Presynaptic mechanism for phorbol ester-induced synaptic potentiation.
(17/1423)
Phorbol ester facilitates transmitter release at a variety of synapses, and the phorbol ester-induced synaptic potentiation (PESP) is a model for presynaptic facilitation. To address the mechanism underlying PESP, we have made paired whole-cell recordings from the giant presynaptic terminal, the calyx of Held, and its postsynaptic target in the medial nucleus of the trapezoid body in rat brainstem slices. Phorbol ester potentiated EPSCs without affecting either presynaptic calcium currents or potassium currents. Protein kinase C inhibitors applied from outside or injected directly into the presynaptic terminal attenuated the PESP. Furthermore, presynaptic loading of a synthetic peptide with the sequence of the N-terminal domain of Doc2alpha interacting with Munc13-1 (Mid peptide) significantly attenuated PESP, whereas mutated Mid peptide had no effect. We conclude that the target of the presynaptic facilitatory effect of phorbol ester resides downstream of calcium influx and may involve both protein kinase C and Doc2alpha - Munc13-1 interaction. (+info)
Mechanisms of induction and expression of long-term depression at GABAergic synapses in the neonatal rat hippocampus.
(18/1423)
Synaptic plasticity at excitatory glutamatergic synapses is believed to be instrumental in the maturation of neuronal networks. Using whole-cell patch-clamp recordings, we have studied the mechanisms of induction and expression of long-term depression at excitatory GABAergic synapses in the neonatal rat hippocampus (LTD(GABA-A)). We report that the induction of LTD(GABA-A) requires a GABA(A) receptor-mediated membrane depolarization, which is necessary to remove the Mg(2+) block from postsynaptic NMDA receptors. LTD(GABA-A) is associated with an increase in the coefficient of variation of evoked GABA(A) receptor-mediated synaptic currents and a decrease in the frequency, but not amplitude, of Sr(2+)-induced asynchronous GABA(A) quantal events. We conclude that LTD(GABA-A) induction requires the activation of both GABA(A) and NMDA postsynaptic receptors and that its expression is likely presynaptic. (+info)
New insights into enzyme catalysis. Ground state tunnelling driven by protein dynamics.
(19/1423)
The wave-particle duality of matter suggests that quantum tunnelling may have a prominent role in enzymatic H-transfer. However, unlike for electron tunnelling, evidence for H-tunnelling in enzyme molecules is extremely limited. The theoretical development, and verification by experiment, of a role for protein dynamics in driving enzymatic H-tunnelling is presented. Dynamic theories of H-tunnelling suggest that the kinetic isotope effect, during rupture of a C-H/C-D bond, for example, can assume values interpreted previously as indicating classical transfer. Vibrationally enhanced ground state tunnelling has been demonstrated for enzymes that cleave stable C-H bonds. This is an attractive mechanism as large activation energies make it energetically unfavourable for a classical, over-the-barrier mode of cleavage. Furthermore, it may be a general strategy used by enzymes for catalysing these 'difficult' transformations. (+info)
Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses.
(20/1423)
Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release. (+info)
Carboxylate binding modes in zinc proteins: a theoretical study.
(21/1423)
The relative energies of different coordination modes (bidentate, monodentate, syn, and anti) of a carboxylate group bound to a zinc ion have been studied by the density functional method B3LYP with large basis sets on realistic models of the active site of several zinc proteins. In positively charged four-coordinate complexes, the mono- and bidentate coordination modes have almost the same energy (within 10 kJ/mol). However, if there are negatively charged ligands other than the carboxylate group, the monodentate binding mode is favored. In general, the energy difference between monodentate and bidentate coordination is small, 4-24 kJ/mol, and it is determined more by hydrogen-bond interactions with other ligands or second-sphere groups than by the zinc-carboxylate interaction. Similarly, the activation energy for the conversion between the two coordination modes is small, approximately 6 kJ/mol, indicating a very flat Zn-O potential surface. The energy difference between syn and anti binding modes of the monodentate carboxylate group is larger, 70-100 kJ/mol, but this figure again strongly depends on interactions with second-sphere molecules. Our results also indicate that the pK(a) of the zinc-bound water ligand in carboxypeptidase and thermolysin is 8-9. (+info)
Proton transfer from histidine 244 may facilitate the 1,2 rearrangement reaction in coenzyme B(12)-dependent methylmalonyl-CoA mutase.
(22/1423)
Methylmalonyl-CoA mutase is an adenosylcobalamin-dependent enzyme that catalyzes the 1,2 rearrangement of methylmalonyl-CoA to succinyl-CoA. This reaction results in the interchange of a carbonyl-CoA group and a hydrogen atom on vicinal carbons. The crystal structure of the enzyme reveals the presence of an aromatic cluster of residues in the active site that includes His-244, Tyr-243, and Tyr-89 in the large subunit. Of these, His-244 is within hydrogen bonding distance to the carbonyl oxygen of the carbonyl-CoA moiety of the substrate. The location of these aromatic residues suggests a possible role for them in catalysis either in radical stabilization and/or by direct participation in one or more steps in the reaction. The mechanism by which the initially formed substrate radical isomerizes to the product radical during the rearrangement of methylmalonyl-CoA to succinyl-CoA is unknown. Ab initio molecular orbital theory calculations predict that partial proton transfer can contribute significantly to the lowering of the barrier for the rearrangement reaction. In this study, we report the kinetic characterization of the H244G mutant, which results in an acute sensitivity of the enzyme to oxygen, indicating the important role of this residue in radical stabilization. Mutation of His-244 leads to an approximately 300-fold lowering in the catalytic efficiency of the enzyme and loss of one of the two titratable pK(a) values that govern the activity of the wild type enzyme. These data suggest that protonation of His-244 increases the reaction rate in wild type enzyme and provides experimental support for ab initio molecular orbital theory calculations that predict rate enhancement of the rearrangement reaction by the interaction of the migrating group with a general acid. However, the magnitude of the rate enhancement is significantly lower than that predicted by the theoretical studies. (+info)
Protein-assisted pericyclic reactions: an alternate hypothesis for the action of quantal receptors.
(23/1423)
The rules for allowable pericyclic reactions indicate that the photoisomerizations of retinals in rhodopsins can be formally analogous to thermally promoted Diels-Alder condensations of monoenes with retinols. With little change in the seven-transmembrane helical environment these latter reactions could mimic the retinal isomerization while providing highly sensitive chemical reception. In this way archaic progenitors of G-protein-coupled chemical quantal receptors such as those for pheromones might have been evolutionarily plagiarized from the photon quantal receptor, rhodopsin, or vice versa. We investigated whether the known structure of bacteriorhodopsin exhibited any similarity in its active site with those of the two known antibody catalysts of Diels-Alder reactions and that of the photoactive yellow protein. A remarkable three-dimensional motif of aromatic side chains emerged in all four proteins despite the drastic differences in backbone structure. Molecular orbital calculations supported the possibility of transient pericyclic reactions as part of the isomerization-signal transduction mechanisms in both bacteriorhodopsin and the photoactive yellow protein. It appears that reactions in all four of the proteins investigated may be biological analogs of the organic chemists' chiral auxiliary-aided Diels-Alder reactions. Thus the light receptor and the chemical receptor subfamilies of the heptahelical receptor family may have been unified at one time by underlying pericyclic chemistry. (+info)
Time-resolved absorption and photothermal measurements with recombinant sensory rhodopsin II from Natronobacterium pharaonis.
(24/1423)
Purified wild-type sensory rhodopsin II from Natronobacterium pharaonis (pSRII-WT) and its histidine-tagged analog (pSRII-His) were studied by laser-induced optoacoustic spectroscopy (LIOAS) and flash photolysis with optical detection. The samples were either dissolved in detergent or reconstituted into polar lipids from purple membrane (PML). The quantum yield for the formation of the long-lived state M(400) was determined as Phi(M) = 0.5 +/- 0.06 for both proteins. The structural volume change accompanying the production of K(510) as determined with LIOAS was DeltaV(R,1) /= Phi(M), indicating that the His tag does not influence this early step of the photocycle. The medium has no influence on DeltaV(R,1), which is the largest so far measured for a retinal protein in this time range (<10 ns). This confirms the occurrence of conformational movements in pSRII for this step, as previously suggested by Fourier transform infrared spectroscopy. On the contrary, the decay of K(510) is an expansion in the detergent-dissolved sample and a contraction in PML. Assuming an efficiency of 1.0, DeltaV(R,2) = -3 ml/mol for pSRII-WT and -4.6 ml/mol for pSRII-His were calculated in PML, indicative of a small structural difference between the two proteins. The energy content of K(510) is also affected by the tag. It is E(K) = (88 +/- 13) for pSRII-WT and (134 +/- 11) kJ/mol for pSRII-His. A slight difference in the activation parameters for K(510) decay confirms an influence of the C-terminal His on this step. At variance with DeltaV(R,1), the opposite sign of DeltaV(R,2) in detergent and PML suggests the occurrence of solvation effects on the decay of K(510), which are probably due to a different interaction of the active site with the two dissolving media. (+info)