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.     

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+.     

Divalent cation conduction in the ryanodine receptor channel of sheep cardiac muscle sarcoplasmic reticulum

The conduction properties of the alkaline earth divalent cations were determined in the purified sheep cardiac sarcoplasmic reticulum ryanodine receptor channel after reconstitution into planar phospholipid bilayers. Under bi-ionic conditions there was little difference in permeability among Ba2+, Ca2+, Sr2+, and Mg2+. However, there was a significant difference between the divalent cations and K+, with the divalent cations between 5.8- and 6.7-fold more permeant. Single-channel conductances were determined under symmetrical ionic conditions with 210 mM Ba2+ and Sr2+ and from the single-channel current-voltage relationship under bi-ionic conditions with 210 mM divalent cations and 210 mM K+. Single-channel conductance ranged from 202 pS for Ba2+ to 89 pS for Mg2+ and fell in the sequence Ba2+ greater than Sr2+ greater than Ca2+ greater than Mg2+. Near-maximal single-channel conductance is observed at concentrations as low as 2 mM Ba2+. Single-channel conductance and current measurements in mixtures of Ba(2+)-Mg2+ and Ba(2+)-Ca2+ reveal no anomalous behavior as the mole fraction of the ions is varied. The Ca(2+)-K+ reversal potential determined under bi-ionic conditions was independent of the absolute value of the ion concentrations. The data are compatible with the ryanodine receptor channel acting as a high conductance channel displaying moderate discrimination between divalent and monovalent cations. The channel behaves as though ion translocation occurs in single file with at most one ion able to occupy the conduction pathway at a time.     

Studies of the cation binding properties of an oligomeric derivative of prostaglandin B1

The ionophoretic activity of PGBx, an oligomeric mixture synthesized from 15-dehydro PGB1, with different cations was measured using arsenazo III-entrapped liposomes. The order of ionophoretic activity was Zn2+ greater than Co2+ greater than Mn2+ greater than Cu2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. The intrinsic fluorescence of PGBx was quenched by the binding of divalent cations as well as by La3+ and H+. Quenching by K+ and Na+ was minimal. The order of quenching strength of divalent cations was Zn2+ greater than Co2+ greater than Cu2+ = Mn2+ greater than Ca2+ greater than Ba2+ greater than Sr2+ greater than Mg2+. Binding affinities of these cations determined by a murexide indicator method were in good agreement with that determined by the fluorescence quenching reaction. The cation binding affinity of PGBx in aqueous solutions correlates with the ionophoretic activity in liposomes. The binding affinity for K+ was estimated from the inhibition by K+ of Ca2+ binding by PGBx. Although PGBx has a lower selectivity for divalent cation binding than the ionophore A23187, the characteristics of the binding affinity of these two compounds for various ions were similar. The pK of PGBx as determined by fluorescence quenching was 6.7. The molecular weight of the divalent cation binding unit was estimated to be about 680, with each PGBx molecule having three such binding sites. The binding of Ca2+ to such a site is one-to-one.     

Axonal surface charges: evidence for phosphate structure.

The effect of different extracellular alkaline-earth cations (Ca2+, Mg2+, Sr2+, Ba2+) upon the threshold membrane potential for spike initiation in crayfish axon has been studied by means of intracellular microelectrodes. This was done at the following extracellular concentrations of the divalent uranyl ion (UO2/2+): 1.0 X 10(-6) M, 3.0 X 10(-6) M, and 9.0 X 10(-6) M. At each concentration employed, extensive neutralization of axonal surface charges by UO2/2+ was evidenced by the fact that equal concentrations (50 mM) of alkaline-earth cations did not have the same effect on the threshold potential. The selectivity sequences observed at the different uranyl-ion concentrations were: 1.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Sr2+ greater than Ba2+; 3.0 X 10(-6) M UO2/2+, Ca2+ greater than Mg2+ greater than Ba2+ larger than or equal to Sr2+; 9.0 X 10(-6) M UO2/2+, Ca2+ approximately Ba2+ greater than Sr2+ greater than Mg2+. These selectivity sequences are in accord with the equilibrium selectivity theory for alkaline-earth cations. At each of the concentrations used, uranyl ion did not have any detectable effect on the actual shape of the action potential itself. It is concluded that many (if not most) of the surface acidic groups in the region of the sodium gates represent phosphate groups of membrane phospholipids, but that the m gates themselves are probably protein-aceous in structure.     

Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was approximately 400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt.target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+.syt action.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.     

Calcium-sensitive univalent cation channel formed by lysotriphosphoinositide in bilayer lipid membranes.

A calcium sensitive univalent cation channel could be formed by lysotriphosphoinositide on an artificial bilayer membrane made of oxidized cholesterol. The modified membrane was selectively permeable to univalent cations, but was only very sparingly permeable to anions or divalent cations. Selectivity sequence among group IA cations was Rb+ greater than Cs+ greater than Na+ greater than K+ greater than Li+. The conductance of the membrane was increased up to a value of about 10-2 ohm-1/cm2 with an increase in the concentration of univalent cation, and was drastically depressed by a relatively small increase in the concentration of calcium ion or other divalent cations. The sequence of depressing efficiency among divalent cations was Zn+ greater than Cd2+ greater than Ca2+ greater than Sr2+ greater than Mg2+.     

Cholesterol affects divalent cation-induced fusion and isothermal phase transitions of phospholipid membranes

The influence of cholesterol on divalent cation-induced fusion and isothermal phase transitions of large unilamellar vesicles composed of phosphatidylserine (PS) was investigated. Vesicle fusion was monitored by the terbium/dipicolinic acid assay for the intermixing of internal aqueous contents, in the temperature range 10-40 degrees C. The fusogenic activity of the cations decreases in the sequence Ca2+ greater than Ba2+ greater than Sr2+ much greater than Mg2+ for cholesterol concentrations in the range 20-40 mol%, and at all temperatures. Increasing the cholesterol concentration decreases the initial rate of fusion in the presence of Ca2+ and Ba2+ at 25 degrees C, reaching about 50% of the rate for pure PS at a mole fraction of 0.4. From 10 to 25 degrees C, Mg2+ is ineffective in causing fusion at all cholesterol concentrations. However, at 30 degrees C, Mg2+-induced fusion is observed with vesicles containing cholesterol. At 40 degrees C, Mg2+ induces slow fusion of pure PS vesicles, which is enhanced by the presence of cholesterol. Increasing the temperature also causes a monotonic increase in the rate of fusion induced by Ca2+, Ba2+ and Sr2+. The enhancement of the effect of cholesterol at high temperatures suggests that changes in hydrogen bonding and interbilayer hydration forces may be involved in the modulation of fusion by cholesterol. The phase behavior of PS/cholesterol membranes in the presence of Na+ and divalent cations was studied by differential scanning calorimetry. The temperature of the gel-liquid crystalline transition (Tm) in Na+ is lowered as the cholesterol content is increased, and the endotherm is broadened. Addition of divalent cations shifts the Tm upward, with a sequence of effectiveness Ba2+ greater than Sr2+ greater than Mg2+. The Tm of these complexes decreases as the cholesterol content is increased. Although the transition is not detectable for cholesterol concentrations of 40 and 50 mol% in the presence of Na+, Sr2+ or Mg2+, the addition of Ba2+ reveals endotherms with Tm progressively lower than that observed at 30 mol%. Although the presence of cholesterol appears to induce an isothermal gel-liquid crystalline transition by decreasing the Tm, this change in membrane fluidity does not enhance the rate of fusion, but rather decreases it. The effect of cholesterol on the fusion of PS/phosphatidylethanolamine (PE) vesicles was investigated by utilizing a resonance energy transfer assay for lipid mixing. The initial rate of fusion of PS/PE and PS/PE/cholesterol vesicles is saturated at high Mg2+ concentrations. With Ca2+, saturation is not observed for cholesterol-containing vesicles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of pig oocytes by intracytoplasmic injection of strontium and barium

Ovulated mouse oocytes are activated by exposure to culture medium containing Sr2+ or Ba2+ or by intracytoplasmic injection of the divalent cations. It is known that in vitro matured pig oocytes are activated by the intracytoplasmic injection of Ca2+. In this study, we examined the effect of exposure and of intracytoplasmic injection of Sr2+ or Ba2+ on in vitro matured pig oocytes (MII-oocytes). When MII-oocytes were exposed to the medium containing divalent cations, no oocytes were activated. However, in the case of oocytes that were injected with Sr2+, Ba2+ and Ca2+, at 6 h after injection, 64%, 71% and 86% of the oocytes had been released from MII-arrest, and 51%, 67% and 84% formed female pronuclei, respectively. The initial transient in intracellular Ca2+ concentration ([Ca2+]i) was measured by the Ca2+ indicator dye fluo-4 dextran. Microinjection of Sr2+, Ba2+ or Ca2+ induced a rapid elevation of [Ca2+]i. The exocytosis of cortical granules was examined by staining with fluorescein isothiocyanate (FITC)-labelled peanut agglutinin. After an injection of divalent cations, a release of cortical granules was observed in the oocytes. Maturation promoting factor (MPF) activity declined to a low level after 6 h in all the oocytes injected with divalent cations. To test their developmental ability, injected oocytes were treated with cytochalasin B and then cultured for 168 h in NCSU23 medium. After 168 h, 29% (Sr2+), 29% (Ba2+) and 51% (Ca2+) of the oocytes had developed to the blastocyst stage. These results indicate that intracytoplasmic injection of Sr2+ and Ba2+, like that of Ca2+, induces in vitro matured pig oocytes to be released from MII-arrest and leads them into a series of events related to oocyte activation.     

Effect of bivalent metal ions on the fluctuations of ion currents in rat liver mitochondria

Ions of bivalent metals are shown to arrange in the Sr2+ greater than Ca2+ greater than Ba2+ greater than Mn2+ series as to their ability to induce ion flow vibration in the rat liver mitochondria. Application of Sr2+ results in the most stable prolonged vibrations of ion flows in mitochondria. Ca2+, Ba2+ and Mn2+ induce slightly pronounced and intensively damped vibrations. The studied Mg2+, Co2+, Ni2+, Pb2+ Fe2+ cations have effect on valinomycin-induced K+ transport in mitochondria and do not induce vibrations. It is established that the ability of bivalent cations to induce vibrations is associated with the possibility of their transfer through the mitochondrion membrane and accumulation in the matrix. Inhibitors of the electrogenic Ca2+ transport in mitochondria produce the similar effect on vibrations induced by Sr2+, Ca2+, Ba2+ and Mn2+.     

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombin's catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.     

Fusogenic capacities of divalent cations and effect of liposome size

The initial kinetics of divalent cation (Ca2+, Ba2+, Sr2+) induced fusion of phosphatidylserine (PS) liposomes, LUV, is examined to obtain the fusion rate constant, f11, for two apposed liposomes as a function of bound divalent cation. The aggregation of dimers is rendered very rapid by having Mg2+ in the electrolyte, so that their subsequent fusion is rate limiting to the overall reaction. In this way the fusion kinetics are observed directly. The bound Mg2+, which by itself is unable to induce the PS LUV to fuse, is shown to affect only the aggregation kinetics when the other divalent cations are present. There is a threshold amount of bound divalent cation below which the fusion rate constant f11 is small and above which it rapidly increases with bound divalent cation. These threshold amounts increase in the sequence Ca2+ less than Ba2+ less than Sr2+, which is the same as found previously for sonicated PS liposomes, SUV. While Mg2+ cannot induce fusion of the LUV and much more bound Sr2+ is required to reach the fusion threshold, for Ca2+ and Ba2+ the threshold is the same for PS SUV and LUV. The fusion rate constant for PS liposomes clearly depends upon the amount and identity of bound divalent cation and the size of the liposomes. However, for Ca2+ and Ba2+, this size dependence manifests itself only in the rate of increase of f11 with bound divalent cation, rather than in any greater intrinsic instability of the PS SUV. The destabilization of PS LUV by Mn2+ and Ni2+ is shown to be qualitatively distinct from that induced by the alkaline earth metals.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Calcium-binding of Synaptosomes isolated from rat brain cortex. II. Inhibitory effects of magnesium ions and some other cations.

As in our previous report (Kamino, Uyesaka & Inouye, J. Membrane Biol. 17:13 1974), the absorbance changes of murexide caused by Ca2+ and followed up by a dual wavelength spectrophotometer were applied to measure synaptosomal Ca2+-binding in the presence of cations such as Rb+, Mn2+ or La3+. All the cations tested showed a significant inhibition of synaptosomal Ca2+-binding except Li+. The inhibitory effects could be divided into the following three categories: (1) noncompetive, co-operative K+-type, which includes alkali metal ions. The potency of inhibition is K+ greater than Rb+ greater than Cs+ greater than Li+, Na+ =0; (2) competitive Mn2+ -type which includes many divalent cations. The inhibitory potency was found to be in the following order: Mn2+ greater than Sr2+ greater than Cd2+, Ba2+ greater than Mg2+; (3) nonspecific, noncompetitive La3+ -type; among the cations tested, La3+ and Ce3+ were found to markedly reduce the Ca-binding capacity of synaptosomal particles, resulting in a noncompetitive inhibition, at least in the range of Ca2+ concentration used.     

Action of polyvalent cations on sodium transport across skin of larval and adult Rana catesbeiana

The actions of alkaline earth (AE) and transition element (TE) cations on Na+ transport across skin of larval and adult Rana catesbeiana were compared. Bathed on the outside by Ca+2-free Ringer's, both larval and adult skins maintained a stable short-circuit current (3-4 mu Amps cm-2 for larval skin and 20-30 mu Amps cm-2 for adult skin). Addition of Ca+2 to the external bath reduced the SCC; maximal inhibition was about 36% for larval skin and 22% for adult skin. Other AE divalent cations were also inhibitory. The order of effectiveness was: Ba+2 = Ca+2 greater than Sr+2 greater than Mg+2 for larval skin and Ba+2 greater than Ca+2 = Mg+2 for adult skin. Sodium influx was markedly elevated when Ca+2 was removed from the external medium. Current-voltage analysis indicated that Ca+2 increases the resistance of the active pathway without affecting the shunt resistance or the electromotive force of Na+ transport (ENa) in larval and adult skins. The SCC across adult skin was stimulated by TE cations (Co+2, Cd+2, La+3). These ions were inhibitory on larval skin. The transition in the response occurred at stage XXI. The inhibitory effect of TE on larvel skin resembles that seen in response to AE cations and we postulate a common mechanism. Since larval skin lacks the selective Na+ channels found in apical membranes of adult skin, we infer that the mechanism of inhibition by AE cations is not on these channels. A more general phenomenon such as change in surface charge at the apical membrane seems more reasonable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

The role of calcium in excitation--contraction coupling in crustacean muscle fibers

The evidence that calcium (Ca) plays an important role in electrical activity and an essential role in excitation--contraction (E--C) coupling in crustacean muscles is reviewed. These muscles produce graded electrical and mechanical responses to applied depolarizations. Removal of Ca from the bath solution eliminates both responses. Addition of Ba2+ or Sr2+ to Ca-free saline restores membrane electrogenesis, and all-or-none action potentials can be induced. With Sr2+ vigorous contractions are produced, whereas Ba action potentials evoke minimal or no tension, showing that rapid depolarization of the membrane potential is not sufficient per se for E--C coupling in crab and barnacle muscle. Several inorganic (e.g., multivalent cations) and organic (e.g., aminoglycoside antibiotics) which block membrane Ca channels block electrogenesis and contraction. However, the "Ca antagonists" verapamil and D600 also block Ca uptake at intracellular storage sites, resulting in spontaneous contractions and the delayed relaxation of small contractions associated with residual Ca currents. The evidence that the Ca which enters the fibres needs to release Ca from intracellular storage sites to produce contractions is detailed and discussed. Finally, a model for E--C coupling is discussed. This model includes the sites and mechanisms of action for several chemicals which modify E--C coupling in crustacean muscle fibres.