Background Despite the recognized advantages of Peer-Assisted Learning (PAL) in academic settings... more Background Despite the recognized advantages of Peer-Assisted Learning (PAL) in academic settings, there is a notable absence of research analyzing its effects on students' Academic Burnout. This study aims to cover this gap by assessing the underlying effectiveness of Informal Peer-Assisted Learning as a cooperative learning method, focusing on its potential to mitigate academic burnout among medical students. Methods In 2022, a cross-sectional study was conducted at the School of Medicine, Universidad Central del Caribe, in Puerto Rico. The research team gathered data from 151 participants, being 49.19% of the total student body. This cohort included 76 female students, 71 male students, and 4 individuals saying other. The School Burnout Inventory questionnaire (SBI-9) was employed to assess Academic Burnout, along with an added query about self-reported Informal Peer-Assisted Learning. The School Burnout Inventory questionnaire underwent validation, and from correlated with the Informal Peer-Assisted Learning. Results The validation process of the questionnaire a rmed its alignment with an eight-item inventory, encapsulating two principal factors that elucidate academic burnout. The rst factor pertains to exhaustion, while the second encompasses the combined subscales of cynicism and inadequacy. The questionnaire shows high reliability (Cronbach's alpha = 0.829) and good t indices (CFI= 0.934; TLI= 0.902; SRMR= 0.0495; RMSEA= 0.09791; p-value <0.001). The factors proven in the selected model were used to evaluate the correlation between the Academic Burnout and the Informal Peer-Assisted Learning. Students engaged in Informal Peer-Assisted Learning showed signi cantly lower academic burnout prevalence (p-value < 0.01) compared to those who never taken part in such practices. Furthermore, this group of students displayed lower cynicism/inadequacy compared to its exhaustion with an p-value < 0.001. Conclusions The results of this study underscore a notable issue of academic burnout among medical students within the surveyed cohort. The investigation presents a robust link between Academic Burnout and Informal Peer-Assisted Learning (IPAL), a rming the hypothesis that Informal Peer-Assisted Learning contributes to mitigating Academic Burnout. This correlation suggests that incorporating IPAL strategies may be bene cial in addressing burnout in medical education settings. Background Burnout, characterized by overwhelming mental and physical exhaustion, presents a critical concern within the medical student (MS) community. This phenomenon is strongly associated with reduced feelings of achievement and depersonalization, potentially leading to adverse student outcomes, such as poor academic performance, compromised mental health, increased dropout rates, and even suicidal ideation [1] [2]. The School Burnout Inventory (SBI-9) questionnaire supplies a standardized tool for assessing academic burnout (ABO), encompassing three key sub-scales: exhaustion (EX), cynicism (CY), and inadequacy (IN) [3]. These metrics, along with others, have been instrumental in shaping our understanding of burnout as a psychological syndrome ([4]) and have contributed to the International Classi cation of Diseases-11 de nition, characterizing burnout as an occupational phenomenon resulting from chronic workplace stress that has not been effectively managed [5].
The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine recept... more The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within RM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [ 125 I]-R-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipidexposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the RM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC 50. Taken together, our results suggest that RM3 contributes to the gating machinery of the nicotinic ACh receptor and that RM3 is comprised of a mixture of two types of helical structures.
New techniques to control the potential in membrane patches are giving in detail important charac... more New techniques to control the potential in membrane patches are giving in detail important characteristics of the pre- and post-synaptic channels. When used to assess the quantal size regulation during synchronous (evoked) or asynchronous (spontaneous) release of neurotransmitter can provide valuable information above this process. The macropatch technique (a version of the original patch clamp technique), has advantages in comparison with other macroscopic voltage clamp techniques to record the miniature end-plate current, mepc. During this focal recording, the puzzle size variation of mepc's amplitudes produced, from events generated over distant parts of the cell, are removed. The quantal response is an important feature during synaptic plasticity. Therefore, in this review we focus on pre- and post-synaptic aspects from the quantal asynchronous release of neurotransmitter, that occurs in the neuromuscular junction. We take emphasis on the caudal muscle of tadpoles from amphibian.
The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using p... more The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.
A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differen... more A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues aS287, aC412, bY441, cM299, cS460, dM293, dS297 and dN305 in the Torpedo AChR were replaced with those found in the muscletype receptor. Mutant receptor expression was measured in Xenopus oocytes using [ 125 I]-a-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5-to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC 50), while three mutant combinations resulted in 1.7-to 2-fold increases in ACh EC 50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.
A recent tryptophan scanning of the R-TM3 domain of the Torpedo californica AChR demonstrated tha... more A recent tryptophan scanning of the R-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [
We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome pre... more We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome presenting in infancy in a single individual as progressive weakness and impaired neuromuscular transmission without overt degeneration of the motor endplate. Prolonged low-amplitude synaptic currents in biopsied anconeus muscle at 9 years of age suggested a kinetic disorder of the muscle acetylcholine receptor. Ultrastructural studies at 16 months, at 9 years, and at 15 years of age showed none of the typical degenerative changes of the endplate associated with the slow-channel congenital myasthenic syndrome, and acetylcholine receptor numbers were not significantly reduced. We identified a novel C-toT substitution in exon 8 of the δ-subunit that results in a serine to phenylalanine mutation in the region encoding the second transmembrane domain that lines the ion channel. Using Xenopus oocyte in vitro expression studies we confirmed that the δS268F
Quarterly journal of experimental physiology, Jul 16, 1984
Single fibres from the sartorius muscle of the toad Bufo marinus were used to study ionic current... more Single fibres from the sartorius muscle of the toad Bufo marinus were used to study ionic currents, using a fast voltage-clamp method. Sodium currents were analysed following Hodgkin-Huxley formulations. Internal caesium fluoride blocks delayed currents but produced shifts in the equilibrium potential of the early channel due to sodium accumulation. Delayed channels are very unstable when fluoride is used inside the fibre. These instabilities and equilibrium potential shifts were eliminated on replacing fluoride by aspartate in the internal solution. Late peaks of inward current, probably associated with activity at the tubular system, were occasionally observed for small depolarizations. The results are compared with those obtained for other amphibian species and used to explain more general electrophysiological properties of muscle fibres of this tropical toad.
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V → F substitution in the M1 domain of the β subunit (βV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation βV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the βV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the βV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the βV229F mutation on the miniature endplate currents and thus are direct evidence that the βV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the ac... more We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the TM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [ 125 I]-R-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the TM3 domain was consistent with an R-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed R-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica subunit.
The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis h... more The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the ␥M4 transmembrane domain (␥TM4) by introducing tryptophan, at residues 451-462, along the ␥TM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [ 125 I]␣-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC 50 , and Hill coefficient values for WT are 1.8 ؎ 0.4 fmol, 30.3 ؎ 1.1 M, and 1.8 ؎ 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 ؎ 0.5, 3.6 ؎ 0.6, and 3.0 ؎ 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC 50 (19.5 ؎ 1.7, 11.4 ؎ 0.7, 16.4 ؎ 3.8, and 19.1 ؎ 2.6 M, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC 50 (42.8 ؎ 6.8 M) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC 50 and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the ␥TM4.
The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distr... more The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distribution of ion channels in adult amphibia that mediate extracellular K+ spatial buffering. 94% of the total membrane conductance of these cells resides in inward rectifier K+ channels in the endfoot processes apposed to the vitreous humour. We now report that this regional specialization is found in Müller cells isolated from adult (&gt;120 day old) bullfrogs but to a far less extent in those from 10-20 day old tadpoles (stages 34-36). Using the cell attached configuration of the patch-clamp technique, we found, in agreement with previous studies in salamanders, that the endfoot of adult cells had 19.2+/-2.4 (mean +/- S.E., n = 81) channels/patch, whereas the soma had 1.81+/-0.28 (n = 21) channels/patch. In the tadpole, the respective values were 4.29+/-0.26 (n = 79) for the endfoot and 2.26+/-0.24 (n = 27) for the soma. The slope conductance of the inward rectifier K+ channel in 115 mM K+, 19.2+/-0.25 pS (n = 205), channel kinetics and the resting membrane potential (-69+/-2.7 mV, n = 224) were similar at both the endfoot and soma of both adults and embryos. We conclude that during development, the K+ conductance of the Müller cell endfoot, but not of the soma, increases due to a selective clustering of inwardly rectifying K+ channels in that specific region of the cell membrane. The properties of the channels change little during the transformation from tadpole to adult bullfrog.
Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of... more Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltageclamped oocytes. For all the substitutions the concentration for half-maximal activation, EC 50 , is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2-to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC 50 s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs.
The International Journal of Developmental Biology, 2009
Thyroid hormones-particularly triiodothyronine, T 3-play a critical role in the morphological tra... more Thyroid hormones-particularly triiodothyronine, T 3-play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre-or pro-metamorphic stages. Exposure to T 3 in the water induced a rapid (beginning within 5 min) and significant decrease (~20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI< 2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T 3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI≥2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.
A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or ... more A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least acr...
Abstract. A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveal... more Abstract. A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues aS287, aC412, bY441, cM299, cS460, dM293, dS297 and dN305 in the Torpedo AChR were replaced with those found in the muscletype receptor. Mutant receptor expression was measured in Xenopus oocytes using [ 125 I]-a-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46 % decrease in the ACh 50% inhibitory concentration (EC 50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC 50. Finally, seven mutant combin...
Background Despite the recognized advantages of Peer-Assisted Learning (PAL) in academic settings... more Background Despite the recognized advantages of Peer-Assisted Learning (PAL) in academic settings, there is a notable absence of research analyzing its effects on students' Academic Burnout. This study aims to cover this gap by assessing the underlying effectiveness of Informal Peer-Assisted Learning as a cooperative learning method, focusing on its potential to mitigate academic burnout among medical students. Methods In 2022, a cross-sectional study was conducted at the School of Medicine, Universidad Central del Caribe, in Puerto Rico. The research team gathered data from 151 participants, being 49.19% of the total student body. This cohort included 76 female students, 71 male students, and 4 individuals saying other. The School Burnout Inventory questionnaire (SBI-9) was employed to assess Academic Burnout, along with an added query about self-reported Informal Peer-Assisted Learning. The School Burnout Inventory questionnaire underwent validation, and from correlated with the Informal Peer-Assisted Learning. Results The validation process of the questionnaire a rmed its alignment with an eight-item inventory, encapsulating two principal factors that elucidate academic burnout. The rst factor pertains to exhaustion, while the second encompasses the combined subscales of cynicism and inadequacy. The questionnaire shows high reliability (Cronbach's alpha = 0.829) and good t indices (CFI= 0.934; TLI= 0.902; SRMR= 0.0495; RMSEA= 0.09791; p-value <0.001). The factors proven in the selected model were used to evaluate the correlation between the Academic Burnout and the Informal Peer-Assisted Learning. Students engaged in Informal Peer-Assisted Learning showed signi cantly lower academic burnout prevalence (p-value < 0.01) compared to those who never taken part in such practices. Furthermore, this group of students displayed lower cynicism/inadequacy compared to its exhaustion with an p-value < 0.001. Conclusions The results of this study underscore a notable issue of academic burnout among medical students within the surveyed cohort. The investigation presents a robust link between Academic Burnout and Informal Peer-Assisted Learning (IPAL), a rming the hypothesis that Informal Peer-Assisted Learning contributes to mitigating Academic Burnout. This correlation suggests that incorporating IPAL strategies may be bene cial in addressing burnout in medical education settings. Background Burnout, characterized by overwhelming mental and physical exhaustion, presents a critical concern within the medical student (MS) community. This phenomenon is strongly associated with reduced feelings of achievement and depersonalization, potentially leading to adverse student outcomes, such as poor academic performance, compromised mental health, increased dropout rates, and even suicidal ideation [1] [2]. The School Burnout Inventory (SBI-9) questionnaire supplies a standardized tool for assessing academic burnout (ABO), encompassing three key sub-scales: exhaustion (EX), cynicism (CY), and inadequacy (IN) [3]. These metrics, along with others, have been instrumental in shaping our understanding of burnout as a psychological syndrome ([4]) and have contributed to the International Classi cation of Diseases-11 de nition, characterizing burnout as an occupational phenomenon resulting from chronic workplace stress that has not been effectively managed [5].
The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine recept... more The functional role of the RM3 transmembrane domain of the Torpedo nicotinic acetylcholine receptor (AChR) was characterized by performing tryptophan-scanning mutagenesis at 13 positions within RM3, from residue M278 through I290. The expression of the mutants in Xenopus oocytes was measured by [ 125 I]-R-bungarotoxin binding, and ACh receptor function was evaluated by using a two-electrode voltage clamp. Six mutants (L279W, F280W, I283W, V285W, S288W, and I289W) were expressed at lower levels than the wild type. Most of these residues have been proposed to face the interior of the protein. The I286W mutant was expressed at 2.4-fold higher levels than the wild type, and the two lipidexposed mutations, F284W and S287W, were expressed at similar levels as wild type. Binding assays indicated that the RM3 domain can accommodate bulky groups in almost all positions. Three mutations, M282W, V285W, and I289W, caused a loss of receptor function, suggesting that the tryptophan side chains alter the conformational changes required for channel assembly or ion channel function. This loss of function suggests that these positions may be involved in helix-helix contacts that are critical for channel gating. The lipid-exposed mutation F284W enhances the receptor macroscopic response at low ACh concentrations and decreases the EC 50. Taken together, our results suggest that RM3 contributes to the gating machinery of the nicotinic ACh receptor and that RM3 is comprised of a mixture of two types of helical structures.
New techniques to control the potential in membrane patches are giving in detail important charac... more New techniques to control the potential in membrane patches are giving in detail important characteristics of the pre- and post-synaptic channels. When used to assess the quantal size regulation during synchronous (evoked) or asynchronous (spontaneous) release of neurotransmitter can provide valuable information above this process. The macropatch technique (a version of the original patch clamp technique), has advantages in comparison with other macroscopic voltage clamp techniques to record the miniature end-plate current, mepc. During this focal recording, the puzzle size variation of mepc's amplitudes produced, from events generated over distant parts of the cell, are removed. The quantal response is an important feature during synaptic plasticity. Therefore, in this review we focus on pre- and post-synaptic aspects from the quantal asynchronous release of neurotransmitter, that occurs in the neuromuscular junction. We take emphasis on the caudal muscle of tadpoles from amphibian.
The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using p... more The action of ATP on Ca(2+)-dependent K+ channels was studied in fresh human erythrocytes using patch-clamp techniques. Single-channel current was recorded at pH 6.5 from inside-out patches in the presence of symmetrical K+ gluconate solutions, containing both 1 microM free Ca2+ in the bath and 0.5 mM LaCl3 on the pipette side. With no ATP, the electrical activity revealed low-conductance K+ channels (25 pS), which showed inward rectification and an opening kinetics dependent on membrane potential. When ATP (1 mM) and Mg2+ (2 mM) were added together and a depolarizing potential was simultaneously applied, only a high-conductance channel (about 75 pS) was observed. This channel showed no rectifying properties and it was not found if ATP was added in the absence of Mg2+. Channel activity was enhanced by adding fluoride (10 mM) or trifluoperazine (50 microM) whilst it was reduced after incubating with dibutyryl cAMP (50 microM) or alkaline phosphatase (250 U/ml). On the other hand, when fragmented membranes from inside-out vesicles were incubated with gamma-32 P-ATP and 1 microM free Ca2+ under above conditions, only two high-molecular weight polypeptides (235 and 320 kDa) were labelled with 32P. The results suggest that ATP-mediated phosphorylation of Ca(2+)-dependent K+ channels leads to a high-conductance state.
A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differen... more A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues aS287, aC412, bY441, cM299, cS460, dM293, dS297 and dN305 in the Torpedo AChR were replaced with those found in the muscletype receptor. Mutant receptor expression was measured in Xenopus oocytes using [ 125 I]-a-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5-to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46% decrease in the ACh 50% inhibitory concentration (EC 50), while three mutant combinations resulted in 1.7-to 2-fold increases in ACh EC 50. Finally, seven mutant combinations resulted in a decrease in normalized, ACh-induced currents. Our results suggest that these residues, although remote from the ion channel pore, (1) contribute to ion channel gating, (2) may affect trafficking of AChR into specialized membrane domains and (3) account for the functional differences between Torpedo and muscle-type AChR. These findings emphasize the importance of the lipid-protein interface in the functional differences between the Torpedo and muscle-type AChRs.
A recent tryptophan scanning of the R-TM3 domain of the Torpedo californica AChR demonstrated tha... more A recent tryptophan scanning of the R-TM3 domain of the Torpedo californica AChR demonstrated that this domain can modulate ion-channel gating [
We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome pre... more We investigated the basis for a novel form of the slow-channel congenital myasthenic syndrome presenting in infancy in a single individual as progressive weakness and impaired neuromuscular transmission without overt degeneration of the motor endplate. Prolonged low-amplitude synaptic currents in biopsied anconeus muscle at 9 years of age suggested a kinetic disorder of the muscle acetylcholine receptor. Ultrastructural studies at 16 months, at 9 years, and at 15 years of age showed none of the typical degenerative changes of the endplate associated with the slow-channel congenital myasthenic syndrome, and acetylcholine receptor numbers were not significantly reduced. We identified a novel C-toT substitution in exon 8 of the δ-subunit that results in a serine to phenylalanine mutation in the region encoding the second transmembrane domain that lines the ion channel. Using Xenopus oocyte in vitro expression studies we confirmed that the δS268F
Quarterly journal of experimental physiology, Jul 16, 1984
Single fibres from the sartorius muscle of the toad Bufo marinus were used to study ionic current... more Single fibres from the sartorius muscle of the toad Bufo marinus were used to study ionic currents, using a fast voltage-clamp method. Sodium currents were analysed following Hodgkin-Huxley formulations. Internal caesium fluoride blocks delayed currents but produced shifts in the equilibrium potential of the early channel due to sodium accumulation. Delayed channels are very unstable when fluoride is used inside the fibre. These instabilities and equilibrium potential shifts were eliminated on replacing fluoride by aspartate in the internal solution. Late peaks of inward current, probably associated with activity at the tubular system, were occasionally observed for small depolarizations. The results are compared with those obtained for other amphibian species and used to explain more general electrophysiological properties of muscle fibres of this tropical toad.
We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weaknes... more We traced the cause of a slow-channel syndrome (SCS) in a patient with progressive muscle weakness, repetitive compound muscle action potential and prolonged low amplitude synaptic currents to a V → F substitution in the M1 domain of the β subunit (βV229F) of the muscle acetylcholine receptor (AChR). In vitro expression studies in Xenopus oocytes indicated that the novel mutation βV229F expressed normal amounts of AChRs and decreased the ACh EC 50 by 10fold compared to wild type. Kinetic analysis indicated that the mutation displayed prolonged mean open duration and repeated openings during activation. Prolonged openings caused by the βV229F mutation were due to a reduction in the channel closing rate and an increase in the effective channel opening rate. Repeated openings of the channel during activation were caused by a significant reduction in the agonist dissociation constant. In addition, the βV229F mutation produced an increase in calcium permeability. The kinetic and permeation studies presented in this work are sufficient to explain the consequences of the βV229F mutation on the miniature endplate currents and thus are direct evidence that the βV229F mutation is responsible for compromising the safety margin of neuromuscular transmission in the patient.
We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the ac... more We used tryptophan substitutions to characterize the beta M3 transmembrane domain (TM3) of the acetylcholine receptor (AChR). We generated 15 mutants with tryptophan substitutions within the TM3 domain, between residues R282W and I296W. The various mutants were injected into Xenopus oocytes, and expression levels were measured by [ 125 I]-R-bungarotoxin binding. Expression levels of the M288W, I289W, L290W, and F293W mutants were similar to that of wild type, whereas the other mutants (R282W, Y283W, L284W, F286W, I287W, V291W, A292W, S294W, V295W, and I296W) were expressed at much lower levels than that of wild type. None of these tryptophan mutants produced peak currents larger than that of wild type. Five of the mutants, L284W, F286W, I287W, V295W, and I296W, were expressed at levels <15% of the wild type. I296W had the lowest expression levels and did not display any significant ACh-induced current, suggesting that this position is important for the function and assembly of the AChR. Tryptophan substitution at three positions, L284, V291, and A292, dramatically inhibited AChR assembly and function. A periodicity analysis of the alterations in AChR expression at positions 282-296 of the TM3 domain was consistent with an R-helical structure. Residues known to be exposed to the membrane lipids, including R282, M285, I289, and F293, were all found in all the upper phases of the oscillatory pattern. Mutants that were expressed at lower levels are clustered on one side of a proposed R-helical structure. These results were incorporated into a structural model for the spatial orientation of the TM3 of the Torpedo californica subunit.
The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis h... more The periodicity of structural and functional effects induced by tryptophan scanning mutagenesis has been successfully used to define function and secondary structure of various transmembrane domains of the acetylcholine receptor of Torpedo californica. We expand the tryptophan scanning of the AchR of T. californica to the ␥M4 transmembrane domain (␥TM4) by introducing tryptophan, at residues 451-462, along the ␥TM4. Wild type (WT) and mutant AChR were expressed in Xenopus laevis oocytes. Using [ 125 I]␣-bungarotoxin binding assays and voltage clamp, we determined that the nAChR expression, EC 50 , and Hill coefficient values for WT are 1.8 ؎ 0.4 fmol, 30.3 ؎ 1.1 M, and 1.8 ؎ 0.3, respectively. Mutations L456W, F459W, and G462W induce a significant increase in nAChR expression (2.8 ؎ 0.5, 3.6 ؎ 0.6, and 3.0 ؎ 0.5 fmol, respectively) when compared with WT. These data suggest that these residues are important for AChR oligomerization. Mutations A455W, L456W, F459W, and G462W result in a significant decrease in EC 50 (19.5 ؎ 1.7, 11.4 ؎ 0.7, 16.4 ؎ 3.8, and 19.1 ؎ 2.6 M, respectively), thus suggesting a gain in function when compared with WT. In contrast, mutation L458W induced an increase in EC 50 (42.8 ؎ 6.8 M) or loss in function when compared with WT. The Hill coefficient values were the same for WT and all of the mutations studied. The periodicity in function (EC 50 and macroscopic peak current) and nAChR expression reveals an average of 3.3 and 3.0 amino acids respectively, thus suggesting a helical secondary structure for the ␥TM4.
The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distr... more The radial glial cells that span the retina, described by Müller in 1851, have a remarkable distribution of ion channels in adult amphibia that mediate extracellular K+ spatial buffering. 94% of the total membrane conductance of these cells resides in inward rectifier K+ channels in the endfoot processes apposed to the vitreous humour. We now report that this regional specialization is found in Müller cells isolated from adult (&gt;120 day old) bullfrogs but to a far less extent in those from 10-20 day old tadpoles (stages 34-36). Using the cell attached configuration of the patch-clamp technique, we found, in agreement with previous studies in salamanders, that the endfoot of adult cells had 19.2+/-2.4 (mean +/- S.E., n = 81) channels/patch, whereas the soma had 1.81+/-0.28 (n = 21) channels/patch. In the tadpole, the respective values were 4.29+/-0.26 (n = 79) for the endfoot and 2.26+/-0.24 (n = 27) for the soma. The slope conductance of the inward rectifier K+ channel in 115 mM K+, 19.2+/-0.25 pS (n = 205), channel kinetics and the resting membrane potential (-69+/-2.7 mV, n = 224) were similar at both the endfoot and soma of both adults and embryos. We conclude that during development, the K+ conductance of the Müller cell endfoot, but not of the soma, increases due to a selective clustering of inwardly rectifying K+ channels in that specific region of the cell membrane. The properties of the channels change little during the transformation from tadpole to adult bullfrog.
Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of... more Our previous amino-acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha subunit. In this study we have extended the mutagenesis analysis using single tryptophan replacements in seven positions (I288, M291, F292, S294, L296, M299 and N300) near the center of the third transmembrane domain of the gamma subunit (γM3). All the tryptophan substitution mutants were expressed in Xenopus laevis oocytes following mRNA injections at levels close to wild type. The functional response of these mutants was evaluated using macroscopic current analysis in voltageclamped oocytes. For all the substitutions the concentration for half-maximal activation, EC 50 , is similar to wild type using acetylcholine. For F292W, L296W and M299W the normalized macroscopic responses are 2-to 3-fold higher than for wild type. Previous photolabeling studies demonstrated that these three positions were in contact with membrane lipids. Each of these M3 mutations was co-injected with the previously characterized αC418W mutant to examine possible synergistic effects of single lipid-exposed mutations on two different subunits. For the γM3/αM4 double mutants, the EC 50 s were similar to those measured for the αC418W mutant alone. Tryptophan substitutions at positions that presumably face the interior of the protein (S294 and M291) or neighboring helices (I288) did not cause significant inhibition of channel function or surface expression of AChRs.
The International Journal of Developmental Biology, 2009
Thyroid hormones-particularly triiodothyronine, T 3-play a critical role in the morphological tra... more Thyroid hormones-particularly triiodothyronine, T 3-play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre-or pro-metamorphic stages. Exposure to T 3 in the water induced a rapid (beginning within 5 min) and significant decrease (~20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI< 2.8). To determine correlated changes in the neuromuscular junctions during metamorphosis and apoptotic tail loss, miniature endplate currents from tail muscle were recorded during acute exposure to a hypertonic solution, which simulates an apoptotic volume decrease. Our results support a role for T 3 in regulating larval locomotor activity during development, and suggest an enhanced response to volume depletion at the neuromuscular junction of older larvae (DI<2.8) compared to younger animals (DI≥2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.
A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or ... more A product of the immediate early gene Arc (Activity-regulated cytoskeleton-associated protein or Arc protein) of retroviral ancestry resides in the genome of all tetrapods for millions of years and is expressed endogenously in neurons. It is a well-known protein, very important for synaptic plasticity and memory consolidation. Activity-dependent Arc expression concentrated in glutamatergic synapses affects the long-time synaptic strength of those excitatory synapses. Because it modulates excitatory-inhibitory balance in a neuronal network, the Arc gene itself was found to be related to the pathogenesis of epilepsy. General Arc knockout rodent models develop a susceptibility to epileptic seizures. Because of activity dependence, synaptic Arc protein synthesis also is affected by seizures. Interestingly, it was found that Arc protein in synapses of active neurons self-assemble in capsids of retrovirus-like particles, which can transfer genetic information between neurons, at least acr...
Abstract. A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveal... more Abstract. A comparison between the Torpedo and muscle-type acetylcholine receptors (AChRs) reveals differences in several lipid-exposed amino acids, particularly in the polarity of those residues. The goal of this study was to characterize the role of eight lipid-exposed residues in the functional differences between the Torpedo and muscle-type AChRs. To this end, residues aS287, aC412, bY441, cM299, cS460, dM293, dS297 and dN305 in the Torpedo AChR were replaced with those found in the muscletype receptor. Mutant receptor expression was measured in Xenopus oocytes using [ 125 I]-a-bungarotoxin, and AChR ion channel function was evaluated using the two-electrode voltage clamp. Eight mutant combinations resulted in an increase (1.5- to 5.2-fold) in AChR expression. Four mutant combinations produced a significant 46 % decrease in the ACh 50% inhibitory concentration (EC 50), while three mutant combinations resulted in 1.7- to 2-fold increases in ACh EC 50. Finally, seven mutant combin...
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