Modulation of Ca2+ release and Ca2+ oscillations in HeLa cells and fibroblasts by mitochondrial Ca2+ uniporter stimulation

The recent availability of activators of the mitochondrial Ca²⁺ uniporter allows direct testing of the influence of mitochondrial Ca²⁺ uptake on the overall Ca²⁺ homeostasis of the cell. We show here that activation of mitochondrial Ca²⁺ uptake by 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) or kaempferol stimulates histamine-induced Ca²⁺ release from the endoplasmic reticulum (ER) and that this effect is enhanced if the mitochondrial Na⁺–Ca²⁺ exchanger is simultaneously inhibited with CGP37157. This suggests that both Ca²⁺ uptake and release from mitochondria control the ability of local Ca²⁺ microdomains to produce feedback inhibition of inositol 1,4,5-trisphosphate receptors (InsP₃Rs). In addition, the ability of mitochondria to control Ca²⁺ release from the ER allows them to modulate cytosolic Ca²⁺ oscillations. In histamine stimulated HeLa cells and human fibroblasts, both PPT and kaempferol initially stimulated and later inhibited oscillations, although kaempferol usually induced a more prolonged period of stimulation. Both compounds were also able to induce the generation of Ca²⁺ oscillations in previously silent fibroblasts. Our data suggest that cytosolic Ca²⁺ oscillations are exquisitely sensitive to the rates of mitochondrial Ca²⁺ uptake and release, which precisely control the size of the local Ca²⁺ microdomains around InsP₃Rs and thus the ability to produce feedback activation or inhibition of Ca²⁺ release.     

Ca2+-dependent generation of mitochondrial reactive oxygen species serves as a signal for poly(ADP-ribose) polymerase-1 activation during glutamate excitotoxicity

Mitochondrial Ca²⁺ uptake and poly(ADP-ribose) polymerase-1 (PARP-1) activation are both required for glutamate-induced excitotoxic neuronal death. Since activation of the glutamate receptors can induce increased levels of reactive oxygen species (ROS), we investigated the relationship of mitochondrial Ca²⁺ uptake and ROS generation, and the possibility that ROS increase is a required signal for PARP-1 activation in cultured striatal neurons. Based on the spatial profile of NMDA-induced ROS generation, we found that only mitochondria showed a significant ROS increase within 30 min after NMDA receptor activation. This ROS increase was inhibited by the mitochondrial complex inhibitors rotenone and oligomycin, but not by the cytosolic phospholipase A₂ or xanthine oxidase inhibitors. Mitochondrial ROS generation was also inhibited by both removal of Ca²⁺ from extracellular medium and blockage of mitochondrial Ca²⁺ uptake by either a mitochondrial uncoupler or a Ca²⁺ uniporter inhibitor. Furthermore, both DNA damage and PARP-1 activation induced by NMDA treatment was inhibited by blocking mitochondrial Ca²⁺ uptake or by antioxidants. Our results demonstrate that ROS production during the early stage of acute excitotoxicity derives primarily from mitochondria and is Ca²⁺-dependent. More importantly, the increase of mitochondrial ROS serves as a signal for PARP-1 activation, suggesting that concomitant mitochondrial Ca²⁺ uptake and PARP-1 activation constitute a unified mechanism for excitotoxic neuronal death.     

Mitochondria as all-round players of the calcium game

Although it has been known for over three decades that mitochondria are endowed with a complex array of Ca²⁺ transporters and that key enzymes of mitochondrial metabolism are regulated by Ca²⁺, the possibility that physiological stimuli that raise the [Ca²⁺] of the cytoplasm could trigger major mitochondrial Ca²⁺ uptake has long been considered unlikely, based on the low affinity of the mitochondrial transporters and the limited amplitude of the cytoplasmic [Ca²⁺] rises. The direct measurement of mitochondrial [Ca²⁺] with highly selective probes has led to a complete reversion of this view, by demonstrating that, after cell stimulation, the cytoplasmic Ca²⁺ signal is always paralleled by a much larger rise in [Ca²⁺] in the mitochondrial matrix. This observation has rejuvenated the study of mitochondrial Ca²⁺ transport and novel, unexpected results have altered long-standing dogmas in the field of calcium signalling. Here we focus on four main topics: (i) the current knowledge of the functional properties of the Ca²⁺ transporters and of the thermodynamic constraints under which they operate; (ii) the occurrence of mitochondrial Ca²⁺ uptake in living cells and the key role of local signalling routes between the mitochondria and the Ca²⁺ sources; (iii) the physiological consequences of Ca²⁺ transport for both mitochondrial function and the modulation of the cytoplasmic Ca²⁺ signal; and (iv) evidence that alterations of mitochondrial Ca²⁺ signalling may occur in pathophysiological conditions.     

ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond

Summary:  Store-operated Ca²⁺ entry (SOCE) is a mechanism used by many cells types including lymphocytes and other immune cells to increase intracellular Ca²⁺ concentrations to initiate signal transduction. Activation of immunoreceptors such as the T-cell receptor, B-cell receptor, or Fc receptors results in the release of Ca²⁺ ions from endoplasmic reticulum (ER) Ca²⁺ stores and subsequent activation of plasma membrane Ca²⁺ channels such as the well-characterized Ca²⁺ release-activated Ca²⁺ (CRAC) channel. Two genes have been identified that are essential for SOCE: ORAI1 as the pore-forming subunit of the CRAC channel in the plasma membrane and stromal interaction molecule-1 (STIM1) sensing the ER Ca²⁺ concentration and activating ORAI1-CRAC channels. Intense efforts in the past several years have focused on understanding the molecular mechanism of SOCE and the role it plays for cell functions in vitro and in vivo . A number of transgenic mouse models have been generated to investigate the role of ORAI1 and STIM1 in immunity. In addition, mutations in ORAI1 and STIM1 identified in immunodeficient patients provide valuable insight into the role of both genes and SOCE. This review focuses on the role of ORAI1 and STIM1 in vivo , discussing the phenotypes of ORAI1- and STIM1-deficient human patients and mice.     

Metabolic Regulation of Ca2+ Release in Permeabilized Mammalian Skeletal Muscle Fibres

In the present study, the link between cellular metabolism and Ca²⁺ signalling was investigated in permeabilized mammalian skeletal muscle. Spontaneous events of Ca²⁺ release from the sarcoplasmic reticulum were detected with fluo-3 and confocal scanning microscopy. Mitochondrial functions were monitored by measuring local changes in mitochondrial membrane potential (with the potential-sensitive dye tetramethylrhodamine ethyl ester) and in mitochondrial [Ca²⁺] (with the Ca²⁺ indicator mag-rhod-2). Digital fluorescence imaging microscopy was used to quantify changes in the mitochondrial autofluorescence of NAD(P)H. When fibres were immersed in a solution without mitochondrial substrates, Ca²⁺ release events were readily observed. The addition of l -glutamate or pyruvate reversibly decreased the frequency of Ca²⁺ release events and increased mitochondrial membrane potential and NAD(P)H production. Application of various mitochondrial inhibitors led to the loss of mitochondrial [Ca²⁺] and promoted spontaneous Ca²⁺ release from the sarcoplasmic reticulum. In many cases, the increase in the frequency of Ca²⁺ release events was not accompanied by a rise in global [Ca²⁺]_i. Our results suggest that mitochondria exert a negative control over Ca²⁺ signalling in skeletal muscle by buffering Ca²⁺ near Ca²⁺ release channels.     

Checking your SOCCs and feet: the molecular mechanisms of Ca2+ entry in skeletal muscle

It has long been known that skeletal muscle contraction persists in the absence of extracellular Ca²⁺. Nevertheless, recent evidence indicates that multiple distinct Ca²⁺ entry pathways exist in skeletal muscle: one active at negative potentials that requires store depletion (store-operated calcium entry or SOCE) and a second that is independent of store depletion and is activated by depolarization (excitation-coupled calcium entry or ECCE). This review highlights recent findings regarding the molecular identity, subcellular localization, and inter-relationship between SOCE and ECCE in skeletal muscle. The respective roles of ryanodine receptors (RyRs), dihydropyridine receptors (DHPRs), inositol-1,4,5-trisphosphate receptors (IP₃Rs), canonical transient receptor potential channels (TRPCs), STIM1 Ca²⁺ sensor proteins, and Orai1 Ca²⁺ permeable channels in mediating SOCE and ECCE in skeletal muscle are discussed. Differences between SOCE and ECCE in skeletal muscle with Ca²⁺ entry mechanisms in non-excitable cells are also reviewed. Finally, potential physiological roles for SOCE and ECCE in skeletal muscle development and function, as well as other currently unanswered questions and controversies in the field are also considered.     

Properties of spontaneously active cells distributed in the submucosal layer of mouse proximal colon

Intracellular electrical activity was recorded from smooth muscle tissues of the mouse proximal colon, and the impaled cells were visualized by injection of neurobiotin. Slow potentials with initial fast and subsequent plateau components (plateau potentials), generated at a frequency of 14.8 min⁻¹, were recorded from oval-shaped cells with bipolar processes. Periodic bursts of spike potentials (4.6 min⁻¹) and bursts of oscillatory potentials (4.3 min⁻¹) were recorded in circular and longitudinal smooth muscle cells, respectively. Nifedipine (0.1 μ m ) abolished the bursts of spike and oscillatory potentials and reduced the duration of plateau potentials. The plateau potentials were abolished by 1 μ m nifedipine. The plateau potentials were also abolished by cyclopiazonic acid (an inhibitor of Ca²⁺ uptake into internal stores) or 2-aminoethoxydiphenyl borate (an inhibitor of inositol 1,4,5-trisphosphate receptor-mediated Ca²⁺ release), and were inhibited by bis-(aminophenoxy) ethane- N,N,N ', N '-tetraacetic acid acetoxymethyl ester (a chelator of intracellular Ca²⁺). Carbonyl cyanide m -chlorophenylhydrazone (a mitochondrial protonophore) abolished plateau potentials, and its action was not mimicked by oligomycin (an inhibitor of mitochondrial ATPase). It is concluded that in mouse proximal colon, submucosal c-kit-positive bipolar cells spontaneously generate plateau potentials with rhythms different from those generated by smooth muscle cells. The plateau potentials are generated through activation of voltage-gated Ca²⁺ channels, which are coupled to the release of Ca²⁺ from the internal stores and the handling of Ca²⁺ in mitochondria.     

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.     

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.     

Mitochondrial calcium sequestration and protein kinase C cooperate in the regulation of cortical F-actin disassembly and secretion in bovine chromaffin cells

Mitochondria play an important role in the homeostasis of intracellular Ca²⁺ and regulate its availability for exocytosis. Inhibitors of mitochondria Ca²⁺ uptake such as protonophore CCCP potentiate the secretory response to a depolarizing pulse of K⁺. Exposure of cells to agents that directly (cytochalasin D, latrunculin B) or indirectly (PMA) disrupt cortical F-actin networks also potentiate the secretory response to high K⁺. The effects of cytochalasin D and CCCP on secretion were additive whereas those of PMA and CCCP were not; this suggests different mechanisms for cytochalasin D and CCCP and a similar mechanism for PMA and CCCP. Mitochondria were the site of action of CCCP, because the potentiation of secretion by CCCP was observed even after depletion of Ca²⁺ from the endoplasmic reticulum. CCCP induced a small increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_c) that was not modified by the protein kinase C (PKC) inhibitor chelerythrine. Both CCCP and PMA induced cortical F-actin disassembly, an effect abolished by chelerythrine. In addition, rotenone and oligomycin A, two other mitochondrial inhibitors, also evoked cortical F-actin disassembly and potentiated secretion; again, these effects were blocked by chelerythrine. CCCP also enhanced the phosphorylation of PKC and myristoylated alanine-rich C kinase substance (MARCKS), and these were also inhibited by chelerythrine. The results suggest that the rapid sequestration of Ca²⁺ by mitochondria would protect the cell from an enhanced PKC activation and cortical F-actin disassembly, thereby limiting the magnitude of the secretory response.     

Calcium regulation in individual peripheral sensory nerve terminals of the rat

Ca²⁺ is vital for release of neurotransmitters and trophic factors from peripheral sensory nerve terminals (PSNTs), yet Ca²⁺ regulation in PSNTs remains unexplored. To elucidate the Ca²⁺ regulatory mechanisms in PSNTs, we determined the effects of a panel of pharmacological agents on electrically evoked Ca²⁺ transients in rat corneal nerve terminals (CNTs) in vitro that had been loaded with the fluorescent Ca²⁺ indicator, Oregon Green 488 BAPTA-1 dextran or fura-2 dextran in vivo . Inhibition of the sarco(endo)plasmic reticulum Ca²⁺-ATPase, disruption of mitochondrial Ca²⁺ uptake, or inhibition of the Na⁺–Ca²⁺ exchanger did not measurably alter the amplitude or decay kinetics of the electrically evoked Ca²⁺ transients in CNTs. By contrast, inhibition of the plasma membrane Ca²⁺-ATPase (PMCA) by increasing the pH slowed the decay of the Ca²⁺ transient by 2-fold. Surprisingly, the energy for ion transport across the plasma membrane of CNTs is predominantly from glycolysis rather than mitochondrial respiration, as evidenced by the observation that Ca²⁺ transients were suppressed by iodoacetate but unaffected by mitochondrial inhibitors. These observations indicate that, following electrical activity, the PMCA is the predominant mechanism of Ca²⁺ clearance from the cytosol of CNTs and glycolysis is the predominant source of energy.     

Short-term potentiation of mEPSCs requires N-, P/Q- and L-type Ca2+ channels and mitochondria in the supraoptic nucleus

The glutamatergic synapses of the supraoptic nucleus display a unique activity-dependent plasticity characterized by a barrage of tetrodotoxin-resistant miniature EPSCs (mEPSCs) persisting for 5–20 min, causing postsynaptic excitation. We investigated how this short-term synaptic potentiation (STP) induced by a brief high-frequency stimulation (HFS) of afferents was initiated and maintained without lingering presynaptic firing, using in vitro patch-clamp recording on rat brain slices. We found that following the immediate rise in mEPSC frequency, STP decayed with two-exponential functions indicative of two discrete phases. STP depends entirely on extracellular Ca²⁺ which enters the presynaptic terminals through voltage-gated Ca²⁺ channels but also, to a much lesser degree, through a pathway independent of these channels or reverse mode of the plasma membrane Na⁺–Ca²⁺ exchanger. Initiation of STP is largely mediated by any of the N-, P/Q- or L-type channels, and only a simultaneous application of specific blockers for all these channels attenuates STP. Furthermore, the second phase of STP is curtailed by the inhibition of mitochondrial Ca²⁺ uptake or mitochondrial Na⁺–Ca²⁺ exchanger. mEPSCs amplitude is also potentiated by HFS which requires extracellular Ca²⁺. In conclusion, induction of mEPSC-STP is redundantly mediated by presynaptic N-, P/Q- and L-type Ca²⁺ channels while the second phase depends on mitochondrial Ca²⁺ sequestration and release. Since glutamate influences unique firing patterns that optimize hormone release by supraoptic magnocellular neurons, a prolonged barrage of spontaneous excitatory transmission may aid in the induction of respective firing activities.     

Propagation of slow waves requires IP3 receptors and mitochondrial Ca2+ uptake in canine colonic muscles

In the gastrointestinal (GI) tract electrical slow waves yield oscillations in membrane potential that periodically increase the open probability of voltage-dependent Ca²⁺ channels and facilitate phasic contractions. Slow waves are generated by the interstitial cells of Cajal (ICC), and these events actively propagate through ICC networks within the walls of GI organs. The mechanism that entrains spontaneously active pacemaker sites throughout ICC networks to produce regenerative propagation of slow waves is unresolved. Agents that block inositol 1,4,5-trisphosphate (IP₃) receptors and mitochondrial Ca²⁺ uptake were tested on the generation of slow waves in the canine colon. A partitioned chamber apparatus was used to test the effects of blocking slow-wave generation on propagation. We found that active propagation occurred along strips of colonic muscle, but when the pacemaker mechanism was blocked in a portion of the tissue, slow waves decayed exponentially from the point where the pacemaker mechanism was inhibited. An IP₃ receptor inhibitor, mitochondrial inhibitors, low external Ca²⁺, and divalent cations (Mn²⁺ and Ni²⁺) caused exponential decay of the slow waves in regions of muscle exposed to these agents. These data demonstrate that the mechanism that initiates slow waves is reactivated from cell-to-cell during the propagation of slow waves. Voltage-dependent conductances present in smooth muscle cells are incapable of slow-wave regeneration. The data predict that partial loss of or disruptions to ICC networks observed in human motility disorders could lead to incomplete penetration of slow waves through GI organs and, thus, to defects in myogenic regulation.     

Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse

Hypoglossal motoneurones (HMN) are selectively damaged in both human amyotrophic lateral sclerosis (ALS) and corresponding mouse models of this neurodegenerative disease, a process which has been linked to their low endogenous Ca²⁺ buffering capacity and an exceptional vulnerability to Ca²⁺-mediated excitotoxic events. In this report, we investigated local Ca²⁺ profiles in low buffered HMNs by utilizing multiphoton microscopy, CCD imaging and patch clamp recordings in slice preparations. Bath application of caffeine induced highly localized Ca²⁺ release events, which displayed an initial peak followed by a slow 'shoulder' lasting several seconds. Peak amplitudes were paralleled by Ca²⁺-activated, apamin-sensitive K⁺ currents ( I _KCa), demonstrating a functional link between Ca²⁺ stores and HMN excitability. The potential involvement of mitochondria was investigated by bath application of CCCP, which collapses the electrochemical potential across the inner mitochondrial membrane. CCCP reduced peak amplitudes of caffeine responses and consequently I _KCa, indicating that functionally intact mitochondria were critical for store-dependent modulation of HMN excitability. Taken together, our results indicate localized Ca²⁺ release profiles in HMNs, where low buffering capacities enhance the role of Ca²⁺-regulating organelles as local determinants of [Ca²⁺]_i. This might expose HMN to exceptional risks during pathophysiological organelle disruptions and other ALS-related, cellular disturbances.     

Orai, STIM1 and iPLA2beta: a view from a different perspective

The mechanism of store-operated Ca²⁺ entry (SOCE) remains one of the intriguing mysteries in the field of Ca²⁺ signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca²⁺ sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA₂β as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA₂β, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA₂β in the SOCE pathway.     

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.     

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.     

Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D

Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca²⁺-induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca²⁺-induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 versus 475 ± 33 nmol (mg protein)⁻¹ for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca²⁺ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca²⁺ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.     

Distinct contributions of small and large conductance Ca2+-activated K+ channels to rat Purkinje neuron function

The cerebellum is important for many aspects of behaviour, from posture maintenance and goal-oriented reaching movements to timing tasks and certain forms of learning. In every case, information flowing through the cerebellum passes through Purkinje neurons, which receive input from the two primary cerebellar afferents and generate continuous streams of action potentials that constitute the sole output from the cerebellar cortex to the deep nuclei. The tonic firing behaviour observed in Purkinje neurons in vivo is maintained in brain slices even when synaptic inputs are blocked, suggesting that Purkinje neuron activity relies to a significant extent on intrinsic conductances. Previous research has suggested that the interplay between Ca²⁺ currents and Ca²⁺-activated K⁺ channels (K_Ca channels) is important for Purkinje cell activity, but how many different K_Ca channel types are present and what each channel type contributes to cell behaviour remains unclear. In order to better understand the ionic mechanisms that control the behaviour of these neurons, we investigated the effects of different Ca²⁺ channel and K_Ca channel antagonists on Purkinje neurons in acute slices of rat cerebellum. Our data show that Ca²⁺ entering through P-type voltage-gated Ca²⁺ channels activates both small-conductance (SK) and large-conductance (BK) K_Ca channels. SK channels play a role in setting the intrinsic firing frequency, while BK channels regulate action potential shape and may contribute to the unique climbing fibre response.