Solubilization and properties of the soluble axonal cholinergic binding macromolecule

Lysolecithin has been used to solubilize the axon plasma membrane preparation from lobster walking legs. This was accomplished with complete recovery of activity of the axonal cholinergic binding macromolecule and retention of the basic properties of the membrane-bound macromolecule. Sedimentation of the soluble protein through a sucrose gradient containing [³H]nicotine enabled the separation of the axonal cholinergic binding macromolecule from acetylcholinesterase and demonstrated the apparent dissociation of the axonal cholinergic binding macromolecule in low ionic strength solutions.     

Effects of external ions on membrane potentials of a lobster giant axon

The effects of varying external concentrations of normally occurring cations on membrane potentials in the lobster giant axon have been studied and compared with data presently available from the squid giant axon. A decrease in the external concentration of sodium ions causes a reversible reduction in the amplitude of the action potential and its rate of rise. No effect on the resting potential was detected. The changes are of the same order of magnitude, but greater than would be predicted for an ideal sodium electrode. Increase in external potassium causes a decrease in resting potential, and a decrease in potassium causes an increase in potential. The data so obtained are similar to those which have been reported for the squid giant axon, and cannot be exactly fitted to the Goldman constant field equation. Lowering external calcium below 25 mM causes a reduction in resting and action potentials, and the occasional occurrence of repetitive activity. The decrease in action potential is not solely attributable to a decrease in resting potential. Increase of external calcium from 25 to 50 mM causes no change in transmembrane potentials. Variations of external magnesium concentration between zero and 50 mM had no measurable effect on membrane potentials. These studies on membrane potentials do not indicate a clear choice between the use of sea water and Cole's perfusion solution as the better external medium for studies on lobster nerve.     

Sodium flux through the sodium channels of axon membrane fragments isolated from lobster nerves

The efflux of 22Na from vesicles formed by axolemma fragments isolated from lobster nerves was studied in the presence and in the absence of drugs having well-known action on the sodium channels. The vesicles were equilibrated 12-14 h at 4 degrees C with 22Na in lobster solution containing 1 mM ouabain. Afterwards the suspension was divided: one portion was used as control and the others were treated with veratrine (0.025-0.50 mg/ml), tetrodotoxin (1-2,000 nM) in the presence of veratrine, or tetrodotoxin alone. After 3 h at 20-22 degrees C, the suspensions were diluted into nonradioactive solutions and the 22Na efflux followed by a rapid filtration technique. The results revealed that veratrine increases the efflux rate and the additional application of tetrodotoxin abolishes it, e.g., 0.50 mg of veratrine/ml increases the rate, expressed in 10(-2) min(-1), from 0.59 +/- 0.04 (mean +/- SEM; n = 13) to 0.86 +/- 0.05 (n = 13), and the addition of 100 nM tetrodotoxin diminishes it to 0.48 +/- 0.07 (n = 4). This increase and diminution are statistically significant (P less than 0.005), but this is not the case between the control and the veratrine plus tetrodotoxin values (P greater than 0.05). 50% of the diminution is produced by 11.9 +/- 2.4 nM tetrodotoxin. Tetrodotoxin alone produces a slight diminution of the 22Na efflux. Batrachotoxin (0.50 muM) has an action similar to veratrine's. These findings are considered evidence of the presence of functioning sodium channels in the isolated axolemma fragments.     

The action of certain polyvalent cations on the voltage-clamped lobster axon

Calcium appears to be an essential participant in axon excitation processes. Many other polyvalent metal ions have calcium-like actions on axons. We have used the voltage-clamped lobster giant axon to test the effect of several of these cations on the position of the peak initial (sodium) and steady-state (potassium) conductance vs. voltage curves on the voltage axis as well as on the rate parameters for excitation processes. Among the alkaline earth metals, Mg⁺² is a very poor substitute for Ca⁺², while Ba⁺² behaves like "high calcium" when substituted for Ca⁺² on a mole-for-mole basis. The transition metal ions, Ni⁺², Co⁺², and Cd⁺² also act like high calcium when substituted mole-for-mole. Among the trivalent ions, La⁺³ is a very effective Ca⁺² replacement. Al⁺³ and Fe⁺³ are extremely active and seem to have some similar effects. Al⁺³ is effective at concentrations as low as 10^-5 M. The data suggest that many of these ions may interact with the same cation-binding sites on the axon membrane, and that the relative effects on the membrane conductance and rate parameters depend on the relative binding constants of the ions. The total amount of Na⁺ transferred during a large depolarizing transient is nearly independent of the kind or amount of polyvalent ion applied.     

Action of the sterol-binding form of filipin on the lobster axon membrane

The present work demonstrates by electron microscopy and spectrofluorimetry the interaction of the sterol-binding form of filipin with the axolemma of lobster nerves. The results also indicate that the axolemma cholesterol accessible to filipin seems to play no specific role in the functioning of the tetrodotoxin receptors and sodium channels.     

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane

Summary The role of phospholipids in the binding of [³H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A₂ (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A₂, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A₂ but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A₂. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.     

Effects of external ions on membrane potentials of a crayfish giant axon

Transmembrane potentials in the crayfish giant axon have been investigated as a function of the concentration of normally occurring external cations. Results have been compared with data already available for the lobster and squid giant axons. The magnitude of the action potential was shown to be a linear function of the log of the external sodium concentration, as would be predicted for an ideal sodium electrode. The resting potential is an inverse function of the external potassium concentration, but behaves as an ideal potassium electrode only at the higher external concentrations of potassium. Decrease in external calcium results in a decrease in both resting potential and action potential; an increase in external calcium above normal has no effect on magnitude of transmembrane potentials. Magnesium can partially substitute for calcium in the maintenance of normal action potential magnitude, but appears to have very little effect on resting potential. All ionic effects studied are completely reversible. The results are in generally good agreement with data presently available for the lobster giant axon and for the squid giant axon.     

Interactions between cations in modifying the binding of hexokinases I and II to mitochondria

Summary Interactions between cations in modifying the binding of hexokinases I and II to mitochondria was examined with reference to the intracellular condition. Mitochondria-binding of either of hexokinases I and II, both prepared from mouse ascites ELD cells, was markedly increased by Mg2+ as has been known well. However, even in the absence of Mg^s+, marked binding was attained by 100 mM K⁺ alone especially for hexokinase I, which seemed generally more ready to bind to mitochondria. On the other hand, the effect of Mg²⁺ to increase the binding was reduced by the addition of K⁺, and the decreasing effect of K⁺ was much more marked for hexokinase II than I. These results indicate that, in addition to Mg²⁺, monovalent cations as represented by K⁺, also have marked effect on the binding, and the effect is different for each of hexokinases I and II, which may be responsible for the difference in the intracellular distribution between these hexokinases.     

Mechanisms of axon guidance: membrane dynamics and axonal transport in semaphorin signalling

The intricate geometry of neuronal networks poses many unique cell-biological problems regarding the way a growing axon responds to its environment. Several groups of ligand-receptor pairs have been identified to regulate such processes. In this study, we take class 3 semaphorins as an example and review what is known about the intracellular movements of semaphorins throughout neuronal cells, transport support structures and location of release sites. We discuss how their receptor trafficking may contribute to regulate membrane dynamics underlying growth cone motility and the physiological contribution made by class 3 semaphorins-induced acceleration of axoplasmic transport on neurite development.     

Interactions between NRP1 and VEGFR2 molecules in the plasma membrane

Here we use a quantitative FRET approach, specifically developed to probe membrane protein interactions, to study the homo-association of neuropilin 1 (NRP1) in the plasma membrane, as well as its hetero-interactions with vascular endothelial growth factor receptor 2 (VEGFR2). Experiments are performed both in the absence and presence of the soluble ligand vascular endothelial growth factor A (VEGFA), which binds to both VEGFR2 and NRP1. We demonstrate the presence of homo-interactions between NRP1 molecules, as well as hetero-interactions between NRP1 and VEGFR2 molecules, in the plasma membrane. Our results underscore the complex nature of the interactions between self-associating receptors, co-receptors, and their ligands in the plasma membrane. They also highlight the need for new methodologies that capture this complexity, and the need for precise physiological measurements of local receptor surface densities in the membrane of cells. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo.     

Effects of ATP on calcium binding to synaptic plasma membrane

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

The effects of some ions on the membrane potential of the giant axon of Myxicola

The effects of various ions on the resting membrane potential of the giant axons of Myxicola were determined. The mean resting potential in artificial sea water is 69 mv, inside negative. The membrane potential decreases with incresing external potassium concentrations, while changes in the sodium and chloride concentrations have little or no effect. For potassium concentrations greater than 50 mM/L the relation between membrane potential and concentration approximates that of a perfectly selective potassium electrode. The data for the whole range of concentrations examined can be well fitted by the equation: It was pointed out that the Myxicola giant axons can be studied under space voltage clamp and can be made available in the laboratory for 12 months out of the year. Myxicola then should become a very useful preparation for the study of membrane phenomena.     

Effects of cations on the plasma membrane of Blastocladiella emersonii zoospores. An ESR study

The physical properties of the plasma membrane of the aquatic phycomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (12-NS), and 2,2,6,6-tetramethylpiperidine-1-oxyl (Tempo). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2T_|, order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (T_L and T_H) were found to be independent of the external K⁺, Ca²⁺, or Mg²⁺ concentrations. They were similar to the break points found in aqueous dispersions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (T_M) was markedly influenced by the external Ca²⁺ concentration. Ca²⁺ increased T_M from 12°C (no Ca²⁺ added) to 22°C (10 mM Ca²⁺), i.e., growth temperature. K⁺ reversed this Ca²⁺ effect, downshifting T_M from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in T_M, is closely correlated with the temperature dependence and physiological effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the plasma membrane and be involved in regulating the initial changes during zoospore encystment.     

Interactions between the endoplasmic reticulum, mitochondria, plasma membrane and other subcellular organelles

Several recent works show structurally and functionally dynamic contacts between mitochondria, the plasma membrane, the endoplasmic reticulum, and other subcellular organelles. Many cellular processes require proper cooperation between the plasma membrane, the nucleus and subcellular vesicular/tubular networks such as mitochondria and the endoplasmic reticulum. It has been suggested that such contacts are crucial for the synthesis and intracellular transport of phospholipids as well as for intracellular Ca²⁺ homeostasis, controlling fundamental processes like motility and contraction, secretion, cell growth, proliferation and apoptosis. Close contacts between smooth sub-domains of the endoplasmic reticulum and mitochondria have been shown to be required also for maintaining mitochondrial structure. The overall distance between the associating organelle membranes as quantified by electron microscopy is small enough to allow contact formation by proteins present on their surfaces, allowing and regulating their interactions. In this review we give a historical overview of studies on organelle interactions, and summarize the present knowledge and hypotheses concerning their regulation and (patho)physiological consequences.     

Influence of high sodium ions and osmolality on the membrane potential of the perfused squid giant axon

• 1.1. The effects of hypertonic and high sodium solution on the membrane potential of squid giant axon were studied.
• 2.2. The excitability of the intracellularly perfused axon was completely maintained in the hypertonic solution, at least three times as high as that of isotonic solution.
• 3.3. The amount of overshoot was increased by a large increase in the external sodium, ranging from 100 to 1700 mM, and the values are in close agreement with the potential calculated from the equation of ionic theory.
• 4.4. The voltage-clamp current was also measured in the perfused axon with hypertonic high sodium concentration. The peak inward current was increased in amplitude as the external sodium increased. The results of hypertonic high sodium experiments are in general agreement with the prediction of the ionic theory.