Petersen, Ole

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J Physiol 595.10 (2017) pp 3107–3108 3107TheJournalofPhysiologyPERSPECT IVESThe effects of Ca2+ buffers oncytosolic Ca2+ signallingOle H. Petersen1,21School of Biosciences, Sir Martin EvansBuilding, Cardiff University, Cardiff, UK2Systems Immunity Research Institute,Cardiff University, Cardiff, UKEmail: petersenoh@cardiff.ac.ukCa2+ is one of the most important anduniversal intracellular signalling agents,controlling a multitude of vitally importantcellular processes (Petersen & Verkhratsky2016). The regulating cytosolic Ca2+ signalsvary enormously with regard to timing,localization and spatial extent, dependingon the specific cell type and the functionto be controlled (Petersen & Verkhratsky,2016). Because of the many differenttypes of Ca2+ sensors (Ca2+-bindingproteins) found at different locationsinside cells, there is often a requirementfor Ca2+ signals to be strictly localized.A well-known example of this is thecontrol of neurotransmitter release by Ca2+entry through voltage-gated Ca2+ channels,generating short-lived local nano-domainsof high Ca2+ concentration ([Ca2+]i)(Meinrenken et al. 2003). A more recent,and very interesting, example is the physio-logical activation of two Ca2+-dependenttranscription isoforms, NFAT1 and NFAT4.This occurs through two coincident, butspatially segregated, intracellular Ca2+signals, namely in the immediate vicinityof Ca2+ release-activated Ca2+ channels inthe plasma membrane and close to inositoltrisphosphate receptors in the inner nuclearmembrane (Kar et al. 2016).Different spatio-temporal Ca2+ signalpatterns can occur in the same celldepending on the type, intensity andduration of stimulation (Petersen 1992).In pancreatic acinar cells, both local Ca2+spiking in the region of secretory controland global [Ca2+]i elevations can begenerated, which is important because Ca2+signals control not only secretion but alsomany other functions, including growth(cell division) (Petersen, 1992). In nerveterminals, functions other than neuro-transmitter secretion may depend on theglobal [Ca2+]i. There has therefore, for along time, been much interest in measuringthe volume-averaged [Ca2+]i (Meinrenkenet al. 2003), which can also be particularlyhelpful for a precise quantitative evaluationof Ca2+ handling.The nature and concentration of intra-cellular Ca2+ buffers play an important rolein determining the timing and spreading ofCa2+ released into the cytosol by openingof Ca2+ channels either in the plasmamembrane or in organelle membranes. Anearly example of experiments showing thisphenomenon was the demonstration, inwhole-cell patch clamp current recordingstudies on pancreatic acinar cells, thatintracellular addition of a highly mobilelow-affinity Ca2+ buffer, for examplecitrate, transformed ACh-evoked local andshort-lasting Ca2+ spikes into global andmuch more prolonged Ca2+ transients(Petersen, 1992). The timing and spatialextension of physiological Ca2+ signalstherefore depends not only on the strengthand type of stimulation but also on theaffinities, mobilities and concentrations ofthe various intracellular Ca2+ buffers, whichcan vary significantly between differentcell types. There is also a practical issuerelating to the ability of Ca2+ buffers toinfluence intracellular Ca2+ signals. Becauseall Ca2+-sensitive fluorescent probes areCa2+ buffers, they can distort the [Ca2+]isignals they are designed to monitor.It has been challenging to obtain reliableestimates for the parameters that define thedynamics of physiological [Ca2+]i changes.In this issue of The Journal of Physio-logy, Erwin Neher and his colleagues (Linet al. 2017) now describe the currentlymost precise quantitative approach tosolving this problem by once more takingadvantage of the calyx of Held, a giantmammalian glutamatergic nerve terminalwhich – following the pioneering workof Ian Forsythe (Forsythe, 1994) – hasbeen extensively studied by several groups(Meinrenken et al. 2003). Lin et al.(2017) describe the dynamic changes ofglobal [Ca2+]i during single and repetitivevoltage-clamp depolarizations and providequantitative data on Ca2+ inflow, Ca2+buffering and Ca2+ clearance.Using low concentrations of thelow-affinity Ca2+ indicator Fura-6F,in order not to overwhelm the endogenousCa2+ buffers, Lin et al. (2017) studiedthe voltage-clamped nerve terminals withpatch pipettes containing solutions withminimal Ca2+ buffer concentrations. Thisallowed them to determine the Ca2+binding properties of the endogenousfixed buffers and also the Ca2+ clearancemechanism. With regard to the latter,a comparison was made between theresults obtained with Cs+- or K+-basedpipette solutions. The data from theseexperiments confirmed the importanceof K+-dependent Na+–Ca2+ exchange forCa2+ extrusion (Schnetkamp, 2004). Inother experiments, Lin et al. (2017) usedpipette solutions with 500 µM of the widelyused Ca2+ chelator EGTA, determiningits Ca2+ binding characteristics underrealistic intracellular conditions. It turnedout that the Ca2+ dissociation constant ofEGTA is more than 3 times higher thanthe value previously obtained in vitro.This result is of great practical importanceas EGTA has been, and no doubt willcontinue to be, a useful tool as a slowCa2+ chelator buffering global rather thanlocal [Ca2+]i.Overall, the major importance of the workreported by Lin et al. (2017) is that, basedon very sensitive experimental protocols,they have been able to generate a consistentset of parameters for modelling [Ca2+]itransients in a mammalian presynapticnerve terminal. Estimates of some of theparameters determining [Ca2+]i dynamicshave been reported previously, but theywere based on experiments carried outunder a variety of different conditions,whereas the new study by Lin et al. (2017)has resulted in a comprehensive set ofparameters valid for recording conditionsgenerally used for studies of the calyx ofHeld. The results of the work of Lin et al.(2017) that ‘one set of parameters accuratelydescribes [Ca2+]i measurements coveringa wide range of amplitudes and obtainedusing quite different stimulation protocolsand ionic conditions’ is remarkable andpromises that this set will turn out to beof real help as a firm quantitative basis forfurther studies in this field.ReferencesForsythe ID (1994). Direct patch recording fromidentified presynaptic terminals mediatingglutamatergic EPSCs in the rat CNS, in vitro.J Physiol 479, 381–387.C© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society DOI: 10.1113/JP273852This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distributionand reproduction in any medium, provided the original work is properly cited.3108 Perspectives J Physiol 595.10Kar P, Mirams GR, Christian HC & Parekh AB(2016). Control of NFAT isoform activationand NFAT-dependent gene expressionthrough two coincident and spatiallysegregated intracellular Ca2+ signals. Mol Cell64, 746–759.Lin K-H, Taschenberger H & Neher E (2017).Dynamics of volume-averaged intracellularCa2+ in a rat CNS nerve terminal duringsingle and repetitive voltage-clampdepolarizations. J Physiol 595, 3219–3236.Meinrenken CJ, Borst JGG & Sakmann B (2003)Local routes revisited: the space and timedependence of the Ca2+ signal for phasictransmitter release at the rat calyx of Held.J Physiol 547, 665–689.Petersen OH (1992). Stimulus-secretioncoupling: cytoplasmic calcium signals and thecontrol of ion channels in exocrine acinarcells. J Physiol 448, 1–51.Petersen OH & Verkhratsky A (2016). Calciumand ATP control multiple vital functions.Philos Trans R Soc Lond B Biol Sci 371,20150418.Schnetkamp PPM (2004). The SLC24Na+/Ca2+-K+ exchanger family: vision andbeyond. Pflügers Arch 447, 683–688.Additional informationCompeting interestsNo competing interests declared.FundingThe author is a Medical Research CouncilProfessor (G19/22/2) and is supported by aMedical Research Council Programme grant(MR/J002771/1).C© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society

The effects of Ca2+ buffers on cytosolic Ca2+ signalling

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The effects of Ca2+ buffers on cytosolic Ca2+ signalling

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