The anterior/posterior polarity of somites is disrupted in paraxis-deficient mice.

Establishing the anterior/posterior (A/P) boundary of individual somites is important for setting up the segmental body plan of all vertebrates. Resegmentation of adjacent sclerotomes to form the vertebrae and selective migration of neural crest cells during the formation of the dorsal root ganglia and peripheral nerves occur in response to differential expression of genes in the anterior and posterior halves of the somite. Recent evidence indicates that the A/P axis is established at the anterior end of the presomitic mesoderm prior to overt somitogenesis in response to both Mesp2 and Notch signaling. Here, we report that mice deficient for paraxis, a gene required for somite epithelialization, also display defects in the axial skeleton and peripheral nerves that are consistent with a failure in A/P patterning. Expression of Mesp2 and genes in the Notch pathway were not altered in the presomitic mesoderm of paraxis(-/-) embryos. Furthermore, downstream targets of Notch activation in the presomitic mesoderm, including EphA4, were transcribed normally, indicating that paraxis was not required for Notch signaling. However, genes that were normally restricted to the posterior half of somites were present in a diffuse pattern in the paraxis(-/-) embryos, suggesting a loss of A/P polarity. Collectively, these data indicate a role for paraxis in maintaining somite polarity that is independent of Notch signaling.

Kinetic basis for activation of CDK2/cyclin A by phosphorylation.

The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr(160) in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group.

Transport mechanisms in acetylcholine and monoamine storage.

Sequence-related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure-function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single-site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence-defined superfamily, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage.

Modification of cysteines reveals linkage to acetylcholine and vesamicol binding sites in the vesicular acetylcholine transporter of Torpedo californica.

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.

A critical histidine in the vesicular acetylcholine transporter.

The role of proton binding sites in the vesicular acetylcholine transporter was investigated by characterization of the pH dependence for the binding of [3H]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pKa 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol binding. The histidine-selective reagent diethylpyrocarbonate causes time-dependent inhibition of [3H]vesamicol binding with a rate constant only about 20-fold lower than for reaction with free histidine. Because its pH titration has a simple, ideal shape, this residue probably controls all pH effects in the transporter between pH 6-8. Inhibition of [3H]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, which binds to a separate site. The data suggest that a critical histidine with a pKa of 7.1 is unhindered when reacting with diethylpyrocarbonate. A conformational model for the histidine is proposed to explain why acetylcholine competes with protons but not with diethylpyrocarbonate. A conserved histidine in transmembrane helix VIII possibly is the histidine detected here.

Synthesis and biological characterization of stable and radioiodinated (+/-)-trans-2-hydroxy-3-P[4-(3-iodophenyl)piperidyl]-1,2,3,4-tetrahydronaphthalene (3'-IBVM).

The vesamicol analogue (+/-)-trans-2-Hydroxy-3-[4-(3-iodophenyl)piperidyl]-1,2,3,4-tetrahydronaphthalene (3'-IBVM), a potent ligand for the vesicular acetylcholine transporter (VAChT), was evaluated as a potential radiotracer for studying VAChT density in vivo. In radioligand binding experiments, 3'-IBVM displays subnanomolar affinity for VAChT and 100-fold selectivity for VAChT over sigma1 and sigma2 receptors. Consistent with this profile, radioiodinated (+/-)-3'-IBVM distributed heterogenously in the rat brain following a bolus IV injection, displaying high concentrations in the striatum and moderate to low concentrations in the cortex and cerebellum, respectively. However, co-injection of the radiotracer with the sigma ligand haloperidol resulted in significant reductions of radiotracer levels in all brain regions examined. Therefore, radioiodinated (+/-)-IBVM appears to bind to both VAChT and sigma receptors in vivo.

N-(3-Iodophenyl)trozamicol (IPHT) and related inhibitors of vesicular acetylcholine transport: synthesis and preliminary biological characterization.

Four isomeric N-(halophenyl)trozamicol analogues (6a-d) were synthesized and evaluated as potential vesicular acetylcholine transporter (VAChT) ligands. Of the four compounds, N-(3-bromophenyl) trozamicol (6b) and N-(3-iodophenyl)trozamicol (6d) displayed the highest affinity for the VAChT in vitro, whereas the para-substituted compound 6c showed the lowest affinity for this transporter. Tissue distribution studies of N-(3-[125I]iodophenyl)trozamicol ([125I]6d, [125I)IPHT) suggest that the central distribution of the latter is consistent with cholinergic innervation. However, only moderate target-to-background ratios were obtained, suggesting little improvement over the N-(halobenzyl)trozamicols described previously.

Differential regulation of epaxial and hypaxial muscle development by paraxis.

In vertebrates, skeletal muscle is derived from progenitor cell populations located in the epithelial dermomyotome compartment of the each somite. These cells become committed to the myogenic lineage upon delamination from the dorsomedial and dorsolateral lips of the dermomyotome and entry into the myotome or dispersal into the periphery. Paraxis is a developmentally regulated transcription factor that is required to direct and maintain the epithelial characteristic of the dermomyotome. Therefore, we hypothesized that Paraxis acts as an important regulator of early events in myogenesis. Expression of the muscle-specific myogenin-lacZ transgene was used to examine the formation of the myotome in the paraxis-/- background. Two distinct types of defects were observed that mirrored the different origins of myoblasts in the myotome. In the medial myotome, where the expression of the myogenic factor Myf5 is required for commitment of myoblasts, the migration pattern of committed myoblasts was altered in the absence of Paraxis. In contrast, in the lateral myotome and migratory somitic cells, which require the expression of MyoD, expression of the myogenin-lacZ transgene was delayed by several days. This delay correlated with an absence of MyoD expression in these regions, indicating that Paraxis is required for commitment of cells from the dorsolateral dermomyotome to the myogenic lineage. In paraxis-/-/myf5-/- neonates, dramatic losses were observed in the epaxial and hypaxial trunk muscles that are proximal to the vertebrae in the compound mutant, but not those at the ventral midline or the non-segmented muscles of the limb and tongue. In this genetic background, myoblasts derived from the medial (epaxial) myotome are not present to compensate for deficiencies of the lateral (hypaxial) myotome. Our data demonstrate that Paraxis is an important regulator of a subset of the myogenic progenitor cells from the dorsolateral dermomyotome that are fated to form the non-migratory hypaxial muscles.

Control of the binding of a vesamicol analog to the vesicular acetylcholine transporter.

4-Aminobenzovesamicol was used to test whether activation of protein kinase C protects the vesicular acetylcholine transporter from interaction with vesamicol-like drugs. The essentially irreversible vesamicol analog inhibits the release of newly synthesized [3H]acetylcholine from stimulated hippocampal slices. Prior activation of protein kinase C with a phorbol ester prevented the inhibition of [3H]acetylcholine release, but activation of protein kinase C after the exposure to the irreversible analog did not prevent the effect of the drug. Binding of 4-aminobenzovesamicol in hippocampal synaptosomes, assayed using [3H]vesamicol and back-titration, was decreased by activation of protein kinase C prior to analog exposure but not by activation subsequent to exposure. We propose that phosphorylation of the vesicular acetylcholine transporter prevents the binding of vesamicol-like drugs.

Hydroxylated decahydroquinolines as ligands for the vesicular acetylcholine transporter: synthesis and biological evaluation.

Analogues of the potent anticholinergic 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) in which the cyclohexyl fragment was replaced with an N-acyl or N-alkyl trans-decahydroquinolyl moiety were synthesized and evaluated as potential ligands for the vesicular acetylcholine transporter (VAChT). The binding of compounds, such as 18, 20, and 21, was both stereospecific and of comparable magnitude to that of the closely related vesamicol analogue 2,3-trans-4a, 8a-trans-3-hydroxy-2-(4-phenylpiperidino)-1,2,3,4,5,6,7, 8-decahydronaphthalene (6) which displays subnanomolar affinity for this transporter. However, these compounds also demonstrated high affinities for sigma(1) and sigma(2) receptors and thus failed to show significantly improved selectivity over previously reported vesamicol analogues.

Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine.

The vesicular acetylcholine transporter (VAChT) mediates ACh storage in synaptic vesicles by exchanging cytoplasmic ACh with vesicular protons. This study sought to determine the stoichiometry of exchange by analysis of ligand binding and transport kinetics. The effects of different pH values inside and outside, external ACh concentrations, and electrical potential gradients on ACh transport by vesicles isolated from the electric organ of Torpedo were determined using a pH-jump protocol. The equilibrium binding of a high-affinity analogue of ACh is inhibited by protonation with a pKa of 7.4 +/- 0.3. A two-proton model fits the transport data much better than a one-proton model does, and uptake increases at more positive internal electrical potential, as expected for the two-proton model. Thus, the results support the two-proton model. The transport cycle begins with binding of external ACh to outwardly oriented site 2 (KACho = 20 mM) and protonation of inwardly oriented site 1 (pKa1 = 4.73 +/- 0.05). Loaded VAChT reorients quickly (73 000 min-1) and releases ACh to the inside (KAChi = 44 000 mM) and the proton to the outside. Unloaded, internally oriented site 2 binds a proton (pKa2 = 7.0), after which VAChT reorients (150 +/- 20 min-1) in the rate-limiting step and releases the proton to the outside to complete the cycle. Rate constants for the reverse direction also were estimated. Two protons provide a thermodynamic driving force beyond that utilized in vivo, which suggests that vesicular filling is regulated. Other phenomena related to VAChT, namely the time required to fill synaptic vesicles, the fractional orientation of the ACh binding site toward cytoplasm, orientational lifetimes, and the rate of nonquantal release of ACh from cholinergic nerve terminals, were computer-simulated, and the results are compared with physiological observations.

Substructure and responses of cholinergic synaptic vesicles in the atomic force microscope.

The substructure and responses of individual 100-nm synaptic vesicles to osmotic stress have been probed with an atomic force microscope (AFM) operating in tapping mode. Cholinergic synaptic vesicles from the electric organ of Torpedo californica were imaged continuously as the osmolarity of the buffer was decreased. Vesicles in hyposmotic buffer lysed to form flat circular structures on the mica surface with a diameter about two times that of intact vesicles and a thickness of 7.2 +/- 1.7 nm, which can accommodate the lipid bilayer plus the internal proteoglycan. Images of intact vesicles in air reveal creases in the membrane surface. Phase mode AFM images of lysed vesicles in air show the presence of a material not seen on intact vesicles that might be intravesicular proteoglycan released from the membrane at very low osmotic and ionic strength.

N-hydroxyalkyl derivatives of 3 beta-phenyltropane and 1-methylspiro[1H-indoline-3,4'-piperidine]: vesamicol analogues with affinity for monoamine transporters.

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Properties of biomolecules measured from atomic force microscope images: a review.

AFM images can be used to obtain quantitative or qualitative information about the properties of biomaterials. Examples presented here are: (1) Persistence length measurements of moving and stationary DNA molecules. (2) Force mapping to measure properties such as the elasticity of cells and vesicles. (3) Phase mode imaging to detect variations in materials and properties of the sample surface. (4) Imaging of surfaces at different constant forces.

Imaging of cholinergic terminals using the radiotracer [18F](+)-4-fluorobenzyltrozamicol: in vitro binding studies and positron emission tomography studies in nonhuman primates.

The goal of the present set of studies was to characterize the in vitro binding properties and in vivo tissue kinetics for the vesicular acetylcholine transporter (VAcChT) radiotracer, [18F](+)-4-fluorobenzyltrozamicol ([18F](+)-FBT). In vitro binding studies were conducted in order to determine the affinity of the (+)- and (-)-stereoisomers of FBT for the VAcChT as well as sigma (sigma 1 and sigma 2) receptors. (+)-FBT was found to have a high affinity (Ki = 0.22 nM) for the VAcChT and lower affinities for sigma 1 (21.6 nM) and sigma 2 (35.9 nM) receptors, whereas (-)-FBT had similar affinities for the VAcChT and sigma 1 receptors (approximately 20 nM) and a lower affinity for sigma 2 (110 nM) receptors. PET imaging studies were conducted in rhesus monkeys (n = 3) with [18F](+)-FBT. [18F](+)-FBT was found to have a high accumulation and slow rate of washout from the basal ganglia, which is consistent with the labeling of cholinergic interneurons in this brain region. [18F](+)-FBT also displayed reversible binding kinetics during the 3 h time course of PET and produced radiolabeled metabolites that did not cross the blood-brain barrier. The results from the current in vitro and in vivo studies indicate that [18F](+)-FBT is a promising ligand for studying cholinergic terminal density, with PET, via the VAcChT.

Changes in the elastic properties of cholinergic synaptic vesicles as measured by atomic force microscopy.

Cholinergic synaptic vesicles from Torpedo californica have been probed with the atomic force microscope in aqueous buffers to map and measure their elastic properties. Elastic properties were mapped with a new atomic force microscope technique known as force mapping. Force mapping of vesicles showed that the centers of the vesicles are harder or stiffer than the peripheral areas in the three buffers that were investigated. These were an isoosmotic buffer, a hypoosmotic buffer, and an isoosmotic buffer with 5 mM CaCl2 added. The hardness of the vesicular centers was quantified by calculation of the elastic modulus. Elastic moduli were in the range of 2-13 x 10(5) Pa. Vesicular centers were hardest in calcium-containing buffer and softest in isoosmotic buffer. Hypotheses are presented for the composition and function of the hard centers.

Pharmacological characterization of the vesamicol analogue (+)-[(125)I]MIBT in primate brain.

The vesamicol analogue, meta-[(125)I]iodobenzyltrozamicol [(+)-[(125)I]MIBT] was evaluated as a probe for the in vitro labeling of the vesicular acetylcholine transporter in primate brain. In the striatum, (+)-[(125)I]MIBT bound a single high-affinity site with a Kd value of 4.4 +/- 0.7 nM. Competition for (+)-[(125)I]MIBT binding to the striatum by a group of vesamicol analogues displayed a pharmacological profile similar to the rank order of potency previously observed for the vesicular acetylcholine transporter on Torpedo synaptic vesicles. High-affinity binding of (+)-[(125)I]MIBT in the occipital cortex was characterized by a Kd value of 4.6 +/- 1.1 nM. However, the rank order of potency for inhibition of (+)-[(125)I]MIBT binding to the occipital cortex by the same test compounds differed from that observed in the striatum. The results suggest that (+)-[(125)I]MIBT is a reliable probe of the vesicular acetylcholine transporter in primate striatum, but its binding in primate occipital cortex is more complex.

Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles.

At least two distinguishable forms of synaptic vesicles exist, the active and reserve, but the reserve form is studied most because it has been difficult to purify the active vesicles. In the work reported here the active vesicles (termed VP2) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP1) with the addition of density gradient centrifugation based on Percoll. No significant differences between the vesicular types were found in the amounts of SV1, SV2, and SV4 epitopes and P-type and V-type ATPase activities. The buoyant densities (g/ml) of VP1 and VP2 vesicles were determined by centrifugation in isosmotic sucrose (1.051, 1.069), Percoll (1.034, 1.040), and glycerol (1.087, 1.090) gradients. The radii were determined by dynamic quasi-elastic laser light-scattering to be (56.6 +/- 10.8) nm and (55.0 +/- 12.7) nm. For both vesicular types the volume of excluded sucrose is only about 37% of the volume of excluded Percoll, indicating that the surfaces are rough. Approx. 51% of the VP1 and 32% of the VP2 vesicular volumes are 'osmotically active' water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP1 and VP2 vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP1 and VP2 synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.