MCU (mitochondrial Ca2+ uniporter) makes the calcium go round.

Store-operated Ca2+ entry (SOCE) is a major mechanism controlling Ca2+ signaling and Ca2+-dependent functions and has been implicated in immunity, cancer, and organ development. SOCE-dependent cytosolic Ca2+ signals are affected by mitochondrial Ca2+ transport through several competing mechanisms. However, how these mechanisms interact in shaping Ca2+ dynamics and regulating Ca2+-dependent functions remains unclear. In a recent issue, Yoast et al. shed light on these questions by defining multiple roles of the mitochondrial Ca2+ uniporter in regulating SOCE, Ca2+ dynamics, transcription, and lymphocyte activation.

Loss of tau and Fyn reduces compensatory effects of MAP2 for tau and reveals a Fyn-independent effect of tau on calcium.

Microtubule-associated protein tau associates with Src family tyrosine kinase Fyn and is tyrosine phosphorylated by Fyn. The presence of tyrosine phosphorylated tau in AD and the involvement of Fyn in AD has drawn attention to the tau-Fyn complex. In this study, a tau-Fyn double knockout (DKO) mouse was generated to investigate the role of the complex. DKO mice resembled Fyn KO in novel object recognition and contextual fear conditioning tasks and resembled tau KO mice in the pole test and protection from pentylenetetrazole-induced seizures. In glutamate-induced Ca2+ response, Fyn KO was decreased relative to WT and DKO had a greater reduction relative to Fyn KO, suggesting that tau may have a Fyn-independent role. Since tau KO resembled WT in its Ca2+ response, we investigated whether microtubule-associated protein 2 (MAP2) served to compensate for tau, since the MAP2 level was increased in tau KO but decreased in DKO mice. We found that like tau, MAP2 increased Fyn activity. Moreover, tau KO neurons had increased density of dendritic MAP2-Fyn complexes relative to WT neurons. Therefore, we hypothesize that in the tau KO, the absence of tau would be compensated by MAP2, especially in the dendrites, where tau-Fyn complexes are of critical importance. In the DKO, decreased levels of MAP2 made compensation more difficult, thus revealing the effect of tau in the Ca2+ response.

The BBSome in POMC and AgRP Neurons Is Necessary for Body Weight Regulation and Sorting of Metabolic Receptors.

The BBSome, a complex of eight Bardet-Biedl syndrome (BBS) proteins involved in cilia function, has emerged as an important regulator of energy balance, but the underlying cellular and molecular mechanisms are not fully understood. Here, we show that the control of energy homeostasis by the anorexigenic proopiomelanocortin (POMC) neurons and orexigenic agouti-related peptide (AgRP) neurons require intact BBSome. Targeted disruption of the BBSome by Bbs1 gene deletion in POMC or AgRP neurons increases body weight and adiposity. We demonstrate that obesity in mice lacking the Bbs1 gene in POMC neurons is associated with hyperphagia. Mechanistically, we present evidence implicating the BBSome in the trafficking of G protein-coupled neuropeptide Y Y2 receptor (NPY2R) and serotonin 5-hydroxytryptamine (HT)2C receptor (5-HT2CR) to cilia and plasma membrane, respectively. Consistent with this, loss of the BBSome reduced cell surface expression of the 5-HT2CR, interfered with serotonin-evoked increase in intracellular calcium and membrane potential, and blunted the anorectic and weight-reducing responses evoked by the 5-HT2cR agonist, lorcaserin. Finally, we show that disruption of the BBSome causes the 5-HT2CR to be stalled in the late endosome. Our results demonstrate the significance of the hypothalamic BBSome for the control of energy balance through regulation of trafficking of important metabolic receptors.

Mechanisms underlying mechanical sensitization induced by complement C5a: the roles of macrophages, TRPV1, and calcitonin gene-related peptide receptors.

The complement system significantly contributes to the development of inflammatory and neuropathic pain, but the underlying mechanisms are poorly understood. Recently, we identified the signaling pathway responsible for thermal hypersensitivity induced by the complement system component C5a. Here, we examine the mechanisms of another important action of C5a, induction of mechanical hypersensitivity. We found that intraplantar injection of C5a produced a dose-dependent mechanical sensitization and that this effect was blocked by chemogenetic ablation of macrophages in both male and female mice. Knockout of TRPV1 or pretreatment with the TRPV1 antagonists, AMG9810 or 5'-iodoresiniferatoxin (5'-IRTX), significantly reduced C5a-induced mechanical sensitization. Notably, local administration of 5'-IRTX 90 minutes after C5a injection resulted in a slow, but complete, reversal of mechanical sensitization, indicating that TRPV1 activity was required for maintaining C5a-induced mechanical hypersensitivity. This slow reversal suggests that neurogenic inflammation and neuropeptide release may be involved. Indeed, pretreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (but not an antagonist of the neurokinin 1 receptor) prevented C5a-induced mechanical sensitization. Furthermore, intraplantar injection of CGRP produced significant mechanical sensitization in both wild-type and TRPV1 knockout mice. Taken together, these findings suggest that C5a produces mechanical sensitization by initiating macrophage-to-sensory-neuron signaling cascade that involves activation of TRPV1 and CGRP receptor as critical steps in this process.

Angiotensin II Triggers Peripheral Macrophage-to-Sensory Neuron Redox Crosstalk to Elicit Pain.

Injury, inflammation, and nerve damage initiate a wide variety of cellular and molecular processes that culminate in hyperexcitation of sensory nerves, which underlies chronic inflammatory and neuropathic pain. Using behavioral readouts of pain hypersensitivity induced by angiotensin II (Ang II) injection into mouse hindpaws, our study shows that activation of the type 2 Ang II receptor (AT2R) and the cell-damage-sensing ion channel TRPA1 are required for peripheral mechanical pain sensitization induced by Ang II in male and female mice. However, we show that AT2R is not expressed in mouse and human dorsal root ganglia (DRG) sensory neurons. Instead, expression/activation of AT2R on peripheral/skin macrophages (MΦs) constitutes a critical trigger of mouse and human DRG sensory neuron excitation. Ang II-induced peripheral mechanical pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs. Furthermore, AT2R activation in MΦs triggers production of reactive oxygen/nitrogen species, which trans-activate TRPA1 on mouse and human DRG sensory neurons via cysteine modification of the channel. Our study thus identifies a translatable immune cell-to-sensory neuron signaling crosstalk underlying peripheral nociceptor sensitization. This form of cell-to-cell signaling represents a critical peripheral mechanism for chronic pain and thus identifies multiple druggable analgesic targets.SIGNIFICANCE STATEMENT Pain is a widespread health problem that is undermanaged by currently available analgesics. Findings from a recent clinical trial on a type II angiotensin II receptor (AT2R) antagonist showed effective analgesia for neuropathic pain. AT2R antagonists have been shown to reduce neuropathy-, inflammation- and bone cancer-associated pain in rodents. We report that activation of AT2R in macrophages (MΦs) that infiltrate the site of injury, but not in sensory neurons, triggers an intercellular redox communication with sensory neurons via activation of the cell damage/pain-sensing ion channel TRPA1. This MΦ-to-sensory neuron crosstalk results in peripheral pain sensitization. Our findings provide an evidence-based mechanism underlying the analgesic action of AT2R antagonists, which could accelerate the development of efficacious non-opioid analgesic drugs for multiple pain conditions.

Culture, Transfection, and Immunocytochemical Analysis of Primary Macrophages.

Macrophages are the primary phagocytes of the body and found in every tissue; often with tissue specific subtypes, e.g., microglia or Kupffer Cells. These cells are essential players in host defense, immune regulation, tissue repair, and homeostasis. Consistent with their diverse functions, macrophages display a remarkable level of plasticity and undergo rapid changes in morphology and activation state in response to environmental cues. Polarization of macrophages towards pro-inflammatory (classically activated or M1) or anti-inflammatory (alternatively activated or M2) activation states is highly dependent on their environment. These activation states result in either tissue remodeling and repair (M2) or enhanced inflammation (M1). As macrophages are dependent upon environmental cues for changes in their activation state, primary cell culture offers the ability to study macrophages under highly controlled conditions in which activation states are easily manipulated with specific growth factors, cytokines, or other signaling molecules and are readily examined through powerful tools such as immunostaining, ELISA, and Ca2+ imaging. Additionally, this approach allows the researcher to manipulate gene expression in these cells to better understanding the underlying principles and mechanisms of macrophage biology. Unfortunately, macrophages are resistant to most forms of transfection and researchers have to use either macrophages isolated from transgenic mice or viral delivery of transgenes which slows the study of these diverse cells. In this chapter we describe methods for isolating, culturing, transfecting, and immunostaining primary macrophages. Particular emphasis is placed on culture conditions and transfection protocol as we found these significantly impacted the success of this protocol. Pairing these methods with functional Ca2+ imaging enables investigation of the effects of silencing or overexpressing specific proteins on the functional properties of primary macrophages.

The Complement System Component C5a Produces Thermal Hyperalgesia via Macrophage-to-Nociceptor Signaling That Requires NGF and TRPV1.

UNLABELLED: The complement cascade is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other components of the complement system in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund's adjuvant was accompanied by C5a upregulation and was markedly reduced by C5a receptor (C5aR1) knock-out or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal hyperalgesia, an effect that was absent in TRPV1 knock-out mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca(2+) mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis mice abolished C5a-dependent thermal hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of NGF, a mediator known to sensitize TRPV1. Preinjection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that complement fragment C5a induces thermal hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF, and TRPV1 as key players in this cross-cellular communication. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into how the complement system, a key component of innate immunity, regulates the development of pain hypersensitivity. We demonstrate a crucial role of the C5a receptor, C5aR1, in the development of inflammatory thermal and mechanical sensitization. By focusing on the mechanisms of C5a-induced thermal hyperalgesia, we show that this process requires recruitment of macrophages and initiation of macrophage-to-nociceptor signaling. At the molecular level, we demonstrate that this signaling depends on NGF and is mediated by the heat-sensitive nociceptive channel TRPV1. This deeper understanding of how immune cells and neurons interact to regulate pain processing is expected to facilitate mechanism-based approaches in the development of new analgesics.

Nerve growth factor (NGF) regulates activity of nuclear factor of activated T-cells (NFAT) in neurons via the phosphatidylinositol 3-kinase (PI3K)-Akt-glycogen synthase kinase 3ß (GSK3ß) pathway.

The Ca(2+)/calcineurin-dependent transcription factor nuclear factor of activated T-cells (NFAT) plays an important role in regulating many neuronal functions, including excitability, axonal growth, synaptogenesis, and neuronal survival. NFAT can be activated by action potential firing or depolarization that leads to Ca(2+)/calcineurin-dependent dephosphorylation of NFAT and its translocation to the nucleus. Recent data suggest that NFAT and NFAT-dependent functions in neurons can also be potently regulated by NGF and other neurotrophins. However, the mechanisms of NFAT regulation by neurotrophins are not well understood. Here, we show that in dorsal root ganglion sensory neurons, NGF markedly facilitates NFAT-mediated gene expression induced by mild depolarization. The effects of NGF were not associated with changes in [Ca(2+)]i and were independent of phospholipase C activity. Instead, the facilitatory effect of NGF depended on activation of the PI3K/Akt pathway downstream of the TrkA receptor and on inhibition of glycogen synthase kinase 3ß (GSK3ß), a protein kinase known to phosphorylate NFAT and promote its nuclear export. Knockdown or knockout of NFATc3 eliminated this facilitatory effect. Simultaneous monitoring of EGFP-NFATc3 nuclear translocation and [Ca(2+)]i changes in dorsal root ganglion neurons indicated that NGF slowed the rate of NFATc3 nuclear export but did not affect its nuclear import rate. Collectively, our data suggest that NGF facilitates depolarization-induced NFAT activation by stimulating PI3K/Akt signaling, inactivating GSK3ß, and thereby slowing NFATc3 export from the nucleus. We propose that NFAT serves as an integrator of neurotrophin action and depolarization-driven calcium signaling to regulate neuronal gene expression.

Mitochondria and plasma membrane Ca2+-ATPase control presynaptic Ca2+ clearance in capsaicin-sensitive rat sensory neurons.

The central processes of primary nociceptors form synaptic connections with the second-order nociceptive neurons located in the dorsal horn of the spinal cord. These synapses gate the flow of nociceptive information from the periphery to the CNS, and plasticity at these synapses contributes to centrally mediated hyperalgesia and allodynia. Although exocytosis and synaptic plasticity are controlled by Ca(2+) at the release sites, the mechanisms underlying presynaptic Ca(2+) signalling at the nociceptive synapses are not well characterized. We examined the presynaptic mechanisms regulating Ca(2+) clearance following electrical stimulation in capsaicin-sensitive nociceptors using a dorsal root ganglion (DRG)/spinal cord neuron co-culture system. Cytosolic Ca(2+) concentration ([Ca(2+)]i) recovery following electrical stimulation was well approximated by a monoexponential function with a ∼2 s. Inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase did not affect presynaptic [Ca(2+)]i recovery, and blocking plasmalemmal Na(+)/Ca(2+) exchange produced only a small reduction in the rate of [Ca(2+)]i recovery (∼12%) that was independent of intracellular K(+). However, [Ca(2+)]i recovery in presynaptic boutons strongly depended on the plasma membrane Ca(2+)-ATPase (PMCA) and mitochondria that accounted for ∼47 and 40%, respectively, of presynaptic Ca(2+) clearance. Measurements using a mitochondria-targeted Ca(2+) indicator, mtPericam, demonstrated that presynaptic mitochondria accumulated Ca(2+) in response to electrical stimulation. Quantitative analysis revealed that the mitochondrial Ca(2+) uptake is highly sensitive to presynaptic [Ca(2+)]i elevations, and occurs at [Ca(2+)]i levels as low as ∼200-300 nm. Using RT-PCR, we detected expression of several putative mitochondrial Ca(2+) transporters in DRG, such as MCU, Letm1 and NCLX. Collectively, this work identifies PMCA and mitochondria as the major regulators of presynaptic Ca(2+) signalling at the first sensory synapse, and underlines the high sensitivity of the mitochondrial Ca(2+) uniporter in neurons to cytosolic Ca(2+).

Distinct activation properties of the nuclear factor of activated T-cells (NFAT) isoforms NFATc3 and NFATc4 in neurons.

The Ca(2+)/calcineurin-dependent transcription factor NFAT (nuclear factor of activated T-cells) is implicated in regulating dendritic and axonal development, synaptogenesis, and neuronal survival. Despite the increasing appreciation for the importance of NFAT-dependent transcription in the nervous system, the regulation and function of specific NFAT isoforms in neurons are poorly understood. Here, we compare the activation of NFATc3 and NFATc4 in hippocampal and dorsal root ganglion neurons following electrically evoked elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)). We find that NFATc3 undergoes rapid dephosphorylation and nuclear translocation that are essentially complete within 20 min, although NFATc4 remains phosphorylated and localized to the cytosol, only exhibiting nuclear localization following prolonged (1-3 h) depolarization. Knocking down NFATc3, but not NFATc4, strongly diminished NFAT-mediated transcription induced by mild depolarization in neurons. By analyzing NFATc3/NFATc4 chimeras, we find that the region containing the serine-rich region-1 (SRR1) mildly affects initial NFAT translocation, although the region containing the serine-proline repeats is critical for determining the magnitude of NFAT activation and nuclear localization upon depolarization. Knockdown of glycogen synthase kinase 3ß (GSK3ß) significantly increased the depolarization-induced nuclear localization of NFATc4. In contrast, inhibition of p38 or mammalian target of rapamycin (mTOR) kinases had no significant effect on nuclear import of NFATc4. Thus, electrically evoked [Ca(2+)](i) elevation in neurons rapidly and strongly activates NFATc3, whereas activation of NFATc4 requires a coincident increase in [Ca(2+)](i) and suppression of GSK3ß, with differences in the serine-proline-containing region giving rise to these distinct activation properties of NFATc3 and NFATc4.

Mechanism of neuroprotective mitochondrial remodeling by PKA/AKAP1.

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and non-neuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults.

The spinocerebellar ataxia 12 gene product and protein phosphatase 2A regulatory subunit Bbeta2 antagonizes neuronal survival by promoting mitochondrial fission.

The neurodegenerative disorder spinocerebellar ataxia 12 (SCA12) is caused by CAG repeat expansion in the non-coding region of the PPP2R2B gene. PPP2R2B encodes Bbeta1 and Bbeta2, alternatively spliced and neuron-specific regulatory subunits of the protein phosphatase 2A (PP2A) holoenzyme. We show here that in PC12 cells and hippocampal neurons, cell stressors induced a rapid translocation of PP2A/Bbeta2 to mitochondria to promote apoptosis. Conversely, silencing of PP2A/Bbeta2 protected hippocampal neurons against free radical-mediated, excitotoxic, and ischemic insults. Evidence is accumulating that the mitochondrial fission/fusion equilibrium is an important determinant of cell survival. Accordingly, we found that Bbeta2 expression induces mitochondrial fragmentation, whereas Bbeta2 silencing or inhibition resulted in mitochondrial elongation. Based on epistasis experiments involving Bcl2 and core components of the mitochondrial fission machinery (Fis1 and dynamin-related protein 1), mitochondrial fragmentation occurs upstream of apoptosis and is both necessary and sufficient for hippocampal neuron death. Our data provide the first example of a proapoptotic phosphatase that predisposes to neuronal death by promoting mitochondrial division and point to a possible imbalance of the mitochondrial morphogenetic equilibrium in the pathogenesis of SCA12.

AKAP150-anchored PKA activity is important for LTD during its induction phase.

Protein kinase A (PKA) is thought to tonically maintain an enhanced level of postsynaptic AMPA receptor responses. Injection of PKA inhibitory peptides leads to a run-down of AMPA receptor responses and prevents long-term depression (LTD). This run-down of AMPA receptor activity was proposed to occlude a further reduction that would otherwise constitute LTD. PKA is recruited to postsynaptic sites by the A kinase anchor protein AKAP150. We found that LTD was strongly impaired in acute hippocampal slices from 2-week-old mice in which the PKA binding site on AKAP150 had been genetically deleted (D36 mice). However, basal postsynaptic AMPA and NMDA receptor activity was indistinguishable between D36 and WT mice. During extracellular recordings of field EPSPs and during intracellular recording of EPSCs from hippocampal slices from WT mice, H-89 and KT5720, two structurally different PKA inhibitors, inhibited LTD by more than 70% without affecting basal synaptic transmission or basal phosphorylation of serine 845 on GluR1. Collectively our data indicate that AKAP150-anchored PKA activity is required to induce LTD and not merely to maintain a tonically heightened activity level of AMPA receptors as proposed earlier.

Protein kinase A anchoring via AKAP150 is essential for TRPV1 modulation by forskolin and prostaglandin E2 in mouse sensory neurons.

Phosphorylation-dependent modulation of the vanilloid receptor TRPV1 is one of the key mechanisms mediating the hyperalgesic effects of inflammatory mediators, such as prostaglandin E(2) (PGE(2)). However, little is known about the molecular organization of the TRPV1 phosphorylation complex and specifically about scaffolding proteins that position the protein kinase A (PKA) holoenzyme proximal to TRPV1 for effective and selective regulation of the receptor. Here, we demonstrate the critical role of the A-kinase anchoring protein AKAP150 in PKA-dependent modulation of TRPV1 function in adult mouse dorsal root ganglion (DRG) neurons. We found that AKAP150 is expressed in approximately 80% of TRPV1-positive DRG neurons and is coimmunoprecipitated with the capsaicin receptor. In functional studies, PKA stimulation with forskolin markedly reduced desensitization of TRPV1. This effect was blocked by the PKA selective inhibitors KT5720 [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylicacid hexyl ester] and H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl), as well as by the AKAP inhibitory peptide Ht31. Similarly, PGE(2) decreased TRPV1 desensitization in a manner sensitive to the PKA inhibitor KT5720. Both the forskolin and PGE(2) effects were strongly impaired in DRG neurons from knock-in mice that express a mutant AKAP150 lacking the PKA-binding domain (Delta36 mice). Protein kinase C-dependent sensitization of TRPV1 remained intact in Delta36 mice. The PGE(2)/PKA signaling defect in DRG neurons from Delta36 mice was rescued by overexpressing the full-length human ortholog of AKAP150 in these cells. In behavioral testing, PGE(2)-induced thermal hyperalgesia was significantly diminished in Delta36 mice. Together, these data suggest that PKA anchoring by AKAP150 is essential for the enhancement of TRPV1 function by activation of the PGE(2)/PKA signaling pathway.

Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP.

Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7-12 but not 4-week-old mice. Inhibitors of PKA and of GluR2-lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4-week-old WT mice. Inhibitors of GluR2-lacking AMPA receptors also prevented LTP in 2 but not 3-week-old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2-lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150-anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8-week-old mice.

Modulating Ca2+ clearance from neurons.

Neurons are exquisitely sensitive to the duration, amplitude and localization of transient increases in intracellular Ca2+ concentration ([Ca2+]i). Modulation of Ca2+ uptake into the mitochondrion and endoplasmic reticulum, and efflux via the plasma membrane Ca2+ pump and Na+/Ca2+ exchange profoundly affect the shape of [Ca2+]i signals. Ca2+ clearance mechanisms are modulated by other signaling pathways, are sensitive to metabolic state and have a memory of the recent history of cell activation. We present here examples of pharmacologic and endogenous regulation of Ca2+ sequestration and efflux in neurons. Ca2+ clearance mechanisms differentially shape [Ca2+]i signals based on their affinity, capacity and location; their modulation alters specific neuronal functions. The increasingly apparent diversity of the molecular entities that make up the [Ca2+]i regulatory system reveals new sites for modulation. Specialized Ca2+ clearance mechanisms participate in unique cellular functions and thus, are important targets for pharmacological and physiological regulation of the neuron.

Bradykinin and ATP accelerate Ca(2+) efflux from rat sensory neurons via protein kinase C and the plasma membrane Ca(2+) pump isoform 4.

Modulation of Ca(2+) channels by neurotransmitters provides critical control of neuronal excitability and synaptic strength. Little is known about regulation of the Ca(2+) efflux pathways that counterbalance Ca(2+) influx in neurons. We demonstrate that bradykinin and ATP significantly facilitate removal of action potential-induced Ca(2+) loads by stimulating plasma membrane Ca(2+)-ATPases (PMCAs) in rat sensory neurons. This effect was mimicked in the soma and axonal varicosities by phorbol esters and was blocked by antagonists of protein kinase C (PKC). Reduced expression of PMCA isoform 4 abolished, and overexpression of isoform 4b enhanced, PKC-dependent facilitation of Ca(2+) efflux. This acceleration of PMCA4 underlies the shortening of the action potential afterhyperpolarization produced by activation of bradykinin and purinergic receptors. Thus, isoform-specific modulation of PMCA-mediated Ca(2+) efflux represents a novel mechanism to control excitability in sensory neurons.