Sea urchin sperm head plasma membranes: characteristics and egg jelly induced Ca2+ and Na+ uptake

Sea urchin sperm respond to egg factors with changes in the ionic permeability of their plasma membrane. It has been previously shown that plasma membranes isolated preferentially from sea urchin sperm flagella respond to egg jelly increasing their Ca2+ and Na+ uptake (Darszon et al. (1984) Eur. J. Biochem. 144, 515-522). However, the egg jelly induced acrosome reaction occurs in the sperm head, and there is evidence for an heterogeneous distribution of plasma membrane components within the various regions of this cell. We here report a method for purifying sperm head membranes using positively charged beads according to Jacobson (1977) Biochim. Biophys. Acta 471, 331-335). Under the transmission electron microscope these membranes appeared homogeneous and apparently free of internal membranes. The yield of the preparation was 0.9% of the total protein in the sperm homogenate. The preparation contained less than 5% of the mitochondrial marker cytochrome oxidase, and 10% of the total DNA/mg protein. Surface labeling with 125I indicated a 2.5-3-fold enrichment in specific activity of the head membranes with respect to whole sperm. The SDS band pattern and the lipid composition of this preparation were different from those of isolated flagellar membranes. Phosphatidylcholine was higher in the head membranes, while phosphatidylserine and phosphatidylethanolamine were lower. The head membranes displayed a 1.7-2.3-fold higher Ca2+-ATPase activity and a 2.5-fold lower Na+/K+-ATPase activity, than the flagellar membranes. These results are consistent with a heterogeneous distribution of membrane components along the sea urchin sperm plasma membranes. Isolated head membranes sonicated in the presence of soybean phospholipid liposomes responded to egg jelly with a species-specific increase in Ca2+ and Na+ uptake. As in whole sperm, Ca2+ uptake was inhibited by the Ca2+ channel blocker nisoldipine. A close analog of this compound, [3H]nitrendipine, binds with high affinity to head membranes in a saturable, reversible manner, showing a Kd and Bmax of 31 nM and 5.3 pmol/mg protein, respectively.     

Ion channels in boar sperm plasma membranes: characterization of a cation selective channel.

Plasma membranes isolated from cauda epididymal and ejaculated boar sperm were inserted into planar lipid bilayers and examined for the presence of ion channels. Channel fusion was frequently observed; the most prominent was a nonselective cation channel which conducted K, Na, Cs, Ca, and Ba. Channel opening did not show a strict dependence on voltage but was partially blocked by verapamil, nitrendipine, and ruthenium red. A channel with these characteristics was observed when plasma membranes were isolated by high-pressure nitrogen cavitation (650 psi, 78% sperm head plasma membranes) or at very low nitrogen pressures (50 psi, 90% sperm head plasma membranes), suggesting that this channel may be present in the plasma membrane overlying the sperm head.     

Separate Binding and Functional Sites for ω co-Conotoxin and Nitrendipine Suggest Two Types of Calcium Channels in Bovine Chromaffin Cells

Purified adrenomedullary plasma membranes contain two high-affinity binding sites for 125I-omega-conotoxin, with KD values of 7.4 and 364 pM and Bmax values of 237 and 1,222 fmol/mg of protein, respectively. Dissociation kinetics showed a biphasic component and a high stability of the toxin-receptor complex, with a t1/2 of 81.6 h for the slow dissociation component. Unlabeled omega-conotoxin inhibited the binding of the radioiodinated toxin, adjusting to a two-site model with Ki1 of 6.8 and Ki2 of 653 pM. Specific binding was not affected by Ca2+ channel blockers or activators, cholinoceptor antagonists, adrenoceptor blockers, Na+ channel activators, dopaminoceptor blockers, or Na+/H+ antiport blockers, but divalent cations (Ca2+, Sr2+, and Ba2+) inhibited the toxin binding in a concentration-dependent manner. The binding of the dihydropyridine [3H]nitrendipine defined a single specific binding site with a KD of 490 pM and a Bmax of 129 fmol/mg of protein. At 0.25 microM, omega-conotoxin was not able to block depolarization-evoked Ca2+ uptake into cultured bovine adrenal chromaffin cells depolarized with 59 mM K+ for 30 s, whereas under the same conditions, 1 microM nitrendipine inhibited uptake by approximately 60%. When cells were hyperpolarized with 1.2 mM K+ for 5 min and then Ca2+ uptake was subsequently measured during additions of 59 mM K+. Omega-conotoxin partially inhibited Ca2+ uptake in a concentration-dependent manner. These results suggest that two different types of Ca2+ channels might be present in chromaffin cells. However, the molecular identity of omega-conotoxin binding sites remains to be determined.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Plasma membrane calcium ATPase is concentrated in the head of sea urchin spermatozoa

Plasma membrane Ca2+ATPases (PMCAs) export Ca2+ from cells in a highly regulated manner, providing fine-tuning to the maintenance of intracellular Ca2+ concentrations. There are few studies of PMCAs in spermatozoa, which is surprising considering the importance of this enzyme in all cell types. Here we describe the primary structure and localization of the PMCA of sea urchin spermatozoa (suPMCA). The suPMCA is 1,154 amino acids and has 56% identity and 76% similarity to all 4 human PMCA isoforms. The suPMCA shares the features of a typical PMCA, including domains for calmodulin binding, ATP binding, ATPase phosphorylation, and 10 putative transmembrane segments with two large cytoplasmic loops. Southern blots show that suPMCA is a single copy gene. Treatment of live sea urchin sperm with the PMCA inhibitor, 5-(-6)-carboxyeosin, results in elevations of intracellular Ca2+ and loss of flagellar motility. Immunoblotting and immunoflorescence show that suPMCA is concentrated in the sperm head plasma membrane. In previous work, we showed that a plasma membrane K+ dependent Na+/Ca2+ exchanger (suNCKX), which also keeps Ca2+ low in these cells, is concentrated in the sperm flagellum. Thus, the sperm head and flagellum localize different gene products, both functioning to keep intracellular Ca2+ low, while the sperm swims in seawater containing 10 mM Ca2+.     

Evidence for the presence of ATP-dependent calcium pump and ATPase activities in bull sperm head membranes

Biochemical studies were carried out to demonstrate for the first time direct evidence for the presence of ATP-dependent calcium uptake activity in plasma membrane isolated from the head of bull spermatozoa. The purified plasma membrane vesicles contain also Na+-K+-ATPase, Mg2+-ATPase and Ca2+-ATPase activities. All the activities mentioned were followed in parallel in isolated plasma membranes from the sperm tail. These results together with others, suggest the involvement of the ATP-dependent calcium pump in regulation of intracellular calcium in the process of capacitation and acrosome reaction.     

猪精子体外获能前后离子成分变化

应用扫描电镜和镜射电镜能谱技术,为猪精子获能前后质膜表面和内部的离子成分进行了研究,结果表明,猪精子获能后质膜表面的Na~+和Al~(3+)升高,而Cl~-和Ca~(2+)降低;精子顶体内Na~+和Cl~-降低,Ca~(2+)、K~+和Fe~(2+)升高;中段线粒体内的Na~+、Ca~(2+)和Fe~(2+)升高,而K~+和Cl~-降低。文章分析了精子获能后顶体内Na~+、Cl~-、K~+、Ca~(2+)和Fe~(2+)变化的浓度比和摩尔比。     

Sperm motility in fishes. (II) Effects of ions and osmolality: a review

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa are released into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationships between seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (K+, Na+, and Ca2+), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions on sperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition. Results in the literature show that: 1. K+ is a key ion controlling sperm motility in Salmonidae and Acipenseridae in combination with osmotic pressure; this control is more simple in other fish species: sperm motility is prevented when the osmotic pressure is high (Cyprinidae) or low (marine fishes) compared to that of the seminal fluid. 2. Cations (mostly divalent, such as Ca2+) are antagonistic with the inhibitory effect of K+ on sperm motility. 3. In many species, Ca2+ influx and K+ or Na+ efflux through specific ionic channels change the membrane potential and eventually lead to an increase in cAMP concentration in the cell, which constitutes the initiation signal for sperm motility in Salmonidae. 4. Media that are hyper- and hypo-osmotic relative to seminal fluid trigger sperm motility in marine and freshwater fishes, respectively. 5. The motility of fish spermatozoa is controlled through their sensitivity to osmolality and ion concentrations. This phenomenon is related to ionic channel activities in the membrane and governs the motility mechanisms of axonemes.     

Single-channel activity of bilayers derived from sea urchin sperm plasma membranes at the tip of a patch-clamp electrode

Changes in the plasma membrane permeability of echinoderm sperm play a fundamental role in the acrosome reaction. During the reaction there is an increase in intracellular Ca2+ and Na+ and an efflux of H+ and K+. We have formed bilayers at the tip of patch pipets from a mixture of lipid vesicles and sea urchin sperm plasma membranes (12-50 microgram protein/ml). We observed three types of K+ channels (conductances: 22, 46, and 82 pS), two of which are partially blocked by TEA, and one Cl- channel (148 pS). The presence of K+ channels in sperm plasma membranes is consistent with the inhibition by TEA of the acrosome reaction in whole sperm and the membrane potential change that occurs during the reaction.     

Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis

The patch-clamp technique is used to investigate divalent ion block of the large-conductance K+ channel from Chara australis. Block by Ba2+, Ca2+, Mg2+, and Pt(NH3)4(2+) from the vacuolar and cytoplasmic sides is used to probe the structure of, and ion interactions within, the pore. Five divalent ion binding sites are detected. Vacuolar Ca2+ reduces channel conductance by binding to a site located 7% along the membrane potential difference (site 1, delta = 0.07; from the vacuolar side); it also causes channel closures with mean a duration of approximately 0.1-1 ms by binding at a deeper site (site 2, delta = 0.3). Ca2+ can exit from site 2 into both the vacuolar and cytoplasmic solutions. Cytoplasmic Ca2+ reduces conductance by binding at two sites (site 3, delta = -0.21; site 4, delta = -0.6; from the cytoplasmic side) and causes closures with a mean duration of 10-100 ms by binding to site 5 (delta = -0.7). The deep sites exhibit stronger ion specificity than the superficial sites. Cytoplasmic Ca2+ binds sequentially to sites 3-5 and Ca2+ at site 5 can be locked into the pore by a second Ca2+ at site 3 or 4. Ca2+ block is alleviated by increasing [K+] on the same side of the channel. Further, Ca2+ occupancy of the deep sites (2, 4, and 5) is reduced by K+, Rb+, NH4+, and Na+ on the opposite side of the pore. Their relative efficacy correlates with their relative permeability in the channel. While some Ca2+ and K+ sites compete for ions, Ca2+ and K+ can simultaneously occupy the channel. Ca2+ binding at site 1 only partially blocks channel conduction. The results suggest the presence of four K+ binding sites on the channel protein. One cytoplasmic facing site has an equilibrium affinity of 10 mM (site 6, delta = -0.3) and one vacuolar site (site 7, delta less than 0.2) has low affinity (greater than 500 mM). Divalent ion block of the Chara channel shows many similarities to that of the maxi-K channel from rat skeletal muscle.     

Evidence against parallel operation of sodium/calcium antiport and ATP-driven calcium transport in plasma membrane vesicles from kidney tubule cells

The present study aimed to clarify the existence of a Na+/Ca2+ antiport device in kidney tubular epithelial cells discussed in the literature to represent the predominant mechanistic device for Ca2+ reabsorption in the kidney. Inside-out oriented plasma membrane vesicles from tubular epithelial cells of guinea-pig kidney showed an ATP-driven Ca2+ transport machinery similar to that known to reside in the plasma membrane of numerous cell types. It was not affected by digitalis compounds which otherwise are well-documented inhibitors of Ca2+ reabsorption. The vesicle preparation contained high, digitalis-sensitive (Na+ + K+)-ATPase activities indicating its origin from the basolateral portion of plasma membrane. The operation of a Na+/Ca2+ antiport device was excluded by the findings that steep Ca2+ gradients formed by ATP-dependent Ca2+ accumulation in the vesicles were not discharged by extravesicular Na+, and did not drive 45Ca2+ uptake into the vesicles via a Ca2+-45Ca2+ exchange. The ATP-dependent Ca2+ uptake into the vesicles became increasingly depressed with time by extravesicular Na+. This was not due to an impairment of the Ca2+ pump itself, but caused by Na+/Ca2+ competition for binding sites on the intravesicular membrane surface shown to be important for high Ca2+ accumulation in the vesicles. Earlier observations on Na+-induced release of Ca2+ from vesicles pre-equilibrated with Ca2+, seemingly favoring the existence of a Na+/Ca2+ antiporter in the basolateral plasma membrane, were likewise explained by the occurrence of Na+/Ca2+ competition for binding sites. The weight of our findings disfavors the transcellular pathway of Ca2+ reabsorption through tubule epithelium essentially depending on the operation of a Na+/Ca2+ antiport device.     

Ion channel activity of membrane vesicles released from sea urchin sperm during the acrosome reaction

The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome reaction vesicles (ARVs), using synaptosome-associated protein of 25 kDa (SNAP-25) as a marker. Isolated ARVs have a unique protein composition. The exocytosis regulatory proteins vesicle-associated membrane protein and SNAP-25 are inside ARVs, as judged by protease protection experiments, and membrane associated based on Triton X-114 partitioning. ARVs fused with planar bilayers display three main types of single channel activity. The most frequently recorded channel is cationic, weakly voltage dependent and has a low open probability that increases with negative potentials. This channel is activated by cAMP, blocked by Ba2+, and has a PK+/PNa+ selectivity of 4.5. ARVs represent a novel membrane preparation suitable to deepen our understanding of ion channel activity in the AR and during fertilization.     

Coexisting independent sodium-sensitive and sodium-insensitive mechanisms of genetic hypertension in spontaneously hypertensive rats (SHR)

Some essential hypertensive patients and genetic hypertensive rat strains have less than the normal levels of Mg2+ tightly bound to the plasma membranes of their erythrocytes and other cells, i.e., the magnesium binding defect (MgBD). This binding defect appears to cause increased passive permeability of the membrane to Na+ and thereby its increased intracellular concentration, particularly if the Na+-extrusion enzyme systems of the cell are also defective. The Na+-Ca2+ exchange system in the cell membrane exports Na+ and imports Ca2+, increasing the tone of the smooth muscle cell and thus producing hypertension (HTn). This HTn is Na+-sensitive. Evidence supporting this postulate was obtained by determining the intraerythrocyte total concentrations of Na+, Ca2+, K+, and Mg2+ in two strains of spontaneously hypertensive rats (SHR and SS/Jr rats, having the MgBD together with the other requisites of the Na+-sensitive pathway) and their respective controls (WKY and SR/Jr rats, in which this complete pathway is absent). The Na+ and Ca2+ concentrations in the hypertensive rats were increased, and that of K+ was decreased. The concentrations of these cations were very similar in the two hypertensive strains. The level of membrane tightly bound Ca2+ in SHR erythrocyte membranes was significantly higher than those in the other three rat strains, which were not statistically different from each other. These results support previously reported evidence of the existence of a novel HTn-generating mechanism in the SHR rat, in which the intracellular Ca2+ concentration is increased as the result of the enhanced diffusion of this ion into the cell and the accompanying deficiency of the Ca2+ extrusion enzyme systems. This pathway is therefore Na+-insensitive, i.e., Ca2+-sensitive.     

Characterization of high affinity binding sites for charybdotoxin in synaptic plasma membranes from rat brain. Evidence for a direct association with an inactivating, voltage-dependent, potassium channel.

Charybdotoxin (ChTX), a potent peptidyl inhibitor of several types of K+ channels, binds to sites in vascular smooth muscle sarcolemma (Vázquez, J., Feigenbaum, P., Katz, G. M., King, V. F., Reuben, J. P., Roy-Contancin, L., Slaughter, R. S., Kaczorowski, G. J., and Garcia, M. L. (1989) J. Biol. Chem. 265, 20902-20909) which are functionally associated with a high conductance Ca2(+)-activated K+ channel (PK,Ca). 125I-ChTX also binds specifically and reversibly to a single class of sites in plasma membranes prepared from rat brain synaptosomes. These sites exhibit a Kd of 25-30 pM, as measured by either equilibrium or kinetic binding protocols and display a maximum density of about 0.3-0.5 pmol/mg of protein. Competition studies with native ChTX yield a Ki of 8 pM for the noniodinated toxin. The highest density of ChTX sites exists in vesicle fractions of plasma membrane origin. Binding of 125I-ChTX is modulated by metal ions that interact with K+ channels: Ba2+, Ca2+, and Cs+ cause inhibition of ChTX binding; Na+ and K+ stimulate binding at low concentration before producing complete inhibition as their concentration is increased. Stimulation of binding is due to an allosteric interaction that decreases Kd whereas inhibition results from an ionic strength effect. Tetraethylammonium ion has no effect on binding, but tetrabutylammonium ion blocks binding with a Ki of 2.5 mM. Different toxins (i.e. alpha-dendrotoxin, noxiustoxin) that inhibit an inactivating, voltage-dependent K+ channel (PK,V) block 125I-ChTX binding in brain. In marked contrast, iberiotoxin, a selective inhibitor of PK,Ca, has no effect on ChTX binding in this preparation. Inhibition of ChTX binding by alpha-dendrotoxin and noxiustoxin results from an allosteric interaction between separate binding sites for these agents and the ChTX receptor. Taken together, these results suggest that the ChTX sites present in brain are associated with PK,V rather than with PK,Ca. Therefore, 125I-ChTX is a useful probe for elucidating the biochemical properties of a number of different types of K+ channels.     

Constitution and properties of axonal membranes of crustacean nerves.

The purification of axonal membranes of crustaceans was followed by measuring enrichment in [3H]tetrodotoxin binding capacity and in Na+, K+-ATPase activity. A characteristic of these membranes is their high content of lipids and their low content of protein as compared to other types of plasmatic membranes. The axonal membrane contains myosin-like, actin-like, tropomyosin-like, and tubulin-like proteins. It also contains Na+, K+-ATPase and acetylcholinesterase. The molecular weights of these two enzymes after solubilization are 280,000 and 270,000, respectively. The molecular weights of the catalytic subunits are 96,000 for ATPase and 71,000 for acetylcholinesterase. We confirmed the presence of a nicotine binding component in the axonal membrane of the lobster but we have been unable to find [3H]nicotine binding to crab axonal membranes. The binding to axonal membranes og of the sodium channel, has been studied in detail. The dissociation constant for the binding of [3H]tetrodotoxin to the axonal membrane receptor is 2.9 nM at pH 7.4. The concentration of the tetrodotoxin receptor in crustacean membranes is about 10 pmol/mg of membrane protein, 7 times less than the acetylcholinesterase, 30 times less than the Na+, K+-ATPase, and 30 times less than the nicotine binding component in the lobster membrane. A reasonable estimate indicates that approximately only one peptide chain in 1000 constitutes the tetrodotoxin binding part of the sodium channel in the axonal membrane. Veratridine, which acts selectively on the resting sodium permeability, binds to the phospholipid part of the axonal membrane. [3H]Veratridine binding to membranes parallels the electrophysiological effect. Veratridine and tetrodotoxin have different receptor sites. Although tetrodotoxin can repolarize the excitable membrane of a giant axon depolarized by veratridine, veratridine does not affect the binding of [3H]tetrodotoxin to purified axonal membranes. Similarly, tetrodotoxin does not affect the binding of [3H]veratridine to axonal membranes. Scorpion neurotoxin I, a presynaptic toxin which affects both the Na+ and the K+ channels, does not interfere with the binding of [3H]tetrodotoxin or [3H]veratridine to axonal membranes. Tetrodotoxin, veratridine, and scorpion neurotoxin I, which have in common the perturbation of the normal functioning of the sodium channel, act upon three different types of receptor sites.     

Role of Ca2+-activated K+ channel in regulation of NaCl reabsorption in thick ascending limb of Henle's loop

1. Reabsorption of NaCl in the thick ascending limb of Henle's loop involves the integrated function of the Na+,K+,Cl- -cotransport system and a Ca2+-activated K+ channel in the luminal membrane with the Na+,K+-pump and a net Cl- conductance in the basolateral membrane. 2. Assay of K+ channel activity after reconstitution into phospholipid vesicles shows that the K+ channel is stimulated by Ca2+ in physiological concentrations and that its activity is regulated by calmodulin and phosphorylation from cAMP dependent protein kinase. 3. For purification luminal plasma membrane vesicles are isolated and solubilized in CHAPS. K+ channel protein is isolated by affinity chromatography on calmodulin columns. The purified protein has high Ca2+-activated K+ channel activity after reconstitution into vesicles. 4. The purified K+ channel consists of two proteins of 51 and 36 kDa. Phosphorylation from cAMP dependent protein kinase stimulates K+ channel activity and labels the 51 kDa band. The 36 kDa band is rapidly cleaved by trypsin and may be involved in Ca2+ stimulation. 5. Opening of the K+ channel by Ca2+ in physiological concentrations and regulation by calmodulin and phosphorylation by protein kinase may mediate kinetic and hormonal regulation of NaCl transport across the tubule cells in TAL.     

Activation of sperm motility in striped bass via a cAMP-independent pathway

The objective of the present study was to identify the effect of osmolality, ions (K+, H+, Ca2+, Mg2+) and cAMP on the initiation of sperm motility in striped bass (Morone saxatilis). Striped bass spermatozoa remained motile in solutions isotonic to seminal plasma (350 mOsm/kg) until osmolality reached 600 mOsm/kg. K+ (0-100 mM) had no effect ( p>0.05 ) on sperm motility, and sperm displayed a high percentage of motility over a wide range of pH (6.0-8.5). Sperm motility could be initiated in Ca2+-free solutions. In contrast, sperm motility was inhibited (P<0.01) by solutions containing > or =10 mM Ca2+, and sperm could not be reactivated by a Ca2+-free solution. This Ca2+ inhibition was not affected by verapamil, a Ca2+ channel blocker. However, if sperm motility was first initiated in a Ca2+-free solution, the addition of Ca2+ solutions, up to 80 mM, failed to inhibit sperm motility, suggesting that Ca2+ inhibited the initiation of motility, but had no control of motile spermatozoa. Mg2+ solutions had similar inhibitory effects on sperm motility as Ca2+ solutions. Therefore, initiation of motility in striped bass sperm may be related to voltage-gated channels across the cell's plasma membrane. Membrane permeable cAMP did not initiate motility of quiescent, intact striped bass spermatozoa, and motility of demembranated sperm could be activated in the absence of cAMP.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Calcium binding by smooth muscle plasma membranes

Calcium binding by the vesiculate fraction of rabbit small intestine myocyte plasma membranes was studied. It was shown that the membrane fraction as well as the muscle tissue contain two types of Ca2(+)-binding sites with binding constants of 2.3-2.5 x 10(4) and 2.1-1.25 x 10(3) M-1. The number of binding sites and their affinity for Ca2+ depend on the presence in the incubation medium of Mg2+, Na+ and ATP.     

Specific binding of the calcium antagonist [3H]verapamil to membrane fractions from plants

Specific binding of the Ca2+ channel blocker [3H] verapamil to a membrane fraction from plants has been characterized. Binding to zucchini membranes was saturable and reversible. The apparent equilibrium dissociation constant is KD = 102 nM and the maximum number of binding sites is Bmax = 60 pmol/mg of protein. The KD determined from the association and dissociation rate constants is 130 nM. [3H]Verapamil binding to zucchini membranes could not be inhibited by the Ca2+ antagonists nifedipine and diltiazem. However, [3H]verapamil could be displaced by diltiazem but not by nifedipine from corn membranes. Sucrose density fractionation of zucchini membrane preparations revealed that [3H]verapamil binding sites are located primarily at the plasma membrane.