁺ _o and no added Ba²⁺ _i. Adding K⁺, Rb⁺, or Na⁺ to the external solution decreased the fraction of Ba²⁺ blocks with preopenings, with K⁺ and Rb⁺ being more effective than Na⁺. These results are consistent with models in which the blocking Ba²⁺ ion either induces a preopening gate, and then dissociates to the external solution, or moves to a site located on the external side of the Ba²⁺ blocking site and acts directly as the preopening gate.     

Purification and characterization of chitinase from Alcaligenes xylosoxydans

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecularmass of chitinase was estimated to be 45 kDa and44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 °C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l^–1. Cu²⁺ and Na⁺ at 5 mM inhibited chitinase activity to 25% while Ca²⁺, Mg²⁺ and Ba²⁺ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Correlated effects of external magnesium on cation content and DNA synthesis in culture chicken embryo fibroblasts.

Depletion of Mg²⁺ in the growth medium for chicken embryo fibroblasts produces a large decrease in DNA synthesis as measured by ³H-thymidine incorporation, and concomitant decreases in cellular K⁺ and Mg²⁺ and increases in Na⁺ and Ca²⁺. In cells grown in media containing 0.2 mM Ca²⁺, graded reduction of Mg²⁺ from 0.8 mM (control) to 0.016 mM produced graded decreases in DNA synthesis to 10% of control at 0.016 mM Mg²⁺. Concomitantly, cell cations showed graded changes, Na⁺ increasing to 227%, K⁺ decreasing to 52.5%, Mg²⁺ decreasing to 57.5% and Ca²⁺ increasing to 153.5% of control. The effects of Mg²⁺ depletion on DNA synthesis and cell cation content exhibited a dependence on Ca²⁺ concentration, the effects being larger at low Ca²⁺ concentration. Use of inorganic pyrophosphate in the growth medium as a selective complexor of Mg²⁺ caused a marked decrease in DNA synthesis which was accompanied by changes in cellular cation content similar to those produced by direct Mg²⁺ depletion. The effects of Mg²⁺ depletion on cell cation content are explainable in terms of changes in membrane permeability caused by rapid external surface exchange of bound divalent cations. Among the several interpretations of the data in terms of possible mechanisms by which changes in external Mg²⁺ concentration may affect cell metabolism, the most consistent with known properties of the system is the concept of a central role for intracellular free Mg²⁺ in the coordinate control of growth and metabolism in animal cells.