Action potential initiation and propagation in hippocampal mossy fibre axons

Dentate gyrus granule cells transmit action potentials (APs) along their unmyelinated mossy fibre axons to the CA3 region. Although the initiation and propagation of APs are fundamental steps during neural computation, little is known about the site of AP initiation and the speed of propagation in mossy fibre axons. To address these questions, we performed simultaneous somatic and axonal whole-cell recordings from granule cells in acute hippocampal slices of adult mice at ∼23°C. Injection of short current pulses or synaptic stimulation evoked axonal and somatic APs with similar amplitudes. By contrast, the time course was significantly different, as axonal APs had a higher maximal rate of rise (464 ± 30 V s⁻¹ in the axon versus 297 ± 12 V s⁻¹ in the soma, mean ± s.e.m. ). Furthermore, analysis of latencies between the axonal and somatic signals showed that APs were initiated in the proximal axon at ∼20–30 μm distance from the soma, and propagated orthodromically with a velocity of 0.24 m s⁻¹. Qualitatively similar results were obtained at a recording temperature of ∼34°C. Modelling of AP propagation in detailed cable models of granule cells suggested that a ∼4 times higher Na⁺ channel density (∼1000 pS μm⁻²) in the axon might account for both the higher rate of rise of axonal APs and the robust AP initiation in the proximal mossy fibre axon. This may be of critical importance to separate dendritic integration of thousands of synaptic inputs from the generation and transmission of a common AP output.     

Spatial Segregation and Interaction of Calcium Signalling Mechanisms in Rat Hippocampal CA1 Pyramidal Neurons

Postsynaptic [Ca²⁺]_i increases result from Ca²⁺ entry through ligand-gated channels, entry through voltage-gated channels, or release from intracellular stores. We found that these sources have distinct spatial distributions in hippocampal CA1 pyramidal neurons. Large amplitude regenerative release of Ca²⁺ from IP₃-sensitive stores in the form of Ca²⁺ waves were found almost exclusively on the thick apical shaft. Smaller release events did not extend more than 15 μm into the oblique dendrites. These synaptically activated regenerative waves initiated at points where the stimulated oblique dendrites branch from the apical shaft. In contrast, NMDA receptor-mediated increases were observed predominantly in oblique dendrites where spines are found at high density. These [Ca²⁺]_i increases were typically more than eight times larger than [Ca²⁺]_i from this source on the main aspiny apical shaft. Ca²⁺ entry through voltage-gated channels, activated by backpropagating action potentials, was detected at all dendritic locations. These mechanisms were not independent. Ca²⁺ entry through NMDA receptor channels or voltage-gated channels (as previously demonstrated) synergistically enhanced Ca²⁺ release generated by mGluR mobilization of IP₃.     

Spino-dendritic cross-talk in rodent Purkinje neurons mediated by endogenous Ca2+-binding proteins

The range of actions of the second messenger Ca²⁺ is a key determinant of neuronal excitability and plasticity. For dendritic spines, there is on-going debate regarding how diffusional efflux of Ca²⁺ affects spine signalling. However, the consequences of spino-dendritic coupling for dendritic Ca²⁺ homeostasis and downstream signalling cascades have not been explored to date. We addressed this question by four-dimensional computer simulations, which were based on Ca²⁺-imaging data from mice that either express or lack distinct endogenous Ca²⁺-binding proteins. Our simulations revealed that single active spines do not affect dendritic Ca²⁺ signalling. Neighbouring, coactive spines, however, induce sizeable increases in dendritic [Ca²⁺]_i when they process slow synaptic Ca²⁺ signals, such as those implicated in the induction of long-term plasticity. This spino-dendritic coupling is mediated by buffered diffusion, specifically by diffusing calbindin-bound Ca²⁺. This represents a central mechanism for activating calmodulin in dendritic shafts and therefore a novel form of signal integration in spiny dendrites.     

Rate, extent and concentration dependence of histamine-evoked Weibel–Palade body exocytosis determined from individual fusion events in human endothelial cells

The rate, concentration dependence and extent of histamine-evoked Weibel–Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45 ± 0.26 ( s.d. , n = 144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0 ± 4.42 s ( n = 9 cells) at 0.3 μ m histamine and 1.57 ± 0.21 s ( n = 15 cells) at 100 μ m histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20 ± 0.16 WPB s⁻¹ ( n = 9) at 0.3 μ m and 3.66 ± 0.45 WPB s⁻¹ at 100 μ m histamine ( n = 15). These occurred 2–5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca²⁺ indicating that the initial burst of secretion is driven by Ca²⁺ release from internal stores, but sustained exocytosis required external Ca²⁺. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 μ m ), for which there was a delay between calcium elevation and secretion of 1.67 ± 0.24 s ( n = 6), and a peak fusion rate of ∼10 WPB s⁻¹.     

Contribution of a Calcium-Activated Non-Specific Conductance to NMDA Receptor-Mediated Synaptic Potentials in Granule Cells of the Frog Olfactory Bulb

We studied granule cells (GCs) in the intact frog olfactory bulb (OB) by combining whole-cell recordings and functional two-photon Ca²⁺ imaging in an in vitro nose-brain preparation. GCs are local interneurones that shape OB output via distributed dendrodendritic inhibition of OB projection neurones, the mitral-tufted cells (MTCs). In contrast to MTCs, GCs exhibited a Ca²⁺-activated non-specific cation conductance ( I _CAN) that could be evoked through strong synaptic stimulation or suprathreshold current injection. Photolysis of the caged Ca²⁺ chelator o -nitrophenol-EGTA resulted in activation of an inward current with a reversal potential within the range -20 to +10 mV. I _CAN in GCs was suppressed by the intracellular Ca²⁺ chelator BAPTA (0.5–5.0 mM), but not by EGTA (up to 5 mM). The current persisted in whole-cell recordings for up to 1.5 h post-breakthrough, was observed during perforated-patch recordings and was independent of ionotropic glutamate and GABA_A receptor activity. In current-clamp mode, GC responses to synaptic stimulation consisted of an initial AMPA-mediated conductance followed by a late-phase APV-sensitive plateau (100–500 ms). BAPTA-mediated suppression of I _CAN resulted in a selective reduction of the late component of the evoked synaptic potential, consistent with a positive feedback relationship between NMDA receptor (NMDAR) current and I _CAN. I _CAN requires Ca²⁺ influx either through voltage-gated Ca²⁺ channels or possibly NMDARs, both of which have a high threshold for activation in GCs, predicting a functional role for this current in the selective enhancement of strong synaptic inputs to GCs.     

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca²⁺ sparks stimulate nearby Ca²⁺-activated K⁺ (BK) channels that hyperpolarize the membrane and close L-type Ca²⁺ channels. We tested the contribution of L-type Ca_v1.2 channels to Ca²⁺ spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Ca_v1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Ca_v1.2 channel gene inactivation reduced Ca²⁺ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca²⁺ levels and reduced sarcoplasmic reticulum (SR) Ca²⁺ load. Elevating cytosolic Ca²⁺ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Ca_v1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca²⁺ sparks. Instead, Ca_v1.2 channels contribute to global cytosolic [Ca²⁺], which in turn influences luminal SR calcium and thus Ca²⁺ sparks.     

Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic β-cells

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic β-cells by a combination of electrophysiological recordings, intracellular Ca²⁺ ([Ca²⁺]_i) microfluorimetry and insulin release measurements. Addition of palmitate (1 m m , bound to fatty acid-free albumin) to intact islets exposed to 15 m m glucose increased the [Ca²⁺]_i by ∼30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca²⁺]_i and insulin release in the presence of tolbutamide and in islets depolarized by high K⁺ in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K⁺ channels (K_ATP channels). Palmitate (0.5 m m ) augmented exocytosis (measured as an increase in cell capacitance) in single β-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca²⁺ currents rose by ∼25% following addition of 0.5 m m palmitate, an effect that was abolished in the presence of 10 μ m isradipine indicating that the free fatty acid specifically acts on L-type Ca²⁺ channels. The actions of palmitate on exocytosis and Ca²⁺ currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K_ATP channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca²⁺ currents and an increased size of the RRP.     

Genetic deletion of SK2 channels in mouse inner hair cells prevents the developmental linearization in the Ca2+ dependence of exocytosis

Inner hair cells (IHCs), the primary sensory receptors of the mammalian cochlea, fire spontaneous Ca²⁺ action potentials (APs) only before the onset of hearing. Although a role for APs in the developing auditory system has not been determined it could, by analogy with other sensory systems, guide the functional maturation of the cochlea before experience-driven activity begins. Spontaneous APs in immature IHCs are shaped by a variety of ion channels including that of the small conductance Ca²⁺-activated K⁺ current (SK2), which is only transiently expressed in immature cells. Using SK2 knockout mice we found that SK2 channels are not required for generating APs but are essential for sustaining continuous repetitive spontaneous AP activity in pre-hearing IHCs. Therefore we used this mutant mouse as a model to study possible developmental implications of disrupted AP activity. Immature mutant IHCs showed impaired exocytotic responses, which are likely to be due to the expression of fewer Ca²⁺ channels. Exocytosis was also impaired in adult mutant IHCs, although in this case it resulted from a reduced Ca²⁺ efficiency and increased Ca²⁺ dependence of the synaptic machinery. Since SK2 channels can only have a functional influence on IHCs during immature development and are not directly involved in neurotransmitter release, the altered Ca²⁺ dependence of exocytosis in adult IHCs is likely to be a consequence of their disrupted AP activity at immature stages.     

Voltage-dependent inactivation of l-type ca2+ Currents in Guinea-Pig Ventricular Myocytes

The objective of this study was to describe the kinetics of voltage-dependent inactivation of native cardiac L-type Ca²⁺ currents. Whole-cell currents were recorded from guinea-pig isolated ventricular myocytes. Voltage-dependent inactivation was separated from Ca²⁺-dependent inactivation by replacing extracellular Ca²⁺ with Mg²⁺ and recording outward currents through Ca²⁺ channels. Voltage-dependent inactivation accelerated from slow monophasic decay at −30 mV to maximal rapid biphasic decay at +20 mV. Maximal voltage-dependent inactivation occurred with τ_f≈30 ms and τ_s≈300 ms, the fast component of decay accounted for 70 % of the current amplitude. In basal conditions Ca²⁺ current availability was sigmoid. Isoproterenol (isoprenaline) evoked a large increase in a time-independent component of the Ca²⁺ current which also increased with depolarisation. This was responsible for the apparent recovery of Ca²⁺ channel current availability at positive membrane potentials and thus a U-shaped availability-voltage ( A-V) relationship. It is concluded that β-adrenergic stimulation altered the reaction of native cardiac L-type Ca²⁺ channels to membrane voltage. In basal conditions, voltage accelerated inactivation. In isoproterenol, voltage could also reduce inactivation.     

Electrophysiological properties of BK channels in Xenopus motor nerve terminals

Single channel properties of Ca²⁺-activated K⁺ (BK or Maxi-K) channels have been investigated in presynaptic membranes in Xenopus motoneurone–muscle cell cultures. The occurrence and density of BK channels increased with maturation/synaptogenesis and was not uniform: highest at the release face of bouton-like synaptic varicosities in contact with muscle cells, and lowest in varicosities that did not contact muscle cells. The Ca²⁺ affinity of the channel ( K _d= 7.7 μ m at a membrane potential of +20 mV) was lower than those of BK channels that have been characterized in other terminals. Hill coefficients varied between 1.5 and 2.8 at different potentials and open probability increased e-fold per 16 mV change in membrane potential over a range of [Ca²⁺]_i from 1 μ m to 1 m m . The maximal activation rate of ensembled single BK channel currents was in the submillisecond range at ≥+20 mV. The activation rate increased ∼10-fold in response to a [Ca²⁺]_i increase from 1 to 100 μ m , but increased only ∼2-fold with a voltage change from +20 to +130 mV. The fastest activation kinetics of BK channels in cell-attached patches resembled that in inside-out patches with [Ca²⁺]_i of 100 μ m or more, suggesting that many BK channels are located very close to calcium channels. Given the low Ca²⁺ affinity and rapid Ca²⁺ binding/unbinding properties, we conclude that BK channels in this preparation are adapted to play an important role in regulation of neurotransmitter release, and they are ideal reporters of local [Ca²⁺] at the inner membrane surface.     

Functional maturation of the exocytotic machinery at gerbil hair cell ribbon synapses

Auditory afferent fibre activity in mammals relies on neurotransmission at hair cell ribbon synapses. Developmental changes in the Ca²⁺ sensitivity of the synaptic machinery allow inner hair cells (IHCs), the primary auditory receptors, to encode Ca²⁺ action potentials (APs) during pre-hearing stages and graded receptor potentials in adult animals. However, little is known about the time course of these changes or whether the kinetic properties of exocytosis differ as a function of IHC position along the immature cochlea. Furthermore, the role of afferent transmission in outer hair cells (OHCs) is not understood. Calcium currents and exocytosis (measured as membrane capacitance changes: Δ C _m) were measured with whole-cell recordings from immature gerbil hair cells using near-physiological conditions. The kinetics, vesicle pool depletion and Ca²⁺ coupling of exocytosis were similar in apical and basal immature IHCs. This could indicate that possible differences in AP activity along the immature cochlea do not require synaptic specialization. Neurotransmission in IHCs became mature from postnatal day 20 (P20), although changes in its Ca²⁺ dependence occurred at P9–P12 in basal and P12–P15 in apical cells. OHCs showed a smaller Δ C _m than IHCs that was reflected by fewer active zones in OHCs. Otoferlin, the proposed Ca²⁺ sensor in cochlear hair cells, was similarly distributed in both cell types despite the high-order exocytotic Ca²⁺ dependence in IHCs and the near-linear relation in OHCs. The results presented here provide a comprehensive study of the function and development of hair cell ribbon synapses.     

Properties of spontaneous Ca2+ transients recorded from interstitial cells of Cajal-like cells of the rabbit urethra in situ

Interstitial cells of Cajal-like cells (ICC-LCs) in the urethra may act as electrical pacemakers of spontaneous contractions. However, their properties in situ and their interaction with neighbouring urethral smooth muscle cells (USMCs) remain to be elucidated. To further explore the physiological role of ICC-LCs, spontaneous changes in [Ca²⁺]_i (Ca²⁺ transients) were visualized in fluo-4 loaded preparations of rabbit urethral smooth muscle. ICC-LCs were sparsely distributed, rather than forming an extensive network. Ca²⁺ transients in ICC-LCs had a lower frequency and a longer half-width than those of USMCs. ICC-LCs often exhibited Ca²⁺ transients synchronously with each other, but did not often show a close temporal relationship with Ca²⁺ transients in USMCs. Nicardipine (1 μ m ) suppressed Ca²⁺ transients in USMCs but not in ICC-LCs. Ca²⁺ transients in ICC-LCs were abolished by cyclopiazonic acid (10 μ m ), ryanodine (50 μ m ) and caffeine (10 m m ) or by removing extracellular Ca²⁺, and inhibited by 2-aminoethoxydiphenyl borate (50 μ m ) and 3-morpholino-sydnonimine (SIN-1; 10 μ m ), but facilitated by increasing extracellular Ca²⁺ or phenylephrine (1–10 μ m ). These results indicated that Ca²⁺ transients in urethral ICC-LCs in situ rely on both Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx through non-L-type Ca²⁺ channel pathways. ICC-LCs may not act as a coordinated pacemaker electrical network as do ICC in the gastrointestinal (GI) tract. Rather they may randomly increase excitability of USMCs to maintain the tone of urethral smooth muscles.     

β-adrenergic and muscarinic agonists modulate inactivation of l-type ca2+ Channel Currents in Guinea-Pig Ventricular Myocytes

The objective of this study was to examine the effects of isoproterenol (isoprenaline) and carbachol upon voltage-dependent inactivation of L-type Ca²⁺ current ( I _Ca,L). I _Ca,L was recorded in guinea-pig isolated ventricular myocytes in the presence and absence of extracellular Ca²⁺ to separate total inactivation and voltage-dependent inactivation. In the presence of Ca²⁺, isoproterenol and carbachol had 'competitive' effects upon the relationships between membrane voltage and I _Ca,L amplitude and inactivation. Neither agonist had a marked effect upon the decay of inward I _Ca,L carried by Ca²⁺. In the absence of Ca²⁺, isoproterenol severely reduced and slowed I _Ca,L inactivation; this effect was reversed by carbachol. Under control conditions decay was dominated by fast inactivation. Isoproterenol reduced fast-inactivating and increased time-independent currents in a dose-dependent manner. These effects were counteracted by carbachol. There was a reciprocal relationship between the amplitude of fast-inactivating and time-independent currents with agonist stimulation. It is concluded that agonist modulation of rapid voltage-dependent inactivation of L-type Ca²⁺ channels involves an 'on-off' switch.     

mGluR1/5 subtype-specific calcium signalling and induction of long-term potentiation in rat hippocampal oriens/alveus interneurones

Hippocampal inhibitory interneurones demonstrate pathway- and synapse-specific rules of transmission and plasticity, which are key determinants of their role in controlling pyramidal cell excitability. Mechanisms underlying long-term changes at interneurone excitatory synapses, despite their importance, remain largely unknown. We use two-photon calcium imaging and whole-cell recordings to determine the Ca²⁺ signalling mechanisms linked specifically to group I metabotropic glutamate receptors (mGluR1α and mGluR5) and their role in hebbian long-term potentiation (LTP) in oriens/alveus (O/A) interneurones. We demonstrate that mGluR1α activation elicits dendritic Ca²⁺ signals resulting from Ca²⁺ influx via transient receptor potential (TRP) channels and Ca²⁺ release from intracellular stores. By contrast, mGluR5 activation produces dendritic Ca²⁺ transients mediated exclusively by intracellular Ca²⁺ release. Using Western blot analysis and immunocytochemistry, we show mGluR1α-specific extracellular signal-regulated kinase (ERK1/2) activation via Src in CA1 hippocampus and, in particular, in O/A interneurones. Moreover, we find that mGluR1α/TRP Ca²⁺ signals in interneurone dendrites are dependent on activation of the Src/ERK cascade. Finally, this mGluR1α-specific Ca²⁺ signalling controls LTP at interneurone synapses since blocking either TRP channels or Src/ERK and intracellular Ca²⁺ release prevents LTP induction. Thus, our findings uncover a novel molecular mechanism of interneurone-specific Ca²⁺ signalling, critical in regulating synaptic excitability in hippocampal networks.     

Mutation of Walker-A lysine 464 in cystic fibrosis transmembrane conductance regulator reveals functional interaction between its nucleotide-binding domains

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel bears two nucleotide-binding domains (NBD1 and NBD2) that control its ATP-dependent gating. Exactly how these NBDs control gating is controversial. To address this issue, we examined channels with a Walker-A lysine mutation in NBD1 (K464 A) using the patch clamp technique. K464A mutants have an ATP dependence (EC₅₀≈ 60 μ m ) and opening rate at 2.75 m m ATP (∼ 2.1 s⁻¹) similar to wild type (EC₅₀≈ 97 μ m ; ∼ 2.0 s⁻¹). However, K464A's closing rate at 2.75 m m ATP (∼ 3.6 s⁻¹) is faster than that of wild type (∼ 2.1 s⁻¹), suggesting involvement of NBD1 in nucleotide-dependent closing. Delay of closing in wild type by adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable ATP analogue, is markedly diminished in K464A mutants due to reduction in AMP-PNP's apparent on-rate and acceleration of its apparent off-rate (∼ 2- and ∼ 10-fold, respectively). Since the delay of closing by AMP-PNP is thought to occur via NBD2, K464A's effect on the NBD2 mutant K1250A was examined. In sharp contrast to K464A, K1250A single mutants exhibit reduced opening (∼ 0.055 s⁻¹) and closing (∼ 0.006 s⁻¹) rates at millimolar [ATP], suggesting a role for K1250 in both opening and closing. At millimolar [ATP], K464A-K1250A double mutants close ∼ 5-fold faster (∼ 0.029 s⁻¹) than K1250A but open with a similar rate (∼ 0.059 s⁻¹), indicating an effect of K464A on NBD2 function. In summary, our results reveal that both of CFTR's functionally asymmetric NBDs participate in nucleotide-dependent closing, which provides important constraints for NBD-mediated gating models.     

Clathrin-mediated endocytosis in snake motor terminals is directly facilitated by intracellular Ca2+

At the snake neuromuscular junction, low temperature (LT, 5–7°C) blocks clathrin-mediated endocytosis (CME) while exocytosis is largely unaffected. Thus compensatory endocytosis that normally follows transmitter release is inhibited, or 'delayed' until the preparation is warmed to room temperature (RT). This delay was exploited to observe how changes in bulk [Ca²⁺]_i directly affect CME. Motor terminals were loaded with fura-2 to monitor [Ca²⁺]_i. With brief stimulation at LT, [Ca²⁺]_i transiently increased but returned to baseline (∼63 n m ) in < 8 min. After 15 min at LT, [Ca²⁺]_i was altered by incubating preparations in the Ca²⁺ ionophore ionomyocin. Preparations were then warmed to RT to initiate delayed endocytosis, which was quantified as uptake of the fluorescent optical probe sulforhodamine 101. Endocytosis was more rapid when [Ca²⁺]_i increased; the rate at 300 n m Ca²⁺ was ∼double that under basal conditions. Thus the rate of CME – isolated from stimulation, transmitter release, and other forms of endocytosis – is directly influenced by intraterminal Ca²⁺.     

α-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet β-cells

α-Latrotoxin (α-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of α-LT on single human, mouse and canine β-cells. In isolated and combined measurements, α-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca²⁺, into the micromolar range, that are dependent on extracellular Ca²⁺; (ii) large conductance non-selective cation channels; and (iii) Ca²⁺-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, α-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca²⁺. These results strongly support the hypothesis that α-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) α-LT inserts into the plasma membrane to form Ca²⁺ permeable channels and promote Ca²⁺ entry thereby triggering Ca²⁺-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, α-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca²⁺-sensitive pool of insulin granules activated by global cytosolic Ca²⁺ concentrations in the range of ∼1 μ m .     

The sources and sequestration of Ca2+ contributing to neuroeffector Ca2+ transients in the mouse vas deferens

The detection of focal Ca²⁺ transients (called neuroeffector Ca²⁺ transients, or NCTs) in smooth muscle of the mouse isolated vas deferens has been used to detect the packeted release of ATP from nerve terminal varicosities acting at postjunctional P2X receptors. The present study investigates the sources and sequestration of Ca²⁺ in NCTs. Smooth muscle cells in whole mouse deferens were loaded with the Ca²⁺ indicator Oregon Green 488 BAPTA-1 AM and viewed with a confocal microscope. Ryanodine (10 µ m ) decreased the amplitude of NCTs by 45 ± 6 %. Cyclopiazonic acid slowed the recovery of NCTs (from a time course of 200 ± 10 ms to 800 ± 100 ms). Caffeine (3 m m ) induced spontaneous focal smooth muscle Ca²⁺ transients (sparks). Neither of the T-type Ca²⁺ channel blockers NiCl₂ (50 µ m ) or mibefradil dihydrochloride (10 µ m ) affected the amplitude of excitatory junction potentials (2 ± 5 % and −3 ± 10 %) or NCTs (−20 ± 36 % and 3 ± 13 %). In about 20 % of cells, NCTs were associated with a local, subcellular twitch that remained in the presence of the α₁-adrenoceptor antagonist prazosin (100 n m ), showing that NCTs can initiate local contractions. Slow (5.8 ± 0.4 µm s⁻¹), spontaneous smooth muscle Ca²⁺ waves were occasionally observed. Thus, Ca²⁺ stores initially amplify and then sequester the Ca²⁺ that enters through P2X receptors and there is no amplification by local voltage-gated Ca²⁺ channels.     

Ca2+-NFATc1 signaling is an essential axis of osteoclast differentiation

Summary:  Osteoclasts are unique, multinucleated giant cells that decalcify and degrade the bone matrix. They originate from hematopoietic cells and their differentiation is dependent on a tumor necrosis factor (TNF) family cytokine, receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL), as well as macrophage-colony stimulating factor (M-CSF). Recent studies have unveiled the precise molecular mechanism underlying osteoclastogenesis. In particular, the discovery of nuclear factor of activated T cells c1 (NFATc1), the master regulator of osteoclastogenesis, has proven to be a breakthrough in this field. NFATc1 is activated by Ca²⁺ signaling induced by the activation of the immunoglobulin-like receptor signaling associated with immunoreceptor tyrosine-based activation motif (ITAM)-harboring adapters. The long-lasting Ca²⁺ oscillation, which is evident during osteoclastogenesis, may ensure the robust induction of NFATc1 through an autoamplification mechanism. Thus, intracellular Ca²⁺ is a critical attribute of osteoclastogenic signaling. In addition, osteoclasts are exposed to a very high extracellular Ca²⁺ concentration ([Ca²⁺]_o) in the bone microenvironment and respond to the change in [Ca²⁺]_o by increasing the intracellular Ca²⁺, which regulates diverse cellular functions. Investigation of the molecular mechanisms underlying the regulation of intracellular Ca²⁺ dynamics may open up new directions for therapeutic strategies in bone disease.     

Physiological modulation of inactivation in L-type Ca2+ channels: one switch

The relative contributions of voltage- and Ca²⁺-dependent mechanisms of inactivation to the decay of L-type Ca²⁺ channel currents ( I _CaL) is an old story to which recent results have given an unexpected twist. In cardiac myocytes voltage-dependent inactivation (VDI) was thought to be slow and Ca²⁺-dependent inactivation (CDI) resulting from Ca²⁺ influx and Ca²⁺-induced Ca²⁺-release (CICR) from the sarcoplasmic reticulum provided an automatic negative feedback mechanism to limit Ca²⁺ entry and the contribution of I _CaL to the cardiac action potential. Physiological modulation of I _CaL by β-adrenergic and muscarinic agonists then involved essentially more or less of the same by enhancing or reducing Ca²⁺ channel activity, Ca²⁺ influx, sarcoplasmic reticulum load and thus CDI. Recent results on the other hand place VDI at the centre of the regulation of I _CaL. Under basal conditions it has been found that depolarization increases the probability that an ion channel will show rapid VDI. This is prevented by β-adrenergic stimulation. Evidence also suggests that a channel which shows rapid VDI inactivates before CDI can become effective. Therefore the contributions of VDI and CDI to the decay of I _CaL are determined by the turning on, by depolarization, and the turning off, by phosphorylation, of the mechanism of rapid VDI. The physiological implications of these ideas are that under basal conditions the contribution of I _CaL to the action potential will be determined largely by voltage and by Ca²⁺ following β-adrenergic stimulation.