Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca2+]i and Cl− channels

Regenerative potentials were initiated by depolarizing short segments of single bundles of circular muscle isolated from the gastric antrum of guinea-pigs. When changes in [Ca²⁺]_i and membrane potential were recorded simultaneously, regenerative potentials were found to be associated with an increase in [Ca²⁺]_i, with the increase starting after a minimum latency of about 1 s. Although the increase in [Ca²⁺]_i was reduced by nifedipine, the amplitudes of the regenerative responses were little changed. Regenerative responses and associated changes in [Ca²⁺]_i were abolished by loading the preparations with the Ca²⁺ chelator MAPTA-AM. Regenerative potentials were abolished by 2-aminoethoxydiphenyl borate (2APB), an inhibitor of IP₃ induced Ca²⁺ release, by N -ethylamaleimide (NEM), an alkylating agent which blocks activation of G-proteins and were reduced in amplitude by two agents which block chloride (Cl⁻)-selective channels in many tissues. The observations suggest that membrane depolarization triggers IP₃ formation. This causes Ca²⁺ release from intracellular stores which activates Ca²⁺-dependent Cl⁻ channels.     

IP3 receptors: some lessons from DT40 cells

Summary:  Inositol-1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels that are regulated by IP₃ and Ca²⁺ and are modulated by many additional signals. These properties allow them to initiate and, via Ca²⁺-induced Ca²⁺ release, regeneratively propagate Ca²⁺ signals evoked by receptors that stimulate formation of IP₃. The ubiquitous expression of IP₃R highlights their importance, but it also presents problems when attempting to resolve the behavior of defined IP₃R. DT40 cells are a pre-B-lymphocyte cell line in which high rates of homologous recombination afford unrivalled opportunities to disrupt endogenous genes. DT40-knockout cells with both alleles of each of the three IP₃R genes disrupted provide the only null-background for analysis of homogenous recombinant IP₃R. We review the properties of DT40 cells and consider three areas where they have contributed to understanding IP₃R behavior. Patch-clamp recording from the nuclear envelope and Ca²⁺ release from intracellular stores loaded with a low-affinity Ca²⁺ indicator address the mechanisms leading to activation of IP₃R. We show that IP₃ causes intracellular IP₃R to cluster and re-tune their responses to IP₃ and Ca²⁺, better equipping them to mediate regenerative Ca²⁺ signals. Finally, we show that DT40 cells reliably count very few IP₃R into the plasma membrane, where they mediate about half the Ca²⁺ entry evoked by the B-cell antigen receptor.     

Discrete influx events refill depleted Ca2+ stores in a chick retinal neuron

The depletion of ER Ca²⁺ stores, following the release of Ca²⁺ during intracellular signalling, triggers the Ca²⁺ entry across the plasma membrane known as store-operated calcium entry (SOCE). We show here that brief, local [Ca²⁺]_i increases (motes) in the thin dendrites of cultured retinal amacrine cells derived from chick embryos represent the Ca²⁺ entry events of SOCE and are initiated by sphingosine-1-phosphate (S1P), a sphingolipid with multiple cellular signalling roles. Externally applied S1P elicits motes but not through a G protein-coupled membrane receptor. The endogenous precursor to S1P, sphingosine, also elicits motes but its action is suppressed by dimethylsphingosine (DMS), an inhibitor of sphingosine phosphorylation. DMS also suppresses motes induced by store depletion and retards the refilling of depleted stores. These effects are reversed by exogenously applied S1P. In these neurons formation of S1P is a step in the SOCE pathway that promotes Ca²⁺ entry in the form of motes.     

Priming of intracellular calcium stores in rat CA1 pyramidal neurons

Repetitive synaptic stimulation evokes large amplitude Ca²⁺ release waves from internal stores in many kinds of pyramidal neurons. The waves result from mGluR mobilization of IP₃ leading to Ca²⁺-induced Ca²⁺ release. In most experiments in slices, regenerative Ca²⁺ release can be evoked for only a few trials. We examined the conditions required for consistent release from the internal stores in hippocampal CA1 pyramidal neurons. We found that priming with action potentials evoked at 0.5–1 Hz for intervals as short as 15 s were sufficient to fill the stores, while sustained subthreshold depolarization or subthreshold synaptic stimulation lasting from 15 s to 2 min was less effective. A single episode of priming was effective for about 2–3 min. Ca²⁺ waves could also be evoked by uncaging IP₃ with a UV flash in the dendrites. Priming was necessary to evoke these waves repetitively; 7–10 spikes in 15 s were again effective for this protocol, indicating that priming acts to refill the stores and not at a site upstream to the production of IP₃. These results suggest that normal spiking activity of pyramidal neurons in vivo should be sufficient to maintain their internal stores in a primed state ready to release Ca²⁺ in response to an appropriate physiological stimulus. This may be a novel form of synaptic plasticity where a cell's capacity to release Ca²⁺ is modulated by its average firing frequency.     

Buffer Kinetics Shape the Spatiotemporal Patterns of IP3-Evoked Ca2+ Signals

Ca²⁺ liberation through inositol 1,4,5-trisphosphate receptors (IP₃Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca²⁺ signals. IP₃Rs display Ca²⁺-induced Ca²⁺ release (CICR), but are grouped in clusters so that regenerative Ca²⁺ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca²⁺ diffusion and CICR. We used confocal microscopy and photoreleased IP₃ in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca²⁺ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca²⁺ signals and 'balkanized' Ca²⁺ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca²⁺ responses and promoted 'globalization' of spatially uniform Ca²⁺ signals. These actions are likely to arise through differential effects on Ca²⁺ feedback within and between IP₃R clusters, because Ca²⁺ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca²⁺-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP₃-evoked Ca²⁺ signals for specific physiological roles.     

Calcium events in smooth muscles and their interstitial cells; physiological roles of sparks

The observation of spontaneous sporadic releases of packets of stored calcium made 20 years ago has opened up a number of new concepts in smooth muscle physiology: (1) the calcium release sites are ryanodine and inositol 1,4,5-trisphosphate (IP₃) receptor channels which contribute to cell-wide increases in [Ca²⁺]_i in response to cell depolarization, activation of IP₃-generating receptors, or other stimuli; (2) changes in [Ca²⁺]_i act back on the cell membrane to activate or modulate K⁺, Cl⁻ and cation channel activity so affecting contraction, in arterial smooth muscle for example affecting blood pressure; (3) IP₃ production is voltage dependent and is believed to contribute to pacemaker potentials and to refractory periods which control the rhythmical motility of many hollow organs. Most smooth muscle tissues contain interstitial cells (ICs) in addition to contractile smooth muscle cells (SMCs). The interactions of these internal mechanisms, and in turn the interactions of SMCs and ICs in various smooth muscle tissues, are major factors in determining the unique physiological profiles of individual smooth muscles.     

Microheterogeneity of calcium signalling in dendrites

Transient changes in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) originating from voltage- or ligand-gated influx and by ligand- or Ca²⁺-gated release from intracellular stores, trigger or modulate many fundamental neuronal processes, including neurotransmitter release and synaptic plasticity. Of the intracellular compartments involved in Ca²⁺ clearance, the endoplasmic reticulum (ER) has received the most attention because it expresses Ca²⁺ pumps and Ca²⁺ channels, thus endowing it with the potential to act as both an intracellular calcium sink and store. We review here our ongoing work on the role of calcium sequestration into, and release from, ER cisterns and the role that this plays in the generation and termination of free [Ca²⁺]_i transients in dendrites of pyramidal neurons in hippocampal slices during and after synaptic activity. These studies have been approached by combining parallel microfluorometric measurements of free cytosolic [Ca²⁺]_i transients with energy-dispersive X-ray microanalytical measurements of total Ca content within specific dendritic compartments at the electron microscopy level. Our observations support the emerging realization that specific subsets of dendritic ER cisterns provide spatial and temporal microheterogeneity of Ca²⁺ signalling, acting not only as a major intracellular Ca sink involved in active clearance mechanisms after voltage- and ligand-gated Ca²⁺ influx, but also as an intracellular Ca²⁺ source that can be mobilized by a signal cascade originating at activated synapses.     

Capacitative calcium entry supports calcium oscillations in human embryonic kidney cells

Treatment of human epithelial kidney (HEK293) cells with low concentrations of the muscarinic agonist methacholine results in the activation of complex and repetitive cycling of intracellular calcium ([Ca²⁺]_i), known as [Ca²⁺]_i oscillations. These oscillations occur with a frequency that depends on the concentration of methacholine, whereas the magnitude of the [Ca²⁺]_i spikes does not. The oscillations do not persist in the absence of extracellular Ca²⁺, leading to the conclusion that entry of Ca²⁺ across the plasma membrane plays a significant role in either their initiation or maintenance. However, treatment of cells with high concentrations of GdCl₃, a condition which limits the flux of calcium ions across the plasma membrane in both directions, allows sustained [Ca²⁺]_i oscillations to occur. This suggests that the mechanisms that both initiate and regenerate [Ca²⁺]_i oscillations are intrinsic to the intracellular milieu and do not require entry of extracellular Ca²⁺. This would additionally suggest that, under normal conditions, the role of calcium entry is to sustain [Ca²⁺]_i oscillations. By utilizing relatively specific pharmacological manoeuvres we provide evidence that the Ca²⁺ entry that supports Ca²⁺ oscillations occurs through the store-operated or capacitative calcium entry pathway. However, by artificial introduction of a non-store-operated pathway into the cells (TRPC3 channels), we find that other Ca²⁺ entry mechanisms can influence oscillation frequency in addition to the store-operated channels.     

Discrete store-operated calcium influx into an intracellular compartment in rabbit arteriolar smooth muscle

This study tested the hypothesis that store-operated channels (SOCs) exist as a discrete population of Ca²⁺ channels activated by depletion of intracellular Ca²⁺ stores in cerebral arteriolar smooth muscle cells and explored their direct contractile function. Using the Ca²⁺ indicator fura-PE3 it was observed that depletion of sarcoplasmic reticulum (SR) Ca²⁺ by inhibition of SR Ca²⁺-ATPase (SERCA) led to sustained elevation of [Ca²⁺]_i that depended on extracellular Ca²⁺ and slightly enhanced Mn²⁺ entry. Enhanced background Ca²⁺ influx did not explain the raised [Ca²⁺]_i in response to SERCA inhibitors because it had marked gadolinium (Gd³⁺) sensitivity, which background pathways did not. Effects were not secondary to changes in membrane potential. Thus SR Ca²⁺ depletion activated SOCs. Strikingly, SOC-mediated Ca²⁺ influx did not evoke constriction of the arterioles, which were in a resting state. This was despite the fura-PE3-indicated [Ca²⁺]_i rise being greater than that evoked by 20 m m [K⁺]_o (which did cause constriction). Release of endothelial vasodilators did not explain the absence of SOC-mediated constriction, nor did a change in Ca²⁺ sensitivity of the contractile proteins. We suggest SOCs are a discrete subset of Ca²⁺ channels allowing Ca²⁺ influx into a 'non-contractile' compartment in cerebral arteriolar smooth muscle cells.     

Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium-imaging study

The non-linear and spatially inhomogeneous interactions of dendritic membrane potential signals that represent the first step in the induction of activity-dependent long-term synaptic plasticity are not fully understood, particularly in dendritic regions which are beyond the reach of electrode measurements. We combined voltage-sensitive-dye recordings and Ca²⁺ imaging of hippocampal CA1 pyramidal neurons to study large regions of the dendritic arbor, including branches of small diameter (distal apical and oblique dendrites). Dendritic membrane potential transients were monitored at high spatial resolution and correlated with supra-linear [Ca²⁺]_i changes during one cycle of a repetitive patterned stimulation protocol that typically results in the induction of long-term potentiation (LTP). While the increase in the peak membrane depolarization during coincident pre- and post-synaptic activity was required for the induction of supra-linear [Ca²⁺]_i signals shown to be necessary for LTP, the change in the baseline-to-peak amplitude of the backpropagating dendritic action potential (bAP) was not critical in this process. At different dendritic locations, the baseline-to-peak amplitude of the bAP could be either increased, decreased or unaltered at sites where EPSP–AP pairing evoked supra-linear summation of [Ca²⁺]_i transients. We suggest that modulations in the bAP baseline-to-peak amplitude by local EPSPs act as a mechanism that brings the membrane potential into the optimal range for Ca²⁺ influx through NMDA receptors (0 to −15 mV); this may require either boosting or the reduction of the bAP, depending on the initial size of both signals.     

Hypo-osmotic potentiation of acetylcholine-stimulated ciliary beat frequency through ATP release in rat tracheal ciliary cells

The ciliary beat frequency (CBF) of rat tracheal ciliary cells in a slice preparation was measured using video-enhanced contrast (VEC) microscopy. Acetylcholine (ACh) increased CBF mediated via intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. An adequate hypo-osmotic stress (−40 mos m ) potentiated ACh-stimulated CBF increase in tracheal ciliary cells and shifted the ACh dose–response curve to the left (lower concentration side). This potentiation was independent of hypo-osmotic stresses applied ranging from −20 mosM to −90 mosM. A hypo-osmotic stress induces ATP release in many cell types. The present study demonstrated that suramin (an inhibitor of purinergic receptors) and apyrase (an ATPase/ADPase) eliminate the hypo-osmotic potentiation of ACh-stimulated CBF increase and that ATP increased [Ca²⁺]_i and CBF, as well as potentiating ACh-stimulated rises in [Ca²⁺]_i and CBF increase. Moreover, the apical surface of tracheal ciliary cells were stained immunopositive for the P2X₄ purinergic receptor. A hypo-osmotic stress (−40 mosM) transiently increased [Ca²⁺]_i and potentiated the ACh-stimulated [Ca²⁺]_i increase. The hypo-osmotic potentiation of ACh-stimulated CBF increase was not detected under Ca²⁺-free conditions. These observations suggest that a hypo-osmotic stress stimulates ATP release from the trachea. The released ATP may induce further increases in [Ca²⁺]_i and CBF in ACh-stimulated tracheal ciliary cells, which may be mediated by purinergic receptors, such as P2X₄.     

Endocannabinoid signalling triggered by NMDA receptor-mediated calcium entry into rat hippocampal neurons

Endocannabinoids are released from neurons in activity-dependent manners, act retrogradely on presynaptic CB₁ cannabinoid receptors, and induce short-term or long-term suppression of transmitter release. The endocannabinoid release is triggered by postsynaptic activation of voltage-gated Ca²⁺ channels and/or G_q-coupled receptors such as group I metabotropic glutamate receptors (I-mGluRs) and M₁/M₃ muscarinic receptors. However, the roles of NMDA receptors, which provide another pathway for Ca²⁺ entry into neurons, in endocannabinoid signalling have been poorly understood. In the present study, we investigated the possible contribution of NMDA receptors in endocannabinoid production by recording IPSCs in cultured hippocampal neurons. Under the conditions minimizing the activation of voltage-gated Ca²⁺ channels, local application of NMDA (200 μ m ) transiently suppressed cannabinoid-sensitive IPSCs, but not cannabinoid-insensitive IPSCs. This NMDA-induced suppression was abolished by blocking NMDA receptors, CB₁ receptors and diacylglycerol lipase, but not by inhibiting voltage-gated Ca²⁺ channels. When the postsynaptic neuron was dialysed with 30 m m BAPTA, the NMDA-induced suppression was reduced significantly. A lower dose of NMDA (20 μ m ) exerted little effect when applied alone, but markedly enhanced the cannabinoid-dependent suppression driven by muscarinic receptors or I-mGluRs. These data clearly indicate that the activation of NMDA receptors facilitates the endocannabinoid release either alone or in concert with the G_q-coupled receptors.     

Roles of Cav channels and AHNAK1 in T cells: the beauty and the beast

Summary:  T lymphocytes require Ca²⁺ entry though the plasma membrane for their activation and function. Recently, several routes for Ca²⁺ entry through the T-cell plasma membrane after activation have been described. These include calcium release-activated channels (CRAC), transient receptor potential (TRP) channels, and inositol-1,4,5-trisphosphate receptors (IP₃Rs). Herein we review the emergence of a fourth new route for Ca²⁺ entry, composed of Ca_v channels (also known as L-type voltage-gated calcium channels) and the scaffold protein AHNAK1 (AHNAK/desmoyokin). Both helper (CD4⁺) and killer (CD8⁺) T cells express high levels of Ca_v1 α1 subunits (α1S, α1C, α1D, and α1F) and AHNAK1 after their differentiation and require these molecules for Ca²⁺ entry during an immune response. In this article, we describe the observations and open questions that ultimately suggest the involvement of multiple consecutive routes for Ca²⁺ entry into lymphocytes, one of which may be mediated by Ca_v channels and AHNAK1.     

Capacitative calcium entry: from concept to molecules

Summary:  Rapid to moderately rapid changes in intracellular Ca²⁺ concentration, or Ca²⁺ signals, control a variety of critical cellular functions in the immune system. These signals are comprised of Ca²⁺ release from intracellular stores coordinated with Ca²⁺ influx across the plasma membrane. The most common mechanisms by which these two modes of signaling occur is through inositol 1,4,5-trisphosphate (IP₃)-induced release of Ca²⁺ from the endoplasmic reticulum (ER) and store-operated Ca²⁺ entry across the plasma membrane. The latter process was postulated over 20 years ago, and in just the past few years, the key molecular players have been discovered: STIM proteins serve as sensors of Ca²⁺ within the ER which communicate with and activate plasma membrane store-operated channels composed of Orai subunits. The process of store-operated Ca²⁺ entry provides support for oscillating Ca²⁺ signals from the ER and also provides direct activator Ca²⁺ that signals to a variety of downstream effectors.     

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.     

The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex

Loading slices of rat barrel cortex with 50 μ m BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca²⁺ stores with 20 μ m cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca²⁺ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca²⁺ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μ m ryanodine or 10 m m caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca²⁺ release from ryanodine stores. Blocking IP₃ receptors with 14 μ m 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP₃ stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μ m ( RS) -3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP₃ receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine stores after the spontaneous opening of IP₃ stores.     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells

We have investigated the nature of the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca²⁺ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca²⁺]_i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca²⁺. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca²⁺ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca²⁺ extrusion with a high concentration of Gd³⁺, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca²⁺]_i oscillations, and this was reversed by use of high concentrations of Gd³⁺; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca²⁺ entry in HEK293 cells. These findings indicate that the Ca²⁺ entry supporting [Ca²⁺]_i oscillations in HEK293 cells depends upon the Ca²⁺ sensor, Stim1, and calcium release-activated Ca²⁺ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.     

Calmodulin kinase modulates Ca2+ release in mouse skeletal muscle

Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca²⁺ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca²⁺ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro , but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca²⁺] ([Ca²⁺]_i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]_i by ≈25 %, but had no significant effect on the rate of SR Ca²⁺ uptake or the force-[Ca²⁺]_i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca²⁺]_i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca²⁺ release in the basal state and during repeated contractions, providing a positive feedback between [Ca²⁺]_i and SR Ca²⁺ release.     

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum

Unloading of endoplasmic reticulum (ER) Ca²⁺ stores activates influx of extracellular Ca²⁺ through 'store-operated' Ca²⁺ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca²⁺ stores. Here, using high resolution imaging with the membrane-associated Ca²⁺ indicator, FFP-18, it is shown that store-operated Ca²⁺ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane–ER junctions. In the absence of extracellular Ca²⁺, depletion of ER Ca²⁺ stores using cyclopiazonic acid, an ER Ca²⁺-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca²⁺ concentration ([Ca²⁺]_SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca²⁺ causes localized Ca²⁺ influx that first increases [Ca²⁺]_SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs , markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca²⁺ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca²⁺ stores.