Analysis of temperature-dependent abnormal bursting patterns of neurons in Aplysia

Temperature affects the firing pattern and electrical activity of neurons in animals, eliciting diverse responses depending on neuronal cell type. However, the mechanisms underlying such diverse responses are not well understood. In the present study, we performed in vitro recording of abdominal ganglia cells of Aplysia juliana , and analyzed their burst firing patterns. We identified atypical bursting patterns dependent on temperature that were totally different from classical bursting patterns observed in R15 neurons of A. juliana . We classified these abnormal bursting patterns into type 1 and type 2; type 1 abnormal single bursts are composed of two kinds of spikes with a long interspike interval (ISI) followed by short ISI regular firing, while type 2 abnormal single bursts are composed of complex multiplets. To investigate the mechanism underlying the temperature dependence of abnormal bursting, we employed simulations using a modified Plant model and determined that the temperature dependence of type 2 abnormal bursting is related to temperaturedependent scaling factors and activation or inactivation of potassium or sodium channels.

Analysis of temperature-dependent abnormal bursting patterns of neurons in Aplysia

Temperature affects the firing pattern and electrical activity of neurons in animals, eliciting diverse responses depending on neuronal cell type. However, the mechanisms underlying such diverse responses are not well understood. In the present study, we performed in vitro recording of abdominal ganglia cells of Aplysia juliana , and analyzed their burst firing patterns. We identified atypical bursting patterns dependent on temperature that were totally different from classical bursting patterns observed in R15 neurons of A. juliana . We classified these abnormal bursting patterns into type 1 and type 2; type 1 abnormal single bursts are composed of two kinds of spikes with a long interspike interval (ISI) followed by short ISI regular firing, while type 2 abnormal single bursts are composed of complex multiplets. To investigate the mechanism underlying the temperature dependence of abnormal bursting, we employed simulations using a modified Plant model and determined that the temperature dependence of type 2 abnormal bursting is related to temperaturedependent scaling factors and activation or inactivation of potassium or sodium channels.

A Computational Model of the Temperature-dependent Changes in Firing Patterns in Aplysia Neurons

We performed experiments using Aplysia neurons to identify the mechanism underlying the changes in the firing patterns in response to temperature changes. When the temperature was gradually increased from 11degrees C to 31degrees C the firing patterns changed sequentially from the silent state to beating, doublets, beating-chaos, bursting-chaos, square-wave bursting, and bursting-oscillation patterns. When the temperature was decreased over the same temperature range, these sequential changes in the firing patterns reappeared in reverse order. To simulate this entire range of spiking patterns we modified nonlinear differential equations that Chay and Lee made using temperature-dependent scaling factors. To refine the equations, we also analyzed the spike pattern changes in the presence of potassium channel blockers. Based on the solutions of these equations and potassium channel blocker experiments, we found that, as temperature increases, the maximum value of the potassium channel relaxation time constant, taun(t) increases, but the maximum value of the probabilities of openings for activation of the potassium channels, n(t) decreases. Accordingly, the voltage-dependent potassium current is likely to play a leading role in the temperature-dependent changes in the firing patterns in Aplysia neurons.

The Effect of Heat on the Spiking Patterns of the Cells in Aplysia / 의학물리

Fruitful findings have been produced from five out of sixty cells which were obtained from each 63 individual Aplisia caught at the Jeju coast. Spiking patterns of three out of five cells, such as relaxation oscillator, bursting within a short time of the inter-burst interval, chaotic bursting, period doubling sequences, bursting with long trains of action potentials separated by short silent periods, regular repeated beating or elliptic bursting, and silent states had been changed in order as the temperature was lowered to 10 degrees C from 32 degrees C. In the intervals of every about 40 minutes repeated ups and downs of temperature produced similar firing patterns at the allowable temperature ranges. The other two cells showed difference from these. The amplitudes of the action potentials of the two cells will not be highly decreased in 24 hours. Average spike frequencies, the inter-burst interval, peak to peak spike amplitude of action potentials, minimum potential values are compared and analyzed by using the computer programme. The spike frequencies according to temperature show the distribution of bell type, with maximal spike frequencies at intermediate temperatures and minimal ones at either end. The most common pattern consist of high spike frequency during falling and low one during rising temperatures.

Contribution of conserved amino acids at the dimeric interface to the conformational stability and the structural integrity of the active site in ketosteroid isomerase from Pseudomonas putida biotype B.

Ketosteroid isomerase (KSI) from Pseudomonas putida biotype B is a homodimeric enzyme catalyzing an allylic isomerization of Delta(5)-3-ketosteroids at a rate of the diffusion-controlled limit. The dimeric interactions mediated by Arg72, Glu118, and Asn120, which are conserved in the homologous KSIs, have been characterized in an effort to investigate the roles of the conserved interface residues in stability, function and structure of the enzyme. The interface residues were replaced with alanine to generate the interface mutants R72A, E118A, N120A and E118A/N120A. Equilibrium unfolding analysis revealed that the DeltaG(U)(H(2)O) values for the R72A, E118A, N120A, and E118A/N120A mutants were decreased by about 3.8, 3.9, 7.8, and 9.5 kcal/mol, respectively, relative to that of the wild-type enzyme. The interface mutations not only decreased the k(cat)/K(M) value by about 8- to 96-fold, but also increased the K(D) value for d-equilenin, a reaction intermediate analogue, by about 7- to 17.5-fold. The crystal structure of R72A determined at 2.5 A resolution and the fluorescence spectra of all the mutants indicated that the interface mutations altered the active-site geometry and resulted in the decreases of the conformational stability as well as the catalytic activity of KSI. Taken together, our results strongly suggest that the conserved interface residues contribute to stabilization and structural integrity of the active site in the dimeric KSI.

The conserved cis-Pro39 residue plays a crucial role in the proper positioning of the catalytic base Asp38 in ketosteroid isomerase from Comamonas testosteroni.

KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro39 residue was proposed to be involved in the proper positioning of Asp38, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp38, but also to confer a structural rigidity on the local active-site geometry consisting of Asp38, Pro39, Val40, Gly41 and Ser42 at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro39 residue near the active site of KSI, Pro39 was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG(U)H2O (the free-energy change for unfolding in the absence of urea at 25.00+/-0.02 degrees C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 A (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp38, as well as the local active-site geometry near Asp38, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro39 residue plays a crucial role in the proper positioning of the critical catalytic base Asp38 and in the structural integrity of the active site in KSI.

Origin of the different pH activity profile in two homologous ketosteroid isomerases.

Two homologous Delta5-3-ketosteroid isomerases from Comamonas testosteroni (TI-WT) and Pseudomonas putida biotype B (PI-WT) exhibit different pH activity profiles. TI-WT loses activity below pH 5.0 due to the protonation of the conserved catalytic base, Asp-38, while PI-WT does not. Based on the structural analysis of PI-WT, the critical catalytic base, Asp-38, was found to form a hydrogen bond with the indole ring NH of Trp-116, which is homologously replaced with Phe-116 in TI-WT. To investigate the role of Trp-116, we prepared the F116W mutant of TI-WT (TI-F116W) and the W116F mutant of PI-WT (PI-W116F) and compared kinetic parameters of those mutants at different pH levels. PI-W116F exhibited significantly decreased catalytic activity at acidic pH like TI-WT, whereas TI-F116W maintained catalytic activity at acidic pH like PI-WT and increased the kcat/Km value by 2.5- to 4.7-fold compared with TI-WT at pH 3.8. The crystal structure of TI-F116W clearly showed that the indole ring NH of Trp-116 could form a hydrogen bond with the carboxyl oxygen of Asp-38 like that of PI-WT. The present results demonstrate that the activities of both PI-WT and TI-F116W at low pH were maintained by a tryptophan, which was able not only to lower the pKa value of the catalytic base but also to increase the substrate affinity. This is one example of the strategy nature can adopt to evolve the diversity of the catalytic function in the enzymes. Our results provide insight into deciphering the molecular evolution of the enzyme and creating novel enzymes by protein engineering.

Association of human tumor necrosis factor-related apoptosis inducing ligand with membrane upon acidification.

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) has been known to induce tumor-specific apoptosis and to share the structural and functional characteristics with the proteins of TNF family. Recently, the crystal structure of human TRAIL showed that TRAIL is a homotrimeric protein whose subunits contain mainly beta-sheets. We characterized the structural changes of recombinant human TRAIL induced by acidification and the biological implication of the structural characteristics at acidic pH in the interaction with the lipid bilayer. At acidic pH below pH 4.5, TRAIL resulted in substantial structural changes to a molten globule (MG)-like state. Far-UV CD spectrum of TRAIL indicated that the acidification induced alpha-helices that are absent in the native state. TRAIL at acidic pH exhibited significant change of tertiary structures as reflected in the near-UV CD spectrum. Thermal transition curve indicated that there was less cooperation at acidic pH than at neutral pH in the thermal denaturation of TRAIL. Moreover, TRAIL at the MG-like state not only enhanced the binding ability to liposomes, but also increased the release rate of a fluorescent dye, calcein, encapsulated in liposomes. The binding assay with anilinonaphthalene-8-sulfonic acid revealed that the surface hydrophobicity of TRAIL was increased while tryptophan residues became more exposed to solvent as judged by blue shift of the maximum fluorescence wavelength. Taken together, our results demonstrate that the acidification of human TRAIL induces the MG-like state in vitro and makes the membrane permeable through the favorable interaction of TRAIL with the membrane, implicating that general intrinsic properties such as TRAIL, TNF-alpha and lymphotoxin are shared by TNF family members.