Petersen, Ole H.

PubMed

Online Research @ Cardiff

FUNCTION, 2022, 3(5): zqac045https://doi.org/10.1093/function/zqac045Advance Access Publication Date: 5 September 2022PerspectivesPE RSPECTIVE SLocal Ca2+ Signaling Domains: The Balancing Actbetween CRAC Channels and Plasma Membrane Ca2+PumpsOle H. Petersen *School of Biosciences, Cardiff University, Museum Avenue, Cardiff CF10 3AX, UK∗Address correspondence to O.H.P. (e-mail: PetersenOH@cardiff.ac.uk)Key words: calcium signalling; local calcium domains; orai1 calcium channels; calcium release activated calcium channels;calcium pumps; control of gene expressionA Perspective on “Plasma Membrane Ca2+ATPase Activity Enables SustainedStore-operated Ca2+ Entry in the Absence of aBulk Cytosolic Ca2+ Rise”We have known for a long time that the physiologically impor-tant cytosolic Ca2+ signals, which control or influence an aston-ishing range of cellular processes, are localized in specific sub-cellular areas. Astonishingly subtle temporal and spatial regula-tions of Ca2+ transport proteins have been shown to elicit pre-cise sequences of processes that have real physiological impor-tance.1,2 One can distinguish between micro- and nanogra-dients of Ca2+. Microgradients occur within spatial dimen-sions of 1–10 μm, and it became possible to visualize suchevents many years ago. Nanogradients, as the name implies,occur within much smaller spatial dimensions, typically decay-ing with space constants of tens of nanometers. Because thenanogradients may also be short-lasting, it is particularly chal-lenging to visualize them.1 In general, physiological Ca2+ sig-nals are oscillatory and a locally elevated [Ca2+] returns quicklyto the basal level due to diffusion away from the domain,reuptake by Ca2+ pumps into the endoplasmic reticulum (ER),uptake via the Ca2+ uniporter into the mitochondria, and extru-sion to the extracellular space via Ca2+ pumps in the plasmamembrane.1,2In the electrically nonexcitable cells, the original focuswas predominantly on local cytosolic Ca2+ signal generationnear IP3 receptors (IP3Rs) on the ER membrane.3,4 One ofthe consequences of eliciting Ca2+ release from the ER viaIP3Rs is opening of Ca2+-release activated Ca2+ (CRAC) chan-nels in the plasma membrane.5 Tullio Pozzan and his col-laborators provided early evidence for local domains of ele-vated [Ca2+] beneath the plasma membrane, particularly dur-ing activated Ca2+ influx.6 Further studies of the Ca2+ domainsin the subplasmalemmal space near CRAC channels and theprocesses initiated by such local signals have provided impor-tant insights into the control of many different physiologicalevents.1,5As mentioned above, it has generally been thought that phys-iological Ca2+ signals are mostly oscillatory, but, in an importantarticle published in this issue of Function,7 Parekh and collabo-rators have now provided compelling evidence for the view thatCRAC channels can remain open and create a sustained elevated[Ca2+] in the local cytosolic space near the inner mouth of theCRAC channels, in spite of the bulk cytosolic Ca2+ concentra-tion only being minimally elevated due to the operation of theplasma membrane Ca2+ ATPase.The experimental arrangement used by Barak et al.7 is,inevitably, nonphysiological, but has the considerable advan-tage of isolating a few key components of the Ca2+ signalingsystem, thereby creating a situation that allows rigorous test-ing of the hypothesis that a sustained elevated subplasmalem-mal [Ca2+] can be maintained with only minimal elevation ofthe bulk cytosolic [Ca2+], thus creating a standing Ca2+ gradi-ent. In HEK293 cells, thapsigargin was used to arrest the ER Ca2+pumps. Ca2+ therefore leaked out of the ER, gradually emptyingthe store and activated CRAC channels of the Orai1 type in theplasma membrane. In this situation, with the ER Ca2+ pumpsSubmitted: 29 August 2022; Accepted: 30 August 2022C© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society. This is an Open Access article distributed underthe terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution,and reproduction in any medium, provided the original work is properly cited.1Downloaded from https://academic.oup.com/function/article/3/5/zqac045/6692343 by guest on 28 October 20222 Function, 2022, Vol. 3, no. 5out of action, the only transport proteins capable of remov-ing excess Ca2+ from the cytosol would be the Ca2+ pumps inthe plasma membrane. In addition to standard measurementsof the bulk cytosolic [Ca2+], Barak et al.7 were able to assessthe local [Ca2+] in the subplasmalemmal space near the CRAC(Orai1) channels by expressing an Orai1 construct in which theCa2+-sensitive fluorescent protein GECO was tagged to the N-terminus of the channel. The key experiments showed thatthapsigargin elicited major rises in [Ca2+] both in the cytosol atlarge as well as close to the Orai1 channels but, whereas the bulkcytosolic [Ca2+] returned to a near prestimulation basal levelwithin about 10–15 min, the local [Ca2+] close to the Orai1 chan-nels remained markedly elevated even 25 min after the startof thapsigargin application. In spite of the relatively fast returnof the bulk cytosolic [Ca2+] to the basal level, the elevated sub-plasmalemmal [Ca2+] was shown to be capable of driving theslowly developing migration of NFAT into the nucleus.7 Inhibi-tion of the plasma membrane Ca2+ pumps, either by externalLa3+ or by increasing the external pH to 11, very effectively pre-vented the rapid return of the bulk cytosolic [Ca2+] to the basallevel. In contrast, marked reduction of the external [Na+] hadhardly any effect, indicating that the Na+–Ca2+ exchanger in theplasma membrane does not play any important role.7 Clearly,in an experimental situation with nonfunctional Ca2+ pumps inthe ER, the plasma membrane Ca2+ pumps are by far the mostimportant elements disposing of excess cytosolic Ca2+, in com-plete agreement with previous studies on electrically nonex-citable cells.1,2The apparent prolonged existence of nanodomains with ahigh [Ca2+] in the subplasmalemmal space7 would be diffi-cult to explain if the Ca2+ pumps in the plasma membranewere colocalized with the CRAC channels. Indeed, Barak et al.show that during thapsigargin-elicited Ca2+ inflow, there is lit-tle colocalization between these two types of Ca2+ transportersin the HEK293 cells.7 In polarized epithelial cells, for exam-ple, the pancreatic acinar cells, which have a clear physiolog-ical role in the body, there can be an almost complete sep-aration of these transporters, as the plasma membrane Ca2+pumps are concentrated in the apical membrane, whereas theCRAC channels reside in the baso-lateral membrane, an arrange-ment of great physiological importance.2 It has also becomeclear that activated CRAC channels are not uniformly distributedthroughout the baso-lateral membrane, but clustered in cer-tain areas.2 This was also shown to be the case in the HEK293cells.7In order to explain the sustained difference between [Ca2+] inthe subplasmalemmal domains and the bulk of the cytosol, it isnecessary to assume a major “resistance” to Ca2+ movements. Ithas been known for a long time that Ca2+ diffusion in the cytosolis severely restricted.1 In the pancreatic acinar cells, for exam-ple, Ca2+ diffuses more readily inside the ER than in the cytosol,as the Ca2+ binding capacity in the cytosol is orders of magni-tude higher than in the lumen of the ER.8Are the conclusions from the experiments of Barak et al.,7with nonfunctional ER Ca2+ pumps in HEK293 cells, relevant tophysiology? Clearly, the ER Ca2+ pumps, which overall are morepowerful than the plasma membrane Ca2+ pumps,2 can have astrong influence on [Ca2+] in both local domains and the bulk ofthe cytosol, but, as pointed out by Rizzuto and Pozzan,1 the long-term steady-state [Ca2+] in the cytosol depends exclusively onthe equilibrium between Ca2+ entry and Ca2+ outflow across theplasma membrane. By exploiting a conceptually simple exper-imental situation, Barak et al.7 have been able to demonstratehow Ca2+ pumps in the plasma membrane can protect the bulkof the cytosol against toxic elevations of [Ca2+] while still allow-ing a sustained local [Ca2+] elevation near the plasma membraneto drive gene expression. This is an achievement of real physio-logical importance.Data availabilityThere are no data presented in this editorial/perspective.FundingNone declared.Conflict of Interest StatementO.H.P. holds the position of Editor-in-Chief for Function and isblinded from reviewing or making decisions for the manuscript.References1. Rizzuto R, Pozzan T. Microdomains of intracellular Ca2+:molecular determinants and functional consequences. Phys-iol Rev. 2006;86(1):369–408.2. Petersen OH, Gerasimenko JV, Gerasimenko OV,Gryshchenko O, Peng S. The roles of calcium and ATP inthe physiology and pathology of the exocrine pancreas.Physiol Rev. 2021;101(4):1691–1744.3. Rizzuto R, Brini M, Murgia M, Pozzan T. Microdomainswith high Ca2+ close to IP3-sensitive channels that aresensed by neighboring mitochondria. Science. 1993;262(5134):744–747.4. Thorn P, Lawrie AM, Smith PM, Gallacher DV, PetersenOH. Local and global cytosolic Ca2+ oscillations in exocrinecells evoked by agonists and inositol trisphosphate. Cell.1993;74(4):661–668.5. Bakowski D, Murray F, Parekh AB. Store-operated Ca2+channels: mechanism, function, pharmacology, and ther-apeutic targets. Annu Rev Pharmacol Toxicol. 2021;61(1):629–654.6. Marsault R, Murgia M, Pozzan T, Rizzuto R. Domains of highCa2+ beneath the plasma membrane of living A7r5 cells.EMBO J. 1997;16(7):1575–1581.7. Barak P, Kaur S, Scappini E, Tucker CJ, Parekh AB. Plasmamembrane Ca2+ ATPase activity enables sustained store-operated Ca2+ entry in the absence of a bulk cytosolic Ca2+rise. Function. 2022;3(5):zqac040.8. Mogami H, Gardner J, Gerasimenko OV, Camello P, PetersenOH, Tepikin AV. Calcium binding capacity of the cytosol andendoplasmic reticulum of mouse pancreatic acinar cells. JPhysiol. 1999;518(2):463–467.Downloaded from https://academic.oup.com/function/article/3/5/zqac045/6692343 by guest on 28 October 2022

Local Ca2+ signaling domains: The balancing act between CRAC channels and plasma membrane Ca2+ pumps

Petersen, Ole H

PubMed Central