Riccardo Fesce

Allosteric Modulation of Drug Binding to Human Serum Albumin

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its extraordinary ligand binding capacity. The three homologous domains of HSA (labeled I, II, and III), each in turn composed of two subdomains (named A and B), give rise to the three-dimensional structure of HSA. This flexible structural organization allows the protein structure to adapt to a variety of ligands. As conformational adaptability of HSA extends well beyond the immediate vicinity of the binding site(s), cooperativity and allosteric modulation arise among binding sites; this makes HSA similar to a multimeric protein. Although kinetic and thermodynamic parameters for ligand binding to HSA calculated by quantitative structure-activity relationship models are in excellent agreement with those obtained in vitro, cooperative and allosteric equilibria between different binding sites and competition between drugs or between drugs and endogenous ligands make difficult the interpretation of HSA binding properties in vivo. Binding of exogenous and endogenous ligands to HSA appears to be relevant in drug therapy and management. Here, the allosteric modulation of drug binding to HSA is briefly reviewed.

Synapsin I partially dissociates from synaptic vesicles during exocytosis induced by electrical stimulation

The distribution of the synaptic vesicle-associated phosphoprotein synapsin I after electrical stimulation of the frog neuromuscular junction was investigated by immunogold labeling and compared with the distribution of the integral synaptic vesicle protein synaptophysin. In resting terminals both proteins were localized exclusively on synaptic vesicles. In stimulated terminals they appeared also in the axolemma and its infoldings, which however exhibited a lower synapsin I/synaptophysin ratio with respect to synaptic vesicles at rest. The value of this ratio was intermediate in synaptic vesicles of stimulated terminals, and an increased synapsin I labeling of the cytomatrix was observed. These results indicate that synapsin I undergoes partial dissociation from and reassociation with synaptic vesicles, following physiological stimulation, and are consistent with the proposed modulatory role of the protein in neurotransmitter release.

Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin. Optical and NMR spectroscopy characterization.

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme–HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme‐bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10–500‐fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme–HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme–HSA, allowing the Mn(III)‐coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal‐coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.

The effect of potassium on exocytosis of transmitter at the frog neuromuscular junction.

1. Electrophysiology and morphology have been combined to investigate the time course of the exocytosis of quanta of neurotransmitter induced by elevated concentrations of K+ at the frog neuromuscular junction. 2. Replicas of freeze‐fractured resting nerve terminals fixed in the presence of 20 mM‐K+ showed images of fusion of synaptic vesicles with the presynaptic axolemma which were closely associated with the active zones. After 1 min in 20 nM‐K+ fusions appeared also outside the active zones, and by 5 min they became uniformly distributed over the presynaptic membrane. 3. The average total density of fusions was not significantly different at the various times examined since it decreased at the active zones while it increased over the rest of the membrane. 4. Resting terminals fixed in 20 mM‐K+ released 33,000‐45,000 quanta after the addition of fixative; terminals stimulated by 20 mM‐K+ for 1‐5 min released 50,000‐100,000 quanta during fixation. The fixative potentiated K+‐induced transmitter release. 5. Fusions were uniformly distributed in terminals pre‐incubated for 5 min in 20 mM‐K+ without added Ca2+, stimulated by adding Ca2+ for 30 s, and then fixed. Conversely, after 5 min stimulation in hypertonic Ringer solution fusions remained predominantly located near the active zones. A similar distribution was observed after 15 min stimulation by a lower concentration of K+ (15 mM). 6. At all concentrations of K+ tested (10, 15, 20, 25 mM) miniature end‐plate potential (MEPP) rate attained a steady‐state value within 10‐15 min. Values from a single junction were generally lower at higher concentrations of K+, which indicates partial inactivation of the secretion‐recycling process. 7. The data indicate that K+ initially activates exocytosis at the active zones. Subsequently, ectopic exocytosis is activated while sites at the active zones appear to undergo partial inactivation. These phenomena are not related to the intensity or to the amount of previous secretion.

Mitogenic effect of serotonin in human small cell lung carcinoma cells via both 5-HT1A and 5-HT1D receptors

We have recently shown that the mitogenic effect of serotonin (5-hydroxytryptamine, 5-HT) on human small lung carcinoma (SCLC) cells is at least partly due to stimulation of a 5-HT1D receptor type. We now report that the 5-HT1A receptor agonist R(+)-8-hydroxy-2-(di-n- propylamino)tetralin (8-OH-DPAT) is also capable of stimulating [3H]thymidine incorporation into SCLC GLC-8 cells, although with lower efficacy than 5-HT. The simultaneous administration of maximal doses of 8-OH-DPAT and the 5-HT1D receptor agonist sumatriptan reproduced the maximal [3H]thymidine incorporation observed with 5-HT alone. The 5-HT1A receptor antagonists spiperone and SDZ 216-525 completely abolished the effect of 8-OH-DPAT (IC50 30 nM for both drugs) behaving as pure antagonists. Accordingly, the two drugs partially inhibited the mitogenic effect of 5-HT. These data indicate that the mitogenic effect of 5-HT in SCLC cells involves both 5-HT1A and 5-HT1D receptor types.

Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneous pectoris muscles treated with crude black widow spider venom (BWSV) or purified alpha‐latrotoxin (alpha‐LTX) in calcium‐free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times. 2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM‐EGTA and no divalent cations, and quantal secretion was started by applying Ca2(+)‐free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre. 3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 degrees C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3‐4 min and reapplied 35‐40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time. 4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well‐separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/microns. 5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22‐23 degrees C or at 9‐10 degrees C. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross‐sections of the terminals were counted on electron micrographs. The number of vesicles remaining per micrometre of terminal was determined from the number per cross‐section and the section thickness.(ABSTRACT TRUNCATED AT 400 WORDS)

PHOSPHORYLATION-DEPENDENT EFFECTS OF SYNAPSIN IIA ON ACTIN POLYMERIZATION AND NETWORK FORMATION

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin‐based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphotylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Synapsins Contribute to the Dynamic Spatial Organization of Synaptic Vesicles in an Activity-Dependent Manner

The precise subcellular organization of synaptic vesicles (SVs) at presynaptic sites allows for rapid and spatially restricted exocytotic release of neurotransmitter. The synapsins (Syns) are a family of presynaptic proteins that control the availability of SVs for exocytosis by reversibly tethering them to each other and to the actin cytoskeleton in a phosphorylation-dependent manner. Syn ablation leads to reduction in the density of SV proteins in nerve terminals and increased synaptic fatigue under high-frequency stimulation, accompanied by the development of an epileptic phenotype. We analyzed cultured neurons from wild-type and Syn I,II,III−/− triple knock-out (TKO) mice and found that SVs were severely dispersed in the absence of Syns. Vesicle dispersion did not affect the readily releasable pool of SVs, whereas the total number of SVs was considerably reduced at synapses of TKO mice. Interestingly, dispersion apparently involved exocytosis-competent SVs as well; it was not affected by stimulation but was reversed by chronic neuronal activity blockade. Altogether, these findings indicate that Syns are essential to maintain the dynamic structural organization of synapses and the size of the reserve pool of SVs during intense SV recycling, whereas an additional Syn-independent mechanism, whose molecular substrate remains to be clarified, targets SVs to synaptic boutons at rest and might be outpaced by activity.

The relation between charge movement and transport-associated currents in the rat GABA cotransporter rGAT1.

Most cotransporters characteristically display two main kinds of electrical activity: in the absence of organic substrate, transient presteady‐state currents (Ipre) are generated by charge relocation during voltage steps; in the presence of substrate, sustained, transport‐associated currents (Itr) are recorded. Quantitative comparison of these two currents, in Xenopus oocytes expressing the neural GABA cotransporter rGAT1, revealed several unforeseen consistencies between Ipre and Itr, in terms of magnitude and kinetic parameters. The decay rate constant (r) of Ipre and the quantity of charge displaced to an inner position in the transporter (Qin(0)) depended on voltage and ionic conditions. Saturating GABA concentrations, applied under the same conditions, suppressed Ipre (i.e. Qin(∞) = 0) and produced a transport‐associated current with amplitude Itr=Qin(0)r. At non‐saturating levels of GABA, changes of Itr were compensated by corresponding variations in Qin, such that Ipre and Itr complemented each other, according to the relation: Itr= (Qin(0) ‐ Qin) r. Complementarity of magnitude, superimposable kinetic properties and equal dependence on voltage and [Na+]o point to the uniqueness of the charge carrier for both processes, suggesting that transport and charge migration arise from the same molecular mechanism. The observed experimental relations were correctly predicted by a simple three‐state kinetic model, in which GABA binding takes place after charge binding and inward migration have occurred. The model also predicts the observed voltage dependence of the apparent affinity of the transporter for GABA, and suggests a voltage‐independent GABA binding rate with a value around 0.64 μm−1 s−1.

Peeping at the vesicle kiss

Kiss-and-run fusion, a quick way of coupling exocytotic vesicle fusion to vesicle recycling by endocytosis, can allow full discharge of secretory-vesicle contents, such as neurotransmitters, and is more common than classical endocytosis at high calcium concentrations.