Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca2+-channel present in α- and β-cells and the modulation of hormone secretion by Ca2+-dependent and -independent processes. We also consider how subtle changes in Ca2+-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca They do so by secreting two hormones with opposing effects on plasma glucose concentration: insulin and glucagon, which lowers and increases plasma glucose levels, respectively. The pancreatic islets are small aggregates of endocrine cells with a diameter of 100–200 μm and consist of ∼1000 endocrine cells. The three major endocrine cells within the islets are the insulin-producing β-cells, glucagon-secreting α-cells and somatostatin-releasing δ-cells which in man comprise ∼50%, 35% and 15% of the islet cell number, respectively Diabetes mellitus is a major metabolic disorder currently affecting 5–10% of the population in the western societies 2+]i) that culminates in exocytotic fusion of the hormone-containing secretory vesicles.Electrophysiological studies on isolated α- and β-cells from both rodent  and human islets have revealed that they are electrically excitable and that they contain a number of voltage-dependent and -independent ion channels 2Electrical activity from mouse pancreatic β-cells was first reported by Dean and Matthews in 1968 who impaled intact mouse islets with sharp intracellular electrodes Membrane potential recordings with sharp intracellular electrodes also allowed the effects of pharmacological agents like tolbutamide and diazoxide +-channel, postulated on the basis of radioisotopic measurements in the 1970s +-channel (KATP-channel).However, it was not until the patch-clamp technique was applied to pancreatic islet cells in the 1980s that the ion channels underlying β-cell electrical activity could be studied under voltage-clamp control. A breakthrough was the identification glucose-sensitive K2+ and K+-channels involved in β-cell action potential firing Patch-clamp measurements also allowed the characterization of the voltage-dependent CaATP-channel activity. KATP-channel activity maintains a negative membrane potential in mouse β-cells and closure of these channels unmasks the depolarizing influence of an (as yet) poorly characterized conductance, which accounts for the initial depolarization up to the threshold for action potential firing. The action potentials involve activation of voltage-gated L-type Ca2+-channels and the associated Ca2+-entry leads, via elevation of the submembrane [Ca2+]i, to stimulation of Ca2+-dependent exocytosis of the insulin-containing secretory granules Based on these findings, a consensus model for glucose-stimulated insulin secretion from mouse or rat islets was proposed ATP-channels by direct interaction with the channel Sulphonylurea drugs (like tolbutamide and glibenclamide) by-pass glucose metabolism and closes the KATP-channels, glucose also augments insulin release by an effect exerted distal to the elevation of [Ca2+]i. These two effects are referred to as the triggering and amplifying effects of glucose 2+]i but that glucose somehow increases the release competence of the secretory granules and their likelihood of undergoing exocytosis in response to a given increase in [Ca2+]i. The identity of the intracellular second messenger mediating the amplifying effect remains obscure but it is of interest that increases and decreases in ATP and ADP concentration, respectively, mimic the effect of an elevation of glucose on exocytosis In addition to the stimulatory effect mediated by closure of K2+]i oscillations resulting from action potential firing in the individual β-cells are synchronized across the entire islet Because of the electrical coupling in mouse islets, [Ca2+]i2+]i oscillations, similar  to those seen in mouse islets, can be observed in recordings from β-cells within intact human islets  not associated with any detectable stimulation of insulin and somatostatin secretion. This argues that the α-cells possess some sort of intrinsic glucose-sensing which operates independently of paracrine signalling ATP-channels play a similar role in α-cells as in β-cells and that glucose-dependent closure of these channels results in membrane depolarization. Because α- and β-cells express different complements of voltage-gated ion channels, membrane depolarization results in reduced rather than increased electrical excitability (as in β-cells) Compared to the detailed understanding of glucose-sensing in β-cells, the knowledge about this process in glucagon-secreting α-cells may seem rudimentary. Unlike β-cells, α-cells are electrically active in the absence of glucose and generate overshooting action potentials +-channels. The significance of these channels for glucagon secretion is illustrated by the strong inhibitory effect of this blocker on glucagon secretion +-channels exhibit a dual dependence on membrane potential. Whereas short depolarizations increase the open probability, sustained depolarizations (even those that are not large enough to activate the channels) make the channel enter a non-conducting inactivated state. If the depolarization is long enough, it will exert a TTX-like effect on α-cell electrical activity and glucagon secretion. We have found that depolarizations of only a few millivolts are sufficient to reduce action potential height by 10–20 mV. Such an effect can be expected to have a dramatic effect on glucagon secretion because exocytosis declines exponentially by >10% for every millivolt reduction of action potential height once their peak voltage goes below 0 mV The α-cell action potentials involve activation of TTX-sensitive voltage-gated NaIt should be emphasized that ‘intrinsic’ and ‘paracrine’ modes of α-cell regulation are not mutually exclusive. Indeed, there is good evidence for paracrine regulation of glucagon secretion 42+-channels (50% of the whole-cell Ca2+-current), SNX482-sensitive R-type Ca2+-channels (25%) and P/Q-type Ca2+-channels (10–15%) 2+-channels leads to transient inhibition of glucose-induced electrical activity but it subsequently resumes in the continued presence of the blocker 2+-channels containing α1C (Cav1.2) and α1D (Cav1.3) pore-forming subunits 2+-current density between wildtype and Cav1.3/α1D-deficient β-cells 2+-current density was reduced by ∼50% in β-cells from Cav1.2−/− mice and the residual current was resistant to dihydropyridines antagonists (like isradipine) and agonists (like BAYK8644) 2+-channels subtype in mouse β-cells.Mouse β-cells are equipped with dihydropyridine-sensitive Ca2+]i oscillations were maintained in β-cells/islets from mice lacking Cav1.2 (α1C) L-type Ca2+-channels. Nevertheless, glucose-induced insulin secretion is strongly reduced in Cav1.2−/− islets. Glucose-induced insulin secretion follows a biphasic time course consisting of a rapid but transient 1st phase followed by a slower and sustained 2nd phase 2+-influx via α1C-containing L-type Ca2+-channels is tightly linked to insulin exocytosis.Glucose-induced action potential firing and i remains elevated for a considerable period after a membrane depolarization, exocytosis only proceeds during the depolarization, echoing the activity of the Ca2+-channels 2+]i just below the inner mouth of the Ca2+-channels. It is implicit from this concept that only secretory granules within these ‘active zones’ of elevated [Ca2+]i (<10 nm from the inner mouth of the Ca2+-channels) will undergo exocytosis. The finding that rapid exocytosis is resistant to intracellular application of 10 mM EGTA is consistent with this scenario 2+-channel closure, the active zones almost instantly collapse and this accounts for the rapid termination of exocytosis.The Ca2+-channel activity in mouse β-cells have revealed that whereas most (>60%) membrane patches contain no active Ca2+-channels at all, ∼20% of the membrane patches contained 3 active channels 2+-channels and instead argues that the β-cell Ca2+-channels associate into triplets. This conceivably cancels out stochastic variation of channel activity and ensures that a release-competent secretory granule residing close to the Ca2+-channels will be exposed to exocytotic levels of [Ca2+]i long enough to trigger secretion. It is interesting that TIRF imaging of the near-membrane [Ca2+]i transients evoked by brief depolarizations concentrate to a few discrete areas  evokes only a transient stimulation of insulin secretion reminiscent of 1st phase glucose-induced insulin secretion Membrane depolarization alone without concomitant stimulation of β-cell metabolism (e.g. using high-K+) and that elicited by glucose result in the release of distinct pools of secretory granules. This raises the interesting question of how the secretory granules can distinguish between Ca2+ entering the cell in response to the two different stimuli. Factors that may be relevant in this context include the nature of the electrical stimulus  as well as biochemical effects influencing the release probability of the secretory granules.Collectively, the observations summarized above suggests that insulin secretion evoked by membrane depolarization alone  triggered by glucose.The existence of biochemically distinct population of secretory granules is supported by the recent and elegant FRET-based studies by Takahashi et al. 72+-channels  open. In mouse α-cells, L-type Ca2+-channels mediate most of the Ca2+-entry during the action potentials but glucagon secretion evoked by low glucose alone is almost resistant to isradipine or nifedipine 2+-channels, whilst having only a weak effect on [Ca2+]i, inhibits glucagon secretion as strongly as elevation of glucose to >6 mM 2+-channel activity is completely suppressed. In this context it may be pertinent that α-cells in freshly isolated mouse islets are equipped with voltage-gated Na+-channels and that the Na+-current amplitude is 5–10-fold larger than all the Ca2+-currents together. During these action potentials, non-L-type Ca2+-channels may still open and trigger exocytosis of the glucagon vesicles. However, it remains obscure why Ca2+-entry via L-type Ca2+-channels is such a poor initiator of glucagon secretion although L-type Ca2+-channels outnumber non-L-type Ca2+-channels by a factor of four. Perhaps the simplest explanation is that L- and non-L-type Ca2+-channels are spatially segregated so that their active zones do not overlap  be expected to have dramatic effects on glucagon secretion. This explains why tolbutamide inhibits glucagon secretion from both mouse and human islets +-channels) to a marked reduction of spike height 2+-channels do not activate sufficiently to trigger exocytosis  promotes Ca2+-induced Ca2+-release (CICR) from intracellular Ca2+ stores. We speculate that the resulting global elevation of [Ca2+]i within the α-cell triggers exocytosis of any release-competent secretory granules regardless of their proximity to the Ca2+-channels  were analyzed. Likewise, there were no signs of increased β-cell death or reduced insulin content/biosynthesis that would explain the reduction of glucose-induced insulin secretion With the fairly detailed understanding of the stimulus-secretion coupling of the pancreatic β-cell we thought it would be straightforward to pinpoint the changes produced by long-term exposure to FFAs such as palmitate and oleate. Surprisingly, β-cells exposed to FFAs for 72 h performed better than control cells not exposed to FFAs when key functional parameters like glucose metabolism (ATP production), K2+-entry. Whereas depolarization-induced Ca2+-entry is concentrated to discrete areas in control cells  depolarizations. Thus, exocytosis in response to 50-ms depolarizations was strongly reduced. By contrast, consistent with our earlier data ol cells B, it becol cells C. Becaus β-cells D–E. Howe+-channel blocker TEA. This compound blocks the K+-channels involved in action potential repolarization and thereby extends the duration of the action potentials. This allows the Ca2+-channels to stay open longer (mimicking the effects of long voltage-clamp depolarizations), resulting in a more global elevation of [Ca2+]i than in response to brief action potentials. Thus, Ca2+-channel dispersion may underlie the suppression of glucose-induced insulin secretion by long-term FFA exposure also in human islets.Long-term FFA exposure also interfered with glucose-stimulated insulin secretion from human islets. In both mouse and human islets 2+-channel dissociation may be particularly strong in the β-cell because it operates with so few Ca2+-channels. In cells with a higher Ca2+-channel density (like chromaffin cells), dispersion of the Ca2+-channels and dissociation of the granule/Ca2+-channel complexes can be expected to have a much smaller effect because the secretory granules will almost always be situated in the close vicinity of one or several voltage-gated Ca2+-channels.The functional consequences of Ca2+-channel dispersion in response to long-term FFA exposure? We propose that this provides the β-cells with a means to suppress insulin secretion during fasting/starvation. Under these conditions, plasma FFA increases to >1 mM whilst plasma glucose, by activation of glucogenic mechanisms, is maintained >3.5 mM What is the physiological significance of Ca2+]i remains unknown. However, work on β-cells from GK rats, a model of non-obese human T2D with elevated plasma FFA levels 2+]i and only subtle changes in exocytosis manifested as a ∼50% reduction of the Ca2+-sensitivity of exocytosis 2+-channels we observe following long-term FFA exposure can be expected to result in an apparent reduction of the Ca2+-sensitivity of exocytosis but whether this underlies the diminished insulin secretion in GK-rats (and in islets from T2D donors) remains to be demonstrated experimentally.Because of limited availability of islets from donors with type-2 diabetes (T2D), the impact type-2 diabetes on β-cell [Ca92+-signalling in pancreatic α- and β-cells. Such studies may reveal processes that are key to the reciprocal glucose-sensing in the two cell types. It is also important to note that there are numerous and important differences between human and rodent islet cells. This illustrates the potential pitfalls of uncritically extrapolating observations made in rodent systems to the situation in man. Finally, there appears to be some truth to the adage that “you are what you eat”, at least when it comes to Ca2+-signalling in the β-cell. As we have discussed above, diets that lead to protracted exposure of the β-cells to FFAs may exert adverse effects on the β-cell's capacity to secrete insulin in response to glucose.It is rewarding to compare CaThe authors declare that there are no conflicts of interest.


Recent research has spotlighted the role of microRNAs (miRNAs) as critical epigenetic regulators of hematopoietic stem cell differentiation and leukemia development. Despite the recent advances in knowledge surrounding epigenetics and leukemia, the mechanisms underlying miRNAs’ influence on leukemia development have yet to be clearly elucidated. Our aim was to identify high ranking biological pathways altered at the gene expression level and under epigenetic control. Specifically, we set out to test the hypothesis that miRNAs dysregulated in acute myeloid leukemia (AML) converge on a common pathway that can influence signaling related to hematopoiesis and leukemia development. We identified genes altered in AML patients that are under common regulation of seven key miRNAs. By mapping these genes to a global interaction network, we identified the “AML Signalisome”. The AML Signalisome comprises 53 AML-associated molecules, and is enriched for proteins that play a role in the aryl hydrocarbon receptor (AhR) pathway, a major regulator of hematopoiesis. Furthermore, we show biological enrichment for hematopoiesis-related proteins within the AML Signalisome. These findings provide important insight into miRNA-regulated pathways in leukemia, and may help to prioritize targets for disease prevention and treatment. In the past decade, microRNAs (miRNAs) have emerged as major players in cellular regulation, especially related to carcinogenesis. The expression profiles of miRNAs can be used to classify tumor types and tumor differentiation state with high levels of accuracy . HematolCaenorhabditis elegans in 1993 [The first miRNA was discovered in  in 1993 . In 2001 in 1993 ,7. Now s in 1993 . By base in 1993 . BecauseResearch involving epigenetics has recently focused on the study of cancers with high prevalence and/or mortality rates. For example, leukemia is prevalent in the United States, where it is estimated that 1 in 75 people will be diagnosed during their lifetime . Acute mThe mechanisms underlying leukemogenesis often involve changes in cellular signaling, which can inhibit hematopoietic progenitor cells from: (i) responding to normal signals regulating proliferation, and/or (ii) differentiating into mature red blood cells, monocytes, neutrophils, and platelets . Many paet al. [Since the initial miRNA tumor classification studies , many otet al. . Some stet al. .versus non-leukemia cells.We hypothesize that miRNAs likely act in a concert to exert their biological functions. More specifically, we propose that the coordinated action of miRNAs on their transcriptional targets may influence key biological pathways involved in leukemogenesis. A recent paper  in whichWhile previous studies have identified associations between individual miRNAs and leukemia-related gene expression profiles, we employed a systems-level approach to identify converging pathways regulated by several key AML-associated miRNAs. We predicted that the statistical integration of existing databases, along with systems-level pathway analyses, would reveal high ranking biological pathways altered at both the gene expression and epigenetic  level in AML patients. Thus, this study’s goal was to elucidate novel biological relationships that may underlie leukemogenesis.et al., where microarray-based classifiers were identified to predict the risk of AML transformation in patients with myelodysplastic syndrome [In order to identify a genomic signature associated with AML, we performed a statistical analysis comparing gene expression levels between AML patients and non-leukemic individuals. Specifically, a publically available database containing microarray data from patients with AML (n = 202), myelodysplastic syndrome (n = 164), or from non-leukemic, control patients (n = 69) was downloaded from the National Center for Biotechnology Information’s Gene Expression Omnibus . This dasyndrome . Of the syndrome ,23.versus non-leukemic patients, where three statistical requirements were set: (i) a fold change of ≥2.5 or ≤−2.5 (AML versus non-leukemic); (ii) p value <0.01 (ANOVA); and (iii) a false discovery rate corrected q-value <0.01. Analysis of variance (ANOVA) p values were calculated using Partek® Genomics SuiteTM software, version 6.5 [Total RNA from bone marrow mononuclear cells was extracted and hybridized to microarrays, as described previously ,24. Genesion 6.5 . To contsion 6.5 . This crsion 6.5 ,28, wherversus normal cells and their relevance to leukemia development. The studies included were Bousquet et al. [et al. [et al. [et al. [et al. [et al. [A set of miRNAs modulated in AML patients was identified through an extensive literature review. The miRNAs were selected based on their altered expression levels in AML t et al. , Cammara [et al. , Garzon  [et al. , Han et  [et al. , O’Conne [et al. , and Wan [et al. .CT) ≥0.5. This PCT filter controlled for background conservation across mammals by accounting for mutational biases, dinucleotide conservation rates, and individual untranslated region conservation rates [In order to understand the effects that the AML-associated miRNAs (n = 7) may cause at the gene expression level, computational predictions of the transcriptional targets of the AML-associated miRNAs were carried out. Here, TargetScanHuman  algorithon rates .p values <0.05 were considered significant. In a similar fashion, functional enrichment analysis was performed to identify biological functions and disease signatures significantly associated with the AML-associated gene signature predicted to be regulated by AML-associated miRNAs. Significance was calculated in the same manner as the pathway enrichment analysis, where functions with enrichment p values <0.0001 were considered significant.Network analysis was performed to identify biological pathways that are targets for miRNA-mediated control in AML. For this analysis, a list of AML-associated genes predicted to be targeted by at least one of the AML-associated miRNAs was overlaid onto a global interaction network. Networks were algorithmically constructed based on connectivity, as enabled through Ingenuity Pathway Analysis . CanonicIn this study, we predicted that miRNAs act together to control genomic signaling of biological pathways associated with AML. To test this hypothesis, we analyzed genomic data from individuals with or without AML, and integrated the genomic data with miRNAs and their transcriptional targets in a six step process . Throughet al. [versus non-leukemic patients , v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), runt-related transcription factor 1 (RUNX1), and Wilms tumor 1 (WT1).To identify a set of genes differentially expressed in AML patients relative to non-leukemic patients, we performed a statistical analysis using publically available data. Data from Mills et al.  were usepatients . As the . report  and are A comparison between the AML-associated gene signature and a previously published set of AML-related genes was also performed. The gene set to which we compared the AML-associated gene expression signature consisted of 66 genes that have been identified as accurate predictors of overall survival for AML patients . Given tversus non-leukemic samples [Based on previous studies that have highlighted the changes in sets of miRNAs, we hypothesized that miRNAs likely act in a coordinated manner to mediate their regulatory effects through key biological pathways. Thus to identify miRNAs known to be altered in expression in AML patients, a thorough literature review was conducted. A total of seven AML-associated miRNAs were identified, consisting of miRNAs with significant differential expression in blood and/or bone marrow samples collected from AML patients in comparison to non-leukemic patients. To detail, miR-125b, miR-126, miR-142-3p, miR-155, miR-223, miR-29a, and miR-29b have all shown altered expression levels in AML samples  samples ,32,33,34 samples ,42,43,44To understand genomic changes that occur in AML patients likely via changes in miRNA expression, we computationally predicted transcriptional targets of the seven AML-associated miRNAs. Using seed match-based algorithms, 196 genes were predicted to be targeted by miR-125b, 22 by miR-126, 308 by miR-142-3p, 164 by miR-155, 68 by miR-223, 805 by miR-29a, and 179 by miR-29b. In total, 1,589 unique genes were predicted to be targeted by at least one of the seven AML-associated miRNAs  to the 1,589 genes predicted to be targeted by AML-associated miRNAs . This comparison showed that 78 genes contained in the AML-associated gene signature are likely regulated by AML-associated miRNAs  sub-networks were constructed . AhR pathway signaling is also present in the most significant (p < 10−44) sub-network within the AML Signalisome , cancer (p = 3 × 10−6), hematological system development and function (p = 3 × 10−6), and hematopoiesis (p = 3 × 10−6).To further establish the biological relevance of our findings, we performed a biological function and disease signature enrichment analysis. Here, 18 functions or disease signatures were identified as significantly associated with the AML Signalisome  with altered expression levels in AML patients are likely targets of the seven key AML-associated miRNAs.In order to understand epigenetic regulation in leukemia, we next identified miRNAs that are dysregulated in AML patients and predicted their transcriptional targets. Here, miRNAs were selected if they have shown significant differential expression in AML The percentage of genes likely regulated by AML-associated genes may seem low, as mammalian miRNAs are estimated to regulate more than 60% of all protein-coding genes . FurtherWe set out to determine whether the miRNA-regulated AML-associated genes interact and uncovered the AML Signalisome. This AML Signalisome was identified based on molecular interactions between proteins encoded by the 78 genes within the miRNA-mediated AML gene signature. The resulting AML Signalisome includes network interactions of molecules whose transcription are altered in AML cells. The AML Signalisome clearly illustrates that molecular signaling altered in AML cells is likely influenced by the coordinated actions of multiple miRNAs acting on common pathways. This finding provides novel evidence supporting our prediction that miRNAs likely act together to regulate important pathways altered in AML cells.Within the AML Signalisome, there is a significant enrichment for genes involved in AhR signaling, as well as functional enrichment for cellular development, cancer, hematological system development/function, and hematopoiesis. In particular, all seven AML-associated miRNAs, miR-125b, miR-126, miR-142-3p, miR-155, miR-223, miR-29a, and miR-29b, were predicted to regulate the most significant sub-network within the AML Signalisome containing AhR pathway signaling. Interestingly, the AhR pathway is known to play a major role in the regulation of hematopoiesis, where altered AhR signaling can contribute to hematological disorders, including leukemia . AlthougAHR promoter region is frequently hypermethylated in acute lymphoblastic leukemia [Altered AhR signaling, potentially regulated via AML-associated miRNAs, is of high significance and may represent a key mechanism potentially underlying leukemogenesis. It is important to note that the AhR pathway is a key regulator of stress and xenobiotic response . As suchleukemia . To our Other pathways besides AhR signaling have been identified as altered through miRNA regulation in AML. For example, miR-29b has been shown to influence signaling related to NFκB, Sp1 transcription factor (SP1), and histone deacetylase (HDAC) . In addiOur study assessed miRNA-mediated pathway interactions that are altered in AML patients. We identified that multiple miRNAs likely act together to regulate key biological pathways altered in AML. The implications of this finding suggest that AML targeted therapies focusing on single miRNAs may prove ineffective in some cases. Our data suggests that targeting of multiple miRNAs or their downstream pathways may be more effective. Future research will further evaluate transcriptomic changes induced by multiple AML-associated miRNAs, while considering AML patient subtypes as well as potential cytogenetic abnormalities. Understanding the epigenetically modified pathways underlying AML progression is extremely important, as increased pathophysiological understanding will foster the development of new effective therapies.We used a systems-level approach to identify AML-associated biological pathways targeted for transcriptional control by a set of miRNAs. We found that AML-associated transcripts targeted by critical miRNAs interact in a highly significant manner. These interactions, when combined, form the AML Signalisome. Within this AML Signalisome, there is a significant enrichment for proteins of the AhR pathway and within hematopoiesis-related functions. These findings have implications for understanding biological pathways perturbed in disease that may inform targeted therapeutic strategy.


Compared to immature pre-hearing HCs (E10-E12), mature post-hearing HCs (E18-P2) can steadily mobilize a larger readily releasable pool (RRP) of vesicles with faster kinetics and higher Ca2+ efficiency. As assessed by varying the inter-pulse interval of a 100 ms paired-pulse depolarization protocol, the kinetics of RRP replenishment were found much faster in mature HCs. Unlike mature HCs, exocytosis in immature HCs showed large depression during repetitive stimulations. Remarkably, when the intracellular concentration of EGTA was raised from 0.5 to 2 mM, the paired-pulse depression level remained unchanged in immature HCs but was drastically increased in mature HCs, indicating that the Ca2+ sensitivity of the vesicle replenishment process increases during maturation. Concomitantly, the immunoreactivity of the calcium sensor otoferlin and the number of ribbons at the HC plasma membrane largely increased, reaching a maximum level at E18-P2. Our results suggest that the efficient Ca2+-dependent vesicle release and supply in mature HCs essentially rely on the concomitant engagement of synaptic ribbons and otoferlin at the plasma membrane.Auditory hair cells (HCs) have the remarkable property to indefinitely sustain high rates of synaptic vesicle release during ongoing sound stimulation. The mechanisms of vesicle supply that allow such indefatigable exocytosis at the ribbon active zone remain largely unknown. To address this issue, we characterized the kinetics of vesicle recruitment and release in developing chick auditory HCs. Experiments were done using the intact chick basilar papilla from E10 (embryonic day 10) to P2 (two days post-hatch) by monitoring changes in membrane capacitance and Ca Remarkably, this synapse can drive postsynaptic auditory nerve fibers at extremely high instantaneous discharge rates  and, after rapid adaptation, can support sustained discharge rates over several hundred spikes/s during ongoing sound stimulation Notably, the first sound-evoked responses of immature auditory nerve fibers in the developing cochlea display high thresholds of rhythmic bursting activity and are unable to maintain a sustained steady-state response to long duration tone bursts th, 1986 (86/609/EEC) and the University of Bordeaux  permit number B 33075, approved this study). The study included chickens at different stages of embryonic development ranging from E10-E21 as well as two-day post-hatched chickens. Fertilized eggs were incubated at 37°C in a Marsh automatic incubator . Chicken embryos were sacrificed and staged according to their number of somites, and additionally for the late stages as following: from E8-E12 based on visceral arches, feather gems and eyelids: after E12 based on the length of the beak 2, 2 MgCl2, 5 Hepes, and 3 glucose, pH 7.4. The tegmentum vasculosum and the tectorial membrane were removed without any prior enzymatic treatment using a fine minutia needle. Chicken basilar papillae were stored in a 37°C incubator in Minimum Essential Medium (Invitrogen) before recordings from HC in situ. All experiments were performed at room temperature (21–23°C) within 5–45 min of isolation. All reagents were obtained from Sigma Chemicals, unless otherwise specified. Recordings were done in neural  HCs along the basilar papilla. These HCs which are mainly innervated by the afferent fibers correspond to the inner hair cells in mammals. The tonotopic location of HCs was divided in three parts. From the proximal narrow end of the papilla, the first 1/3 part was considered as the high frequency coding region of basal HCs. Low frequency apical HCs were recorded at the top 1/3 part of the wide end of the basilar papilla.The present investigation was performed in accordance with the guidelines of the Animal Care and Use Committee of the European Communities Council Directive of November 24Calcium currents were recorded in whole-cell voltage-clamp configuration using 3–5 MΩ resistance pipettes. Currents were recorded with an EPC 10 amplifier  and filtered at a frequency of 2–5 kHz through a low-pass Bessel filter. The sampling frequency was determined by the protocol used. No online leak current subtraction was made. Only recordings with holding current less than 20 pA were accepted for analyses.m) were recorded using the EPC 10 amplifier. A 2 kHz sine wave of 10 mV was applied to the cells from a holding potential of -90 mV. Capacitance (Cm) signals were low-pass filtered at 80 Hz. Changes in membrane capacitance were measured 0.05–0.5 s after the end of the depolarizing pulse and averaged over a period of 0.2–2 s. Membrane and series resistance (Rm and Rs) were monitored during the course of the experiment. Only recordings with stable Rm and Rs were considered for further analysis. The study included ∼247 cells with Rs within the 5–20 MΩ range. Holding membrane potentials were corrected for liquid junction potentials. Extracellular solution for measuring Ca2+ currents contained (in mM) NaCl/CholineCl 125, KCl 6, CaCl2 5, 25 TEA, 5 4-AP, D-glucose 10, MgCl2 1, HEPES 10, pH 7.3, 310 mOsm. TEA and 4-AP were present in the external medium to block residual inward rectifying K+-currents that may contaminate the measured inward Ca2+ current. Intracellular solution contained (in mM) NMG 75, CsCl 70, Na2ATP 5, MgCl2 2, HEPES 10, EGTA 0.5–10, and glucose 10; pH 7.3, 300 mOsm.Real-time changes in membrane capacitance  for ∼3 hrs, then rinsed and immunostained with a polyclonal antibody directed against mouse CtBP2  and a monoclonal HCS1-antibody  were obtained from fits using Origin software. Time constants were obtained by fitting multiple exponential equations to the activation decay of the current. The equation was of the form:0 is the initial current magnitude, τ1, τ2...τn are the time constants, and A1, A2...An, are the proportionality constants. Synaptic transfer functions relating Ca2+ current (ICa) and ΔCm, or QCa2+ and ΔCm were calculated using an integral of total ICa, including the tail currents. The data was fitted using first-order power functions: The number of cells (n) is given with each data set. Data were analyzed using pClamp10 (Axon Instruments) and Origin7.0 . Pooled data were presented as mean ± SD. Significant difference between groups of cells or between different embryonic stages of development was evaluated using a two-tailed Student's t test; m = ΔCm measured using the first, control pulse, and ΔCm, test = ΔCm measured using test pulse.The % RRP refilling was calculated as:recovered was calculated as:Ca, control, = ICa measured using the first, control pulse, and ICa, test = ICa, test measured using test pulse.The % of I2+-evoked exocytosis was characterized at four developmental periods of cochlear synaptogenesis: embryonic stages (in ovo) E10–11, E12–14, E16–18 and 2 days post-hatching P2. The first embryonic period, E10–11, corresponds to an early stage of synaptogenesis when the first presynaptic specializations (synaptic bodies or ribbons) can be detected in HCs The efficiency of CaCa) and a concomitant increase in membrane capacitance (ΔCm) was recorded when HCs were voltage-stepped from -90 mV to varying depolarized potentials (m displayed a bell shape that followed the ICa activation curve (maximum amplitude near -10 mV), a behavior consistent with ΔCm being activated consecutive to Ca2+ influx. Indeed, complete blockage of ICa by 250 µM Cd2+ eliminated ΔCm (data not shown), confirming that ΔCm is sensitive to Ca2+ entry via VGCC, in agreement with m responses increased with HC maturation (m responses were as follows: (in fF at -10 mV) E10, 10±2 (n = 10); E12, 28±6 (n = 13); E16, 37±7 (n = 11); and P2, 45±6 (n = 9). High frequency HCs recorded at the base of the basilar papilla were also found to undergo a similar increase in ΔCm responses with maturation , 100-ms voltage-step depolarization did not produce significant ΔCm responses in all HCs tested .At all developmental stages from E10 to P2, rapidly activating inward current  was compared at different developmental stages by stepping the cells to various potentials from -60 to -10 mV for a constant 100 ms duration   ; table 1p<0.001, p<0.001; 2+ buffer EGTA from 0.5 (estimated buffer space of ∼ 1000 nm) to 2 mM (estimated buffer space of ∼ 200 nm from the point of Ca2+ source) 2+ entry, the SRP was largely reduced by 2 mM EGTA  for different durations from 20 to 3000 ms . Data po mM EGTA .1 = 0.8±0.1 and τ2 = 6.1±1.4 (n = 5); E16: best fit with a single exponential function τ = 0.7 ±0.1 (n = 5), p<0.05; Ca in immature E12 HCs as compared to mature P2 HCs. The kinetics of RRP recovery in E12 HCs paralleled the time course of ICa recovery , p<0.001; 2+ tail currents below this time frame.Auditory HCs must be able to quickly replenish the RRP to sustain neurotransmitter release recovery , indicat2+-sensitive : 0.5, 95±2 (n = 4); 2, 10±2, (n = 4), p<0.001), 2+-sensitive. The novelty of our findings is that this vesicle supply process in immature HCs and mature HCs shows a different Ca2+ sensitivity.Next, we compared the rate of vesicle supply to the RRP when using an intracellular recording solution containing 2 mM EGTA instead of 0.5 mM . Surprisensitive . By contm responses showed marked depression in immature developing HCs, as indicated by a progressive decrease in ΔCm responses during the repetitive stimuli: In basal high frequency HCs, ΔCm decreased from a mean of 27±4 fF after the first stimulus to 10±3 fF after the 20th one at E12  from RRP remained constant during the repetitive stimulations  at E8, 2.1±1.6 (n = 50) at E12, 3.1±1.4 (n = 50) at E16 and 9.3±2.2 (n = 50) at P2  at P2 . These rWe then used the recently characterized monoclonal antibody HCS-1 in ovo2+ efficiency with maturation. Similar changes have been shown in HCs of pre-hearing animals such as mouse and gerbil 2+-dependent in mature chick HCs, in agreement with previous findings 2+-dependent in turtle auditory HCs This report characterizes the functional changes occurring during progressive maturation of the HC synaptic machinery in a precocial post-hearing vertebrate, the chick, where sound-evoked cochlear nuclei activity can be measured as early as E11 2+ current at this developmental stage. Indeed, the Ca2+ current and the RRP showed similar kinetics of recovery during paired-pulse stimulations. Notably, while the Ca2+ current of mature chick HCs is mainly driven by non-inactivating dihydropyridine-sensitive L-type Ca2+ channels 2+ channels, 2+ currents of immature HCs display strong calmodulin-mediated calcium-dependent inactivation 2+ current inactivation leading to RRP depression could partially explain the transient rhythmic temporal discharge pattern of the auditory nerve fibers observed in young kittens Remarkably, immature chick HCs displayed significant depression in exocytosis during repetitive brief stimuli or paired-pulse stimulation, while mature HCs showed little RRP depression. This exocytotic depression in immature HCs is likely due in part to the rapid inactivating property of the Ca2+ entry, while the reloading sites extend farther than 200 nm. Similar Ca2+ regulation of vesicle replenishment has been shown at the cone ribbon synapses of the retina 2+2+ entry during development. A larger and wider extrasynaptic distribution of Ca2+ channels at the early stage of development, as shown in immature mouse HCs 2+ entry closer to the refilling machinery (reserve pool of vesicles) in immature HCs and in turn make vesicle replenishment less sensitive to EGTA. Notably, we found a positive correlation between the increasing number of ribbons with maturation and the efficiency of vesicle supply and release. Our results obtained in developing HCs are in good agreement with those obtained in a recent study using transgenic mice lacking the presynaptic scaffold protein bassoon, an essential element to dock the ribbon to the active zone 2+ channels and vesicles in the synaptic active zone in order to promote efficient vesicle replenishment.In agreement with 2+-dependent vesicle supply may also progressively take place in mature HCs. Otoferlin, which is considered to be a high affinity Ca2+ sensor that directly triggers SNARE-membrane fusion in vitro2+-evoked membrane fusion at the ribbon synapse of both cochlear 2+-dependent vesicle supply at a large distance from Ca2+ entry. Indeed, we found that the expression of otoferlin increases at the right period of cochlear maturation when vesicle replenishment becomes efficient, suggesting a progressive engagement of this Ca2+ sensor in the ribbon active zone. By progressively replacing other Ca2+ sensors such as synaptotagmins during development In addition to an increased number of ribbons during maturation, the organization of a different Ca2+-sensitive vesicle supply and otoferlin expression, suggests that this multi-C2 protein may also act as a Ca2+ sensor for the recruitment of vesicles located far from the release sites and Ca2+ channels, and probably farther than 200 nm as suggested by its sensitivity to 2 mM EGTA. This hypothesis is also reinforced by the phenotype of the pachanga mouse model, which carries a missense mutation in the C2F domain of otoferlin, and where the replenishment process of synaptic vesicles is affected independently of RRP fusion Contrary to mature auditory HCs, the ribbon synapses of the retina do not express otoferlin


EIA (Equine Infectious Anemia) is a blood-borne disease primarily transmitted by haematophagous insects or needle punctures. Other routes of transmission have been poorly explored. We evaluated the potential of EIAV (Equine Infectious Anemia Virus) to induce pulmonary lesions in naturally infected equids. Lungs from 77 EIAV seropositive horses have been collected in Romania and France. Three types of lesions have been scored on paraffin-embedded lungs: lymphocyte infiltration, bronchiolar inflammation, and thickness of the alveolar septa. Expression of the p26 EIAV capsid (CA) protein has been evaluated by immunostaining. Compared to EIAV-negative horses, 52% of the EIAV-positive horses displayed a mild inflammation around the bronchioles, 22% had a moderate inflammation with inflammatory cells inside the wall and epithelial bronchiolar hyperplasia and 6.5% had a moderate to severe inflammation, with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the pulmonary parenchyma. Changes in the thickness of the alveolar septa were also present. Expression of EIAV capsid has been evidenced in macrophages, endothelial as well as in alveolar and bronchiolar epithelial cells, as determined by their morphology and localization. To summarize, we found lesions of interstitial lung disease similar to that observed during other lentiviral infections such as FIV in cats, SRLV in sheep and goats or HIV in children. The presence of EIAV capsid in lung epithelial cells suggests that EIAV might be responsible for the broncho-interstitial damages observed. Equine Infectious Anemia Virus (EIAV) is a retrovirus belonging to the lentivirus genus, which infects equids worldwide (reviewed in ). PersisEIAV infection is endemic in horse populations throughout the world  includinin vivo, supporting active viral replication during clinical and subclinical infection  formula taking into account the severity of (peri)bronchiolar inflammation [B] and thickness of the alveolar septa [C] as previously reported , 83% of the EIAVPOS horses developed mild to severe lung lesions defined by a score above 0.4. In contrast, only 21% of animals from the EIAVNEG group had mild lesions with a score under 1.5, with no animal with moderate or severe lesions bronchiolar inflammation and thickness of the septa were present in most EIAVes Table . Only miPOS-horses (10.13%) .In order to associate the observed lesions with the expression of the EIAV viral protein, immunostainings for the p26 capsid protein using a monoclonal anti-p26 antibody have been performed on lung sections from EIAVThe p26 capsid was expressed in the infiltrated mononuclear cells within the alveolar septa Figure A and intWe explored the potential of EIAV to induce pulmonary lesions in naturally infected equids from 79 EIAV-seropositive horses collected in Romania and France. We scored three types of lesions on paraffin-embedded sections: lymphocyte infiltration, (peri)bronchiolar inflammation, and thickness of the alveolar septa and analyzed the expression of the p26 EIAV capsid protein. Compared to EIAV-negative horses, over 80% of the EIAV-positive animals displayed lesions of interstitial lung disease, with an histological score above 0.4 varying from from mild inflammation around the bronchioles, to moderate inflammation with inflammatory cells inside the walls and epithelial bronchiolar hyperplasia or severe inflammation with destruction of the bronchiolar epithelium and accumulation of smooth muscle cells within the lung parenchyma. The lesions were often localized sub-pleurally concomitantly with metaplasia of the alveolar epithelium, suggesting a chronic bronchiolitis. Interestingly, the p26 EIAV capsid was strongly expressed in mononuclear cells, endothelial cells as well as alveolar and bronchiolar epithelial cells.POS horses as evidenced by microscopic examination of lung parenchyma and immunostaining for the expression of α-smooth muscle actin. Lentivirus infection of smooth muscle cells may play a role in the induction of pathogenic events as reported for myomatosis in the lung of SRLV infected sheep [Smooth muscle proliferation or myomatosis was present in over 10% of the EIAVed sheep ,13, primed sheep -16, or ied sheep ,18. FromTaking all together, the observed lesions resemble the lung lesions associated with LIP , a lung disease described during lentivirus-infection, and reported in child infected by HIV-1 , cats inIn vivo, equine infectious anemia virus (EIAV) replicates in macrophage-rich tissues [We observed EIAV capsid expression in mononuclear cells as well as in endothelial cells.  tissues ,30,31 an tissues . Infecti tissues . Our res tissues , due to  tissues . Except  tissues , little  tissues ,36. Chro tissues , needs tIn conclusion, we described an interstitial lung disease in EIAV-infected horses associated with capsid-expression in macrophages, endothelial as well as in mature epithelial cells of the distal lung. The detection of infected pulmonary cells and of lesions consistent with lentivirus-induced interstitial lung disease opens the hypothesis of a virus transmission through aerosolized particles as reported for other retroviruses, such as SRLV or JSRV.The authors declare that they have no competing interests.PB collected most tissues, carried out the histopathological analysis and the immunostainings, drafted the manuscript; MN carried out the immunostaining and tissue analysis; JLC participated to the result analysis and to the writing; CC participated to the tissues collection and to the histopathological analysis, FA carried out the fluorescent microscopy observations and helped to draft the manuscript, CD carried out the immunostaining and Western blot analysis; JFM participated to the design, to the interpretation of the results and their analysis and to the final version of the manuscript; CL conceived the study, elaborated its design, coordinated the work and wrote the manuscript. All authors read and approved the final manuscript.


Here we evaluated the function of the R4892W and G4896V RyR1 mutants, both associated with central core disease (CCD) in humans, in myotubes and in adult muscle fibers. For both mutants expressed in RyR1-null (dyspedic) myotubes, voltage-gated Ca2+ release was absent following homotypic expression and only partially restored following heterotypic expression with wild-type (WT) RyR1. In muscle fibers from adult WT mice, both mutants were expressed in restricted regions of the fibers with a pattern consistent with triadic localization. Voltage-clamp-activated confocal Ca2+ signals showed that fiber regions endowed with G4896V-RyR1s exhibited an ∼30% reduction in the peak rate of SR Ca2+ release, with no significant change in SR Ca2+ content. Immunostaining revealed no associated change in the expression of either α1S subunit (Cav1.1) of the dihydropyridine receptor (DHPR) or type 1 sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1), indicating that the reduced Ca2+ release resulted from defective RyR1 function. Interestingly, in spite of robust localized junctional expression, the R4892W mutant did not affect SR Ca2+ release in adult muscle fibers, consistent with a low functional penetrance of this particular CCD-associated mutant.The large and rapidly increasing number of potentially pathological mutants in the type 1 ryanodine receptor (RyR1) prompts the need to characterize their effects on voltage-activated sarcoplasmic reticulum (SR) Ca As a result, SR Ca2+ release is under tight control by the potential across the T-tubular membrane. The fact that several types of muscular disorders  result from either inherited or de novo mutations in the RYR1 gene underscores the critical role that proper RyR1 Ca2+ release channel function plays in coordinating skeletal muscle performance  mice have revealed that mutations associated with a CCD phenotype that do not increase susceptibility for malignant hyperhermia (MHS) result in reduced SR Cain vivo electroporation of cDNA constructs encoding green fluorescent protein (GFP)-tagged RyR channels provides an alternative approach to characterize the functional properties of the expressed channel in fully-differentiated adult skeletal muscle fibers 2+ release in fibers expressing MHS or CCD associated RyR1 mutants, respectively Although knock-in mouse models provide an incredibly powerful means for elucidating the primary defect of RyR1 disease mutations on skeletal muscle function, it is not possible to employ this approach for each of the more than 300 disease-associated RyR1 variants. We recently demonstrated that 2+ release when co-expressed with WT RyR1 in dyspedic myotubes, voltage-gated Ca2+ release was significantly reduced only for the G4896V mutant following transient expression in muscle fibers of adult WT mice.In the present work, we used this approach to explore the functional properties of two CCD-associated RyR1mutants for which there is yet no data available in adult muscle fibers. These two CCD RyR1 mutants (R4892W and G4896V – numbered according to rabbit RyR1 sequence) affect residues lying either within (G4896V) or immediately adjacent (R4892W) to the RyR1 selectivity filter. The corresponding mutations in the human RyR1 sequence are R4893W and G4897V, respectively. In order to avoid confusion, numbering according to the rabbit RyR1 sequence (i.e. R4892W and G4896V) is used throughout rest of this study. We found that while both mutants markedly reduce voltage-gated CaExperiments and procedures were conducted in accordance with the guidelines of the local animal ethics committee of the University of Rochester (2006–114R), University Lyon 1, of the French Ministry of Agriculture (87/848) and of the European Community (86/609/EEC); they were specifically approved by the animal ethics committes of University Lyon 1 and of University of Rochester.The two identified RYR1 mutants (R4892W and G4896V) were introduced into both non-tagged and N-terminal EGFP-tagged versions of a full-length rabbit RYR1 complementary DNA (cDNA) (accession #X15750) using standard two-step site-directed mutagenesis. All sequences generated and modified by polymerase chain reaction were checked for integrity by sequence analysis. Myotubes were prepared from primary cultures of myoblasts obtained from skeletal muscle of newborn RYR1-null (dyspedic) mice as described previously 2+ measurements were obtained from Indo-1 AM-loaded myotubes as described previously 2, 1 MgCl2, 10 HEPES, pH 7.4. Cytosolic dye within a small rectangular region of the expressing myotube was excited at 350±10 nm, fluorescence emission at 405±30 nm (F405) and 485±25 nm (F485) was measured at a 100 Hz sampling frequency with a photomultiplier detection system , and results were presented as the ratio of 405 and 485 nm (F405/F485). A standard excitation-contraction (EC) uncoupling screening protocol was used to sequentially measure electrically-evoked Ca2+ release, agonist-induced RyR1 Ca2+ release and total SR Ca2+ content. This consisted of applying, via a local perfusion system, 30 seconds of control Ringer's solution followed by 10 electrically-evoked release events (1Hz), 10 seconds of Ringer's solution, 30 seconds of 500 µM 4-chloro-m-cresol (4-CMC), 30 seconds of Ringer's solution and 30 seconds of a Ca2+ store release cocktail consisting of 10 µM Ionomycin, 30 µM Cyclopiazonic acid (CPA) and 100 µM EGTA (ICE) in a Ca2+ free Ringer solution (in mM): 145 NaCl, 5 KCl, 3 MgCl2, 10 HEPES, pH 7.4. Peak changes in intracellular Ca2+ were expressed as ΔRatio (Ragonist – Rbaseline). The peak value during 4-CMC application was always taken as the maximum value of the change in indo-1 ratio, whether this occurred during an oscillation or a slowly rising transient. The response to the ICE cocktail was previously shown to be a sensitive index of the SR Ca2+ store capacity with negligible contamination of Ca2+ from outside as the signal is strongly reduced by prior partial SR store depletion with a low concentration of CPA 2+Intracellular Caflexor digitorum brevis (fdb) and interosseus muscles of 6–8 week-old Swiss OF1 male mice using a procedure described previously −1) using a commercial delivery system . Twenty five microliters of a solution containing 2 mg/ml hyaluronidase dissolved in sterile saline was then injected into the footpads of each hind paw. Forty minutes later the mouse was re-anaesthetized by isoflurane inhalation. Plasmid DNAs (EGFP-G4896V or EGFP-R4892W) were then injected into the footpads of the animal at a concentration of 1.5 µg/µl in standard Tyrode solution. A 20 µl total volume of this solution was injected in different locations so as to target both the fdb and the interosseus muscles. Following the injection, two gold-plated stainless steel acupuncture needles connected to the electroporation apparatus were inserted under the skin, near the proximal and distal portion of the foot, respectively. The standard protocol used consisted in 20 pulses of 110 V/cm amplitude and 20 ms duration delivered at a frequency of 2 Hz by a BTX ECM 830 square wave pulse generator . Experimental observations and measurements were carried out 2 weeks later.Exogenous expression of RyR1 mutants by electroporation was performed in the fdb and interosseus muscles using a procedure described previously 2 (see Voltage clamp solutions). The internal solution was introduced within the voltage-clamp pipette, the tip of which was inserted through the silicone, within the insulated part of the fiber. In order to ease intracellular dialysis, the electrode tip was slightly crushed within the silicon-insulated portion of the fiber by moving it back and forth a few times in a non-gentle manner towards the bottom of the chamber. Intracellular equilibration of the solution was allowed for a period of 30 min with the holding voltage held at −80 mV, before initiating measurements. All experiments were performed at room temperature (20–22°C).Single fibers were isolated from 2+ signals in both an EGFP-positive fiber region (i.e. enriched in exogenously expressed RyR1s) and adjacent EGFP-deprived regions where the direct contribution of exogenous RyR1s should be much less. To accomplish this objective, we worked specifically on imaging a silicone-free portion of the fiber that exhibited large and restricted EGFP signals. Intracellular Ca2+-related fluorescence changes were imaged by using the line-scan mode of the confocal system with the line set parallel to the longitudinal fiber axis. The line position was moved after each line-scan image. The majority of images were taken with a scanning frequency of either 1.15 ms or 1.92 ms per line. Image processing and analysis was performed using Image/J  and Microcal Origin .Experiments were performed with a Zeiss LSM 5 Exciter confocal microscope  equipped with a 63× oil immersion objective . EGFP excitation was provided by the 488 nm line of an argon laser and a 505 nm long pass filter was used on the detection channel. For detection of rhod-2 fluorescence, excitation was from the 543 nm line of a HeNe laser and fluorescence was collected above 560 nm. One major aim of the experiments was to simultaneously record intracellular rhod-2 Ca0F/F, where 0F is the resting (or baseline) fluorescence level. Changes in [Ca2+] were calculated from the rhod-2 signals using the previously described pseudo-ratio equation 2+] of 100 nM and a Kd of rhod-2 for Ca2+ of 1.2 µM. An estimation of the Ca2+ release flux underlying the calculated global [Ca2+] transients was performed according to a previously described procedures 2+ ([Ca]T) obtained from the occupancy of intracellular calcium binding sites. The model included troponin C binding sites with a total concentration of sites  of 250 µM, an “on” rate constant  of 0.0575 µM−1.ms−1 and an “off” rate constant  of 0.115 ms−1; Ca-Mg binding sites on parvalbumin with a total concentration of sites  of 2000 µM, “on” rate constant for Ca2+  of 0.125 µM−1.ms−1, “off” rate constant for Ca2+  of 5.10−4 ms−1, “on” rate constant for Mg2+  of 3.3.10−5 µM−1.ms−1, “off” rate constant for Mg2+  of 3.10−3 ms−1. Calcium transport across the SR membrane was included with a rate assumed to be proportional to the fractional occupancy of the SR pump sites with a dissociation constant (pumpKd Ca) of 2 µM and a maximum pump rate of 10 µM.ms−1. Resting [Mg2+] was assumed to be 1.5 mM. The model also included Ca2+-binding sites on EGTA at a concentration of 15 mM, an “on” rate constant  of 0.056 µM−1.ms−1 and an “off” rate constant  of 0.002 ms−1. Under the present recording conditions, calcium binding to EGTA made a predominant contribution to the calculated Ca2+ release flux as compared to the intrinsic Ca2+ -buffering and -removal components of the model. Analyses essentially focused on comparing the properties of Ca2+ transients and Ca2+ release within a given line-scan image between regions of the same fiber with and without detectable EGFP-RyR1 fluorescence.Relative changes in rhod-2 fluorescence were expressed as An RK-400 patch-clamp amplifier  was used in whole-cell voltage-clamp configuration. Fibers were bathed in a TEA-containing extracellular solution (see Voltage clamp solutions). Command voltage pulse generation and data acquisition were achieved with an A/D, D/A converter  controlled by pClamp 9 software (Molecular Devices). Voltage-clamp was performed with the micropipette filled with a rhod-2 and EGTA containing intracellular solution (see Voltage clamp solutions). Analog compensation was systematically used to decrease the effective series resistance. Membrane depolarizing steps of 0.5 s duration were applied from a holding command potential of −80 mV.Fdb and interosseus muscles were electroporated with the EGFP-G4896V RyR1 construct. Two weeks later, single fibers were isolated using collagenase treatment as described above. Muscles were triturated in Tyrode solution onto glass slides. The presence of EGFP-positive fibers on the slides was checked by fluorescence microscopy. Slides were air-dried and muscle fibers were fixed and permeabilized in cold methanol (−20°C) for 10 minutes. Slides were then stored at −80°C. Slides were re-hydrated in PBS buffer. RyR1 labeling was performed as described by Lefebvre et al. 2-ATP, 5 Na2-phosphocreatine, 5.5 MgCl2, 5 glucose, 0.1 rhod-2, 20 EGTA, 8 CaCl2 and 5 HEPES. The extracellular solution contained (in mM) 140 TEA-methanesulfonate, 2.5 CaCl2, 2 MgCl2, 10 TEA-HEPES and 0.002 tetrodotoxin. All solutions were adjusted to pH 7.20.The intracellular voltage-clamp pipette solution contained (in mM) 120 K-glutamate, 5 Nan cells. Statistical significance was determined using a Student's t-test (One-Way Anova for data from cultured myotubes) with significance accepted at P<0.05 (*) and P<0.01 (**).Least-squares fits were performed using a Marquardt-Levenberg algorithm routine included in Microcal Origin . Data values are presented as means ± SEM for 2+ release in a manner that is independent of a detectable change in total SR Ca2+ content, a functional phenotype referred to as “EC uncoupling” 2+ release, maximal 4-chloro-m-cresol-induced (500 µM) Ca2+ release, and total intracellular Ca2+ store content following addition of a Ca2+ release cocktail (ICE) consisting of 10 µM ionomycin and 30 µM cyclopiazonic acid in a Ca2+-free (100 µM EGTA/0Ca2+) Ringer's solution. WT RyR1-expressing myotubes exhibit robust responses to electrical stimulation, 4-CMC application, and ICE-induced liberation of Ca2+ stores  calculated at the two line positions where the EGFP signal was maximal and minimal, respectively (positions are indicated by double-arrows next to the uppermost line-scan image). The rhod-2 signal was averaged over 50 adjacent rows. The right series of traces in 2+ release flux. For both the F/F0 and Ca2+ release flux, traces from the EGFP-positive region of the fiber are displayed in red. For all values of membrane depolarization, the initial peak of the rhod-2 fluorescence transient and of the corresponding Ca2+ release flux were reduced within the EGFP positive region of the fiber. Importantly, as observed previously 2+ release increased with stronger depolarizing pulses, its late decay phase was faster, consistent with SR Ca2+ depletion occurring early during depolarization as peak Ca2+ release increased. As described previously 2+ content due to expression of mutant RyR1s should be reflected as a decrease in the value of total released Ca2+, estimated from the time integral of the Ca2+ release flux during strong depolarizing pulses. In 2+ release in the high-expression region of the fiber  is identical in the two regions of the line-scan. 2+ release flux in fibers expressing EGFP-G4896V. Statistical significance was tested against the hypothesis of the ratio being equal to unity. Throughout the voltage range tested (−40 to +10 mV), all values were significantly lower than unity, with an average corresponding to an ∼30% decrease in peak Ca2+ release flux. Conversely, the corresponding mean ratio for total released Ca2+ yielded values that statistically did not differ from 1 for the largest levels of depolarization  along the lines scanned in EGFP-G4896V-expressing fibers, for the different test potentials. For each image, the initial peak ΔF/F0 value in response to the pulse was calculated at each x position along the line: in order to reduce the noise, the average of consecutive values over a 20 ms time interval centered around the time of the peak of the global transient was used in the calculation. Pairs of ΔF/F0-EGFP values were then sorted and averaged according to values falling within a given range of EGFP amplitudes with an increment arbitrarily set to 0.1 unit of relative EGFP signal, as described by Lefebvre et al. 0 values was then divided by the largest one to give the normalized ΔF/F0 values. These sets of normalized values from the different fibers were then averaged for each membrane potential tested. For all test depolarizations, a clear negative correlation was found between EGFP expression and peak Ca2+ transient amplitude, with a similar negative slope observed for each membrane potential tested.0) calculated within high (red traces) and low (black traces) EGFP-R4892W-expressing regions of the line, whereas the series of traces on the right show the corresponding Ca2+ release flux. Rhod-2 F/F0 transients and corresponding Ca2+ release flux traces were very similar in the two regions.2+ release and for total released Ca2+, respectively, in regions of high and low EGFP-R4892W expression. In contrast with that observed for EGFP-G4896V, a significant change in Ca2+ release was not observed at any test potential in areas high in EGFP-R4892W expression (0) and the corresponding EGFP signal  and DHPR (lower) fluorescence profiles along the box regions highlighted in the anti-DHPR frame. Whereas the EGFP signal almost completely vanished from left to right, the typical periodic DHPR signal intensity remained constant across the same region. Similar results were observed in 4 separate fibers. Parallel experiments using a SERCA1 antibody revealed a similar constant periodic SERCA1 signal intensity across a region of the fiber exhibiting a strong gradient of EGFP-G4896V expression  and G4898 (G4898E/R) do not support voltage-gated Ca2+ release (EC uncoupled) when expressed in dyspedic myotubes  explicitly demonstrates a defective function of these channels, consistent with the muscle weakness observed in patients. Thus, the observation that (unlike G4896V and I4897T) a Ca2+ release defect was not observed following expression of R4892W in adult muscle was unexpected. Assuming that the different mutant channels are produced, processed and targeted in the fibers with a similar efficiency, the lack of defective Ca2+ release associated with R4892W expression in adult muscle could reflect either a specific requirement for incorporation of more mutant monomers into a hetero-tetrameric channel or a compensatory effect due to a maintained Ca2+-sensitivity of the heterotypic channels Here, we found that the dominant-negative effect of the R4892W mutant on voltage-gated CaFigure S1Immunofluorescence labeling of SERCA1 in fibers expressing EGFP-G4896V and EGFP-R4892W mutant RyR1 channels. Examples of local expression of either EGFP-G4896V-RyR1 (top and bottom left panels) or EGFP-R4892W-RyR1 (medium left panel) and the corresponding SERCA1 labeling. The bottom right image shows a negative control image obtained in the absence of primary antibody.(TIF)Click here for additional data file.


We investigated the possible involvement of the mTOR pathway in the age-dependent changes on Ca2+ stores mobilization in colonic smooth muscle cells of young (4 month old) and aged (24 month old) guinea pigs. mTORC1 activity was enhanced in aged smooth muscle, as revealed by phosphorylation of mTOR and its direct substrates S6K1 and 4E-BP1. Mobilization of intracellular Ca2+ stores through IP3R or RyR channels was impaired in aged cells, and it was facilitated by mTOR and by FKBP12, as indicated by the inhibitory effects of KU0063794 (a direct mTOR inhibitor), rapamycin (a FKBP12-mediated mTOR inhibitor) and FK506 (an FKBP12 binding immunosuppressant). Aging suppressed the facilitation of the Ca2+ mobilization by FKBP12 but not by mTOR, without changing the total expression of FKBP12 protein. In conclusion, or study shows that in smooth muscle aging enhances the constitutive activity of mTORC1 pathway and impairs Ca2+ stores mobilization by suppression of the FKBP12-induced facilitation of Ca2+ release.Aging is considered to be driven by the so called senescence pathways, especially the mTOR route, although there is almost no information on its activity in aged tissues. Aging also induces Ca Mobilization of intracellular stores is mediated by IP3R and RyR channels, activated respectively by the intracellular messengers IP3 and cADPribose. The subsequent [Ca2+]i decrease to resting levels is operated by active extrusion to extracellular medium or reuptake into intracellular organelles.One of the functional consequences of aging is the alteration of different aspects of calcium signals, which play a key role in multiple cellular functions, from contraction or secretion to gene regulation and cell fate and proliferation. Therefore, these changes are the basis for important alterations linked to aging -15. Calc2+]i has focused on Ca2+ clearing mechanisms i signals, knowledge is rather superficial, most of the studies addressing only a description of the signal alterations. Some authors have proposed that mitochondrial modifications or redox imbalance are the origin for Ca2+ signal disfunction [2+ release imply that the mTOR pathway could be involved in the age-related [Ca2+]i modifications: Ca2+ release from intracellular stores can be modulated by mTOR kinase i signals induced by mobilization of intracellular Ca2+ pools in smooth muscle cells are characterized by an initial [Ca2+]i peak followed by gradual decline towards pre-stimulus levels, as shown in Figure 2+]i increase which was smaller in aged  than in young cells . Compared to bethanechol, the effect of caffeine in young cells was also transient but larger in amplitude , and showed a similar decrease in aged cells .To study the effects of aging in the mobilization of Caell type ; 34, or ough RyR . Ca2+]i+i2+ from2+ pools mobilization, we used a double pulse protocol: either bethanechol or caffeine was applied twice separated by a 15 minutes interval to allow for recovery of the response and acute application of inhibitors , its inhibitory effect in young cells was qualitatively similar to that of KU0063794 though smaller in amplitude  significantly decreased (p < 0.005) the responses to bethanechol and to caffeine  reduced clearly (p < 0.005) the responses to bethanechol and caffeine by approximately 50 and 60% respectively , reportiewed in ; 23 but iewed in . Resultsle cells ; 46 but le cells . We repo2+ signals. Because its facilitator role in Ca2+ release through IP3R and RyR channels i. A calibration of the ratio for [Ca2+]i was not performed in view of the many uncertainties related to the binding properties of fura 2 with Ca2+ inside of smooth muscle cells.i responses are expressed as increases in the ratio of fura-2 fluorescence (ΔF340/F380). To compare two groups, paired t test was used to assess the effect of treatment. The effects of age and experimental treatments were tested using a two-way analysis of variance, followed by planned comparisons between selected groups. Differences were considered significant at p < 0.05.Results are expressed as means ± standard error of the mean (sem) of n cells or blots. [Ca


Thermobifida fusca (Tf-trHb) over more than 10 time decades (from 1 ps to ∼100 ms) by combining femtosecond transient absorption, nanosecond laser flash photolysis and optoacoustic spectroscopy. Photolysis is followed by a rapid geminate recombination with a time constant of ∼2 ns representing almost 60% of the overall reaction. An additional, small amplitude geminate recombination was identified at ∼100 ns. Finally, CO pressure dependent measurements brought out the presence of two transient species in the second order rebinding phase, with time constants ranging from ∼3 to ∼100 ms. The available experimental evidence suggests that the two transients are due to the presence of two conformations which do not interconvert within the time frame of the experiment. Computational studies revealed that the plasticity of protein structure is able to define a branched pathway connecting the ligand binding site and the solvent. This allowed to build a kinetic model capable of describing the complete time course of the CO rebinding kinetics to Tf-trHb.CO recombination kinetics has been investigated in the type II truncated hemoglobin from  The dynamics of ligand binding after photolysis in heme proteins is a complex phenomenon that entails a sequence of distinct events covering more than 10 time decades. Such a time frame spans from the early quantum events (electron promotion and decay) occurring in the subpicoseconds time scale to ligand motion within the distal heme pocket, falling in the tens of nanoseconds, from protein relaxation typically observed in the microsecond time regime, to ligand rebinding from the solvent, reaching the milliseconds range The overall dynamics of the photodissociated CO ligand has been previously investigated in vertebrate globins: in the case of myoglobin, this has allowed to draw a detailed picture of the dynamics following photoexcitation of the protein Thermobifida fusca (Tf-trHb), the first identified thermostable truncated Hb Tf-trHb TrpG8 and TyrCD1 are mainly involved in the stabilization of exogenous ligands, namely sulfide Mycobacterium tuberculosisBacillus subtilis trHbOs Biological diversity, however, proposes novel challenges in the study of hemoglobin dynamics by offering an amazingly vast array of “variations on the globin theme” in terms of a wide collection of proteins whose biochemical functions are still far from being understood, though most likely unrelated to simple reversible oxygen transport Tf-trHb has been undertaken by combining femtosecond transient absorption (TA), nanosecond laser flash photolysis (LFP), laser-induced optoacoustic spectroscopy (LIOAS) and molecular dynamics (MD) simulations.In the present study, a complete characterization of the dynamics of an acidic surface variant of Tf-trHb at different delay times after laser photolysis are presented in Tf-trHb and of the protein-CO complex. Thus, for delay times ≥40 ps the transient signal is simply composed of an amplitude decay with a fixed cross-zero point, matching the isosbestic point of the two species , the transient signal does not vanish and the remaining 5c-Tf-trHb concentration (nearly 60%) gives rise to the absorbance changes observed in LFP.Multiexponential decay fitting of the kinetic traces at a single wavelength see  yielded bination  e S2. ImTf-trHb were recorded as a function of CO concentration (to distinguish between first- and second-order processes) and temperature .Time-resolved absorbance spectra measured after nanosecond laser photolysis add strong support to the absence of a conformational relaxation upon CO photodissociation see . The SVDOne of the major goals of the present work is the full characterization of the protein dynamics over 10 and more temporal decades and to reproduce a single rebinding curve. The problem of merging two sets of data obtained by TAS, from few picoseconds to 2 nanoseconds, and by LFP, from 20 ns to milliseconds, is to define a proper data treatment without introducing artefacts. The “blind” interval between 2 and 20 ns does not allow a direct junction of the two sets of data.0, i.e., N(t) = α(t)/α(t0), (with t > t0 ), is independent on the excitation and decreases with time from unity. It is also easily seen that N(t) = ΔA(t)/ΔA(t0). On these basis, it is possible to merge the rebinding kinetics measured within different time domains, with the additional assumption that the the primary quantum yield for the Fe-CO photodissociation is Φ = 1 0 = 40 ps, when all the electronical and vibrational relaxations are completed (N(t) = ΔA(t)/ΔA(40 ps)).Although the time-dependent α fraction varies with the experimental excitation conditions, the fraction of photoproduct with respect to that initially generated at time t0. This 50% fraction agrees well with the estimate of surviving 5c-Tf-trHb unligated molecules at the end of the observation time window in the femtosecond photolysis experiment, a fact which confirms that multiple photolysis (i.e. photolysis of those molecules which have bound CO in the geminate phase) is a minor effect. In these conditions α≈1 (within 10%), and the fraction of the 5c-form N(t) can be calculated by considering ΔA(t0) as the steady state difference of the absorbance at 435 nm between the 5c-form and the corresponding ligated species at the concentration c0. With the caution to discard the transient data for t <20 ns as they are strongly influenced by the instrumental pulse width, we have combined the picoseconds TA results with the LFP results. The two branches of the diagram, N(t) vs time, are reported in Tf-trHb along ten temporal decades. The convolution of this diagram with a Gaussian shaped function (FWHM = 12 ns) centered at t = 0 gives as a result a calculated profile which completely overlaps the experimental LFP profile  are reported in Tf-trHb-CO is larger than that observed for the reference compound. This is consistent with an exothermic reaction and/or with the presence of a concomitant structural volume change Tf-trHb-CO is delayed in comparison with that of the calorimetric reference, the delay becoming especially appreciable at low temperatures. This indicates that the observed volume changes have characteristic lifetimes that must be determined according to the deconvolution analysis described in pρ/βC) - pC is the heat capacity at constant pressure, ρ the mass density, and β the cubic expansion coefficient - whereas the amplitude arising from structural volume changes ΔiV will stay unaltered The acoustic waves detected upon photoexcitation of aqueous solutions of i -φ multiplied by the laser energy λE - are plotted vs (pρ/βC). This allows for the simultaneous determination of ΔiV and of the heat release iQ associated to the prompt and to the slow components of the decay . On the other hand, a negative enthalpic contribution is estimated for the slow reaction. Activation parameters are obtained from the Eyring plots in the lower part of Best fit of the signal amplitude was obtained with a sum of two exponential decay functions, with lifetimes falling in the tens and the hundreds of nanoseconds, respectively. Both lifetimes are temperature dependent in the investigated range. A clear linear trend is observed  when thehe decay , as repoTf-trHb with femtosecond laser pulses show three spectral components with time constants of ∼300 fs, 3–4 ps and several nanoseconds. The similarity between the spectral shape and lifetimes of the two faster components with those observed after photoexcitation of Mb Tf-trHb, suggests that the two kinetic phases are entirely accounted for by electronic and vibrational relaxation processes. On the basis of its spectral shape, the slowest phase should be attributed to the geminate recombination of CO. Recently, the initial steps of photodissociation process of CO ligated heme have been investigated by time-dependent density function theory (TDDFT) et al.π→a1u,a2u) charge transfer is the key event in the mechanism of photolysis (<50 fs) of diatomic ligands following a porphyrin ring π→π* transition, then the complex can decay via spin-conversion into the high-spin ground state of the unligated system, i.e. the 5c- form. Although the interpretation of the electronic and vibrational relaxation after photoexcitation of heme proteins has not yet converged to a single model Tf-trHb. Thus, we can assume that complete relaxation of the excited state takes place within a few tens of picoseconds.Sub-nanosecond processes are known to include the formation of transient excited states with the subsequent structural response of the porphyrin macrocycle to the new electronic configuration, followed by the non-radiative relaxation to the ground state Tf-trHb is similar to that reported for the CO complexes of Bacillus subtilis Bs-trHb Micobacterium tubercolosis Mt-trHbO Tf-trHb, Bs-trHb and Mt-trHbO) exhibit very similar distal heme pocket architecture with highly conserved polar residues. These residues are in contact with the iron-bound CO coordination shell in a common pattern defined “ligand inclusive hydrogen bond network”, representing a considerable barrier to ligand escape as demonstrated by the efficient geminate rebinding process After excited state dynamics is complete, the onset of geminate recombination from the distal pocket is observed on the hundreds of picoseconds time scale . The ∼ 2cRhodospirillum rubrumc is best described by a multiphasic recombination with time constants of 16 ps, 120 ps and 1 ns. The authors have attributed the three phases to CO rebinding from different locations within the distal pocket. The high efficiency of the ligand rebinding was seen as a consequence of a sterically hindered “caged” nature of the distal heme pocket, from which it is difficult for the ligand to escape. Similarly, CO geminate rebinding to nitrophorin 4 was shown to occur with a prominent subnanosecond, nonexponential phase, accounting for nearly 70% of the rebinding.Although a sub-nanosecond geminate heme-CO rebinding is not routinely considered, experimental evidence is being accumulated that this kinetic phase should not be overlooked. Picosecond geminate processes were reported for the CO complex of several non-globin heme proteins, including microperoxidase Tf-trHb-CO, nanosecond photolysis experiments show that first order processes are observed also on longer time scale, as demonstrated by the presence of CO concentration independent bands in the MEM lifetime distribution in Tf-trHb shows kinetic features which extend well beyond the picoseconds time scale and suggest that photodissociated ligands may access transient docking sites located farther from the distal pocket.In the case of Tf-trHb suggest that there is just one short tunnel associated with a cavity system through which small ligands can enter and bind to the iron heme group (ILS (Implicit Ligand Sampling) me group . This sh1, and further away (trHb : CO)2 were unambiguously singled out. A further small cavity, (trHb : CO)3, was also observed. However, this cavity is disconnected from the distal site, since TyrCD1is blocking the passage between (trHb : CO)1 and (trHb : CO)3 b : CO)3 .1 and (trHb : CO)3 opens up is observed observed  and attrobserved , indicatILS thus identified an egression pathway for ligands, which has its origin in the distal pocket and reaches the solvent through an opening between helices E and F see . This tuThe model we have chosen to account for the observed kinetics is shown in Scheme 1. In addition to an exit/entry through an on-pathway docking site, the model introduces migration to a secondary, off- pathway, docking site, which is accessible from the primary site in the distal pocket.Tf-trHb. (trHb : CO)1 and (trHb : CO)2 indicate respectively the primary and secondary docking site for the photodissociated CO inside the distal pocket along the exit (entry) pathway to (from) the solvent, while (trHb : CO)3 represents a reaction intermediate with CO in a temporary docking site accessible from the distal site. Two static conformations are differing in the rate constants kc,o2 and kc,o−2. and their relative weights are held identical to those retrieved by the amplitudes of the two exponential decays in the bimolecular rebinding phase.Extended minimal reaction scheme for the observed CO rebinding kinetics to The differential equations associated with Scheme 1 were solved numerically, and the microscopic rate constants were obtained by a simultaneous fit to the recombination kinetics at the same temperature, and different ligand concentration.kc,o2 and kc,o−2), while the remaining rate constants are held as shared parameters. It can describe accurately the rebinding kinetics under all tested experimental conditions, as shown in The model assumes for each of the two conformations distinct values for some of the microscopic rates . Similarly, the geminate phase in the nanosecond experiments is essentially unaffected by temperature. This unambiguously implies that neither the rate k−1 = 3·108 s−1, higher than those typically observed for other heme proteins, including human neuroglobin (Ngb) (k−1 = 1.5·107 s−1) Arabidopsis thaliana  k−1 = 0.13·107 s−1) The rebinding reaction from the primary site occurs with a rate c,ok−2) has similar activation enthalpies and entropies for both open and closed forms, suggesting that the viscosity of the solution determines the barrier k2) do not share the same similarity, being higher in the case of the open conformation.Unlike the case of innermost steps, the exit rate increases with temperature. This finding indicates that ligand exit from the protein matrix is assisted by structural dynamics, which thus appears to tune the exchange of the ligands between the solvent and the reaction site. The exit/entry to/from the solvent are then thermally activated processes and the ligand binding from solution (rate 1 and (trHb : CO)3, responsible for a delayed CO exit to the solvent.The presence of the on- and off-pathway kinetic traps modulates ligand exit to the solvent. The time course of ligand escape to the solvent is monitored by rising portion of the curve describing concentration of the species trHb+CO in Scheme 1 see also . The sigONk, which is 8.7×104 M−1s−1 and 9.3×105 M−1s−1 for the slow (∼80%) and fast (∼20%) rebinding conformation, respectively, at 20°C. These figures increase to 1.63×105 M−1s−1 and 2.75×106 M−1s−1 at 25°C. These values can be compared with the previous determination of ONk for the reaction of oxygen binding to Tf-trHb, estimated equal to 9×105 M−1s−1 at 25°C ONk for oxygen is one order of magnitude larger than that for CO. Previous investigations also indicated that ligand release constants of Tf-trHb were exponentially dependent upon temperature and signal recovery was identical from 20 to 70°C, demonstrating that the protein is capable of exchanging oxygen reversibly at high temperatures and did not denature even at the highest temperature. Moreover, previous results also highlighted that the activation enthalpy for the oxygen-dissociation reaction was identical  to that measured for vertebrate Hbs and Mbs, thus indicating that the bond dissociation energy of the iron-oxygen adduct was the same in all these hemoglobins. On this basis, it is expected that ligand binding behavior at 25°C can be easily extrapolated to 55°C by taking into account linear activation enthalpy and entropy parameters with no need of additional thermal dependent contributions.It is useful to estimate the bimolecular binding rate constant, usually termed 3, and are only partly released at later times. Thus, a lower limit for the prompt reaction volume ΔR,1V, which is related to the measured structural volume change Δ1V through the quantum yield: ΔR,1V =  Δ1V/1Φ, is ∼ 4.5 ml mol−1.The LIOAS data show that photodissociation leads to a prompt (i.e. occurring at the edge or below the 20 ns resolution of the experiment) expansion of the solution. The events integrated on this time scale comprise photodissociation of the Fe-CO bond, non radiative relaxation of the heme excited state, and geminate rebinding. Photodissociation and geminate rebinding are characterized by volume  changes of opposite sign, and are thus expected to cancel. The observed fast expansion thus arises from that fraction of photodissociated CO molecules which survive geminate rebinding and escape to the solvent. Such fraction – which represents an effective quantum yield on the LIOAS time scale - is estimated to be nearly 0.4  and 9. TR,1V, suggesting that the latter, like process 1, ends in the displacement of CO to the solvent. The observed expansions are comparable to the reaction volumes determined by photoacoustic spectroscopy for the complete dissociation of CO from myoglobin, leading to the formation of 5c-myoglobin and CO solvation −1 for the CO complex of sperm whale myoglobin −1 following photoexcitation of the CO complex of horse myoglobin The delayed CO emission may be held, at least partly, responsible for the transient with lifetime in the 100 ns range detected in the photoacoustic experiments  of sodium dithionite in the ferric protein solution at pH 7.2 under CO atmosphere.pump = 400 nm) and the induced absorbance changes were monitored in the 410–650 nm region. Experiments exciting into the Q bands at 560 nm  were also performed . In addition, single value decomposition (SVD) was applied to extract the spectra associated (DAS) to the kinetic components The laser setup was described previously The lifetime distributions associated with the observed kinetics were evaluated using the program MemExp (ver. 2.0) iφ is the pre-exponential factor of the transient with lifetime iτ. The values of iφ and iτ are found from the deconvolution analysis The LIOAS measurements were performed with a previously described setup using as excitation source the II harmonic (532 nm) of a Q-switched Nd:YAG laser (pulse width ∼10 ns) iφ – multiplied by the photon energy λE - were then plotted versus the thermoelastic parameter ratio of the solution. From the linear relation:iQ (from the intercept) and the structural volume change ΔiV, expressed as milliliters per mole of absorbed photons. Separation of the volume change (ΔiV) from the thermal contribution (iQ) can be obtained most easily in aqueous solutions, since the pρ/βC ratio is strongly temperature dependent for water in the ordinary temperature range. The measurements were performed in a phosphate buffer solution with 0.1 M ionic strength, for which the pρ/βC ratio has been previously determined Experiments conducted at multiple temperatures (10–25°C) were used to determine the heat release and the structural volume change for each transient Tf-trHb crystal structure (Protein Data Bank entry 2BMM) as determined by Bonamore et al.et al.2 for all Cα atoms (ii) pressure equilibration of the entire system simulated for 1 ns at 300K with the same restrained atoms. After these two steps an unconstrained 50 ns MD long simulation at constant temperature (300K) was performed. As shown previously, this simulation strategy is adequate for the study of the free energy of ligand migration along the protein tunnel cavity system using the Implicit Ligand Sampling (ILS) method as shown by Forti et al.The starting structure corresponds to The free energy for the CO migration process inside the protein tunnel/cavity system was computed by ILS approach that use computed MD simulation in the absence of the ligand and incorporate it afterwards. This method was thoroughly tested for heme proteins. Figure S1Transient absorption spectra of the 5c-Tf-trHb excited at 400 nm with femtosecond pulses. The absorbance of the sample was 0.8 at the pump wavelength. The delay times between pump and probe pulses were reported in the legend, the value were expressed in picoseconds. B: Bleaching; ESA: excited state absorption.(TIF)Click here for additional data file.Figure S2Results of the SVD analysis of transient absorption spectra in the first ∼2 ns after photoexcitation of the CO complex of the Tf-trHb (upper panel) and of the ferrous 5c-protein (lower panel). The transient absorption spectra have been examined by fitting the kinetic profiles at any given wavelength and by SVD analysis. The latter has allowed to extract the spectra associated with each exponential time (DAS) and the comparison between the behavior observed in the 5c-Tf-trHb and in the CO complex has been useful to disentangle the geminate recombination process. In the CO complex, three DAS have been extracted with the following time constants: 300 fs (red line), 6 ps (blue line) and 2.8 ns (black line). Only two DAS have been observed in the 5c-Tf-trHb with equal time constant and similar spectral shape in comparison with the CO complex. The similarity between the first two components suggests that they are due to the photodynamics of the excited heme. The fastest one shows a broad absorption that can be attributed to the metal-to-ring charge transfer transition, in agreement with the model proposed by Franzen et al.(TIF)Click here for additional data file.Figure S3CO rebinding kinetics to Tf-trHb, at 1 atm CO and 10°C (black), 15°C (red), 20°C (green), 25°C (blue) and 30°C (cyan). Protein concentration was 38 µM.(TIF)Click here for additional data file.Figure S4Spectral component  and amplitude  retrieved from the SVD analysis of time resolved spectra collected between 10 ns and 100 ms. The red solid line shows the absorbance change measured at 435 nm. SVD analysis of time resolved difference absorbance spectra collected after nanosecond laser excitation afforded only one statistically meaningful spectral component. The time course of its amplitude closely matches that of the absorbance change at 435 nm showing that this component is monitoring the binding process. The absence of additional spectral components suggest that no significant structural relaxation is occurring at the heme.(TIF)Click here for additional data file.Figure S5Change in CO concentration in solution as estimated from the analysis of the rebinding kinetics reported in  of the article. Green, simulation of CO concentration, red, fitting with a double exponential rise.(TIF)Click here for additional data file.Figure S6PROBE = 435 nm after photolysis with femtosecond pulses; λPUMP = 400 nm (thick black line) and λPUMP  = 560 nm (thin red line).CO rebinding kinetics at λ The signals are normalized to 1 to the maximum of the intensity. Although the different quality of the results, we can assert that the kinetic profiles are not affected by the excitation wavelength. The behavior observed in the horse heart myoglobin has been also shown in the figure in order to highlight the presence of the ultrafast rebinding component in Tf-trHb. The curve taken in Tf-trHb after excitation at 400 nm has been fitted with a mono-exponential decay function: the time constant results equal to 3.9 ns. It decreases to 2 ns if we include a nonzero asymptotic contribution, kept fixed to 0.38 corresponding to the bimolecular dissociation yield (LFP experiments).(TIF)Click here for additional data file.