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Contents lists available at ScienceDirect

Cell Calcium

jou rn al hom epage: www.elsev ier .com/ locate /ceca

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rganellar channels and transporters

aoxing Xua,∗, Enrico Martinoiab, Ildiko Szaboc,d

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 3089 Natural Science Building (Kraus), 830 North Universityvenue, Ann Arbor, MI 48109-1048, USAInstitute of Plant Biology, University of Zürich, Zollikerstr. 107, CH-8008 Zürich, SwitzerlandDepartment of Biology, University of Padova, Viale G. Colombo 3, 35121 Padova, ItalyCNR Neuroscience Institute, Viale G. Colombo 3, 35121 Padova, Italy

r t i c l e i n f o

rticle history:eceived 17 February 2015ccepted 20 February 2015vailable online xxx

eywords:on channels and transportersrganelle membranes

ntracellular channelsrganellar channel targeting

a b s t r a c t

Decades of intensive research have led to the discovery of most plasma membrane ion channelsand transporters and the characterization of their physiological functions. In contrast, although over80% of transport processes occur inside the cells, the ion flux mechanisms across intracellular mem-branes (the endoplasmic reticulum, Golgi apparatus, endosomes, lysosomes, mitochondria, chloroplasts,and vacuoles) are difficult to investigate and remain poorly understood. Recent technical advances insuper-resolution microscopy, organellar electrophysiology, organelle-targeted fluorescence imaging, andorganelle proteomics have pushed a large step forward in the research of intracellular ion transport. Manynew organellar channels are molecularly identified and electrophysiologically characterized. Addition-ally, molecular identification of many of these ion channels/transporters has made it possible to studytheir physiological functions by genetic and pharmacological means. For example, organellar channelshave been shown to regulate important cellular processes such as programmed cell death and photosyn-

thesis, and are involved in many different pathologies. This special issue (SI) on organellar channels andtransporters aims to provide a forum to discuss the recent advances and to define the standard and openquestions in this exciting and rapidly developing field. Along this line, a new Gordon Research Conferencededicated to the multidisciplinary study of intracellular membrane transport proteins will be launchedthis coming summer.

© 2015 Elsevier Ltd. All rights reserved.

. Introduction

Ion channels are classically understood to mediate the flux ofons across the plasma membrane in response to cellular stimu-ation. However, they also reside on intracellular membranes toegulate various organellar and cellular functions as well [1,2].ntracellular organelles can be arbitrarily divided into two groups3]. The first endocytic, secretory, and autophagic group (group) includes the endoplasmic reticulum (ER), the Golgi apparatus,ndosomes, autophagosomes, phagosomes, lysosomes, secretoryesicles, and vacuoles (see Fig. 1). Group I organelles mediate cargoransport and exchange materials with each other [4]. There are also

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ell-type-specific compartments derived from group I organelles,hich include synaptic vesicles in neurons [5] and various

ysosome-related organelles, such as melanosomes in melanocytes

∗ Corresponding author. Tel.: +1 734 615 2845.E-mail addresses: haoxingx@umich.edu (H. Xu),

nrico.Martinoia@botinst.uzh.ch (E. Martinoia), ildi@bio.unipd.it (I. Szabo).

ttp://dx.doi.org/10.1016/j.ceca.2015.02.006143-4160/© 2015 Elsevier Ltd. All rights reserved.

[6]. Group II intracellular organelles include mitochondria, nucleus,chloroplasts, and peroxisomes, which are dedicated to specificcellular functions such as bioenergetics (mitochondria and chloro-plasts). Ion channels and transporters are functionally present onthe membranes of the aforementioned organelles [1,2].

A major function of organellar ion transport is to regulateintracellular Ca2+ signaling, which plays important roles in bothsignal transduction and membrane trafficking [1,2,4]. Indeed, manygroups I and II intracellular organelles serve as intracellular Ca2+

stores with the luminal Ca2+ concentration ([Ca2+]lumen) rangingfrom micromolar (�M) to millimolar (mM), 10 to 5000-fold higherthan the level of resting cytosolic Ca2+ ([Ca2+]cyt, ∼100 nM) [3].Consistently, many Ca2+ channels and transporters are enrichedin intracellular organelles [2]. For example, the inositol 1,4,5-trisphosphate receptors (IP3-Rs) are Ca2+-permeant channels in theER, the primary Ca2+ store in the cell [7]. IP3-Rs are the essential

nellar channels and transporters, Cell Calcium (2015),

signal transduction player in the phospholipase C (PLC) pathwaythat is stimulated by numerous neurotransmitters and hormones(Mak and Foskett [8] in this SI). Additionally, intracellular transportof other ions such as Na+ and K+ regulates organellar membrane

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Fig. 1. Organellar channels and transporters. Intracellular organelles include endosomes, phagosomes, autophagosomes, lysosomes, mitochondria, chloroplasts, plantvacuoles, Golgi apparatus, the ER, peroxisomes, and the nucleus. Intracellular channels are shown as oval objects while transporters and pumps are rectangular. Chan-nels/transporters are color-coded, with calcium-permeable proteins in blue, chloride in green, sodium in yellow, and potassium in violet. Proteins allowing the passage ofmetabolites and/or several different types of ions are depicted in orange. In the nucleus of plants, castor and pollux proteins may mediate potassium flux. In the ER, severalcalcium transport systems are found (Ryanodine Receptor, IP3 receptor, SERCA pump) as well as cation-permeable channels (TRIC, TRP). Functionally active K+ transport sys-tems are the LETM1K+/H+ antiporter, and potassium channels (KATP, KCa). In the lysosomes, TRPMLs are permeable to Ca2+ and heavy metals; TPCs are Na+-selective channels,but are also permeable to Ca2+; CLCs are Cl-transporters. TRPs, TPCs, and CLCs are also present in the early endosomes. In the plant vacuoles, TPC1 is the putative Ca2+ channels,while CAXs mediate Ca2+ uptake. TPKs are vacuolar K+ channels, while NHXs mediate H+/Na+ or H+/K+ exchange. CLC proteins function as anion transporters. ALMTs maymediate malate transport. In the mitochondria, only the channels mentioned in this SI are shown – for a complete list see Ref. [68]. The MCU complex is responsible for theu (K(ATp In addK

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ptake of calcium. The potassium-permeable pathways in the mitochondria includeore. In chloroplasts, many metabolite transporters have been identified (see [85]).EA family have been identified in chloroplasts.

otential and luminal ionic homeostasis, which are known to affecta2+ signaling indirectly [2]. For instance, ER K+ channels areeported to affect both Ca2+ uptake and release (Kuum et al. [9]n this SI).

The last wave of organellar channel research culminated in thedentification of two ER-localized Ca2+ release channels: IP3-Rs andyanodine receptors (RyRs) [7,10]. Recently, the field has expe-ienced another dramatic development, surmounting technicalimits through new methods like patch clamping of endosomes andysosomes [11–13], and through molecular identification of chan-els affecting cell and bioenergetic activities (e.g., the long-sought

dentity of the mitochondrial calcium uniporter, MCU) [14,15].any new channels and transporters have been discovered in

oth groups I and II intracellular organelles, such as mitochondria,ysosomes, Golgi apparatus, ER, melanosomes, and plant vacuoles.

uch of this work is the basis for the reviews in this SI. Included inhis series are papers on mitochondrial MCU Ca2+ channels (Mur-ia and Rizzuto [16] in this SI), mitochondrial K+ channels (Leanzat al. [17] in this SI), mitochondrial permeability-transition-porePTP) proteins (Rasola and Bernardi [18] in this SI), endosomal Clhannels/transporters (Pusch and Zifarelli [19] in this SI), lysoso-

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al NAADP receptors (Galione [20] in this SI), lysosomal TRP-typea2+ channels (Venkatachalam and Zhu [21] in this SI). Ion channels

n the ER are also discussed, including the IP3R-type Ca2+ channelsMak and Foskett [8] in this SI) and various K+ channels (Kuum

P), K(Ca), Kv1.3 channels, and LETM1K+/H+ antiporter. MPTP is a large, non-specificition, ClC-type Cl channel, TPK3 K+ channels, and members of the K+/H+ antiporter

et al. [9] in this SI). Examples of discovery can be drawn fromchloroplasts, such as the identification of new membrane trans-port proteins through proteomics and transcriptomics (Finazzi et al.[22] in this SI), and the discernment of how subcellular targetingand biogenesis of organellar channels are regulated (Oh and Hwang[23]; von Charpuis et al. [24] in this SI). So far, organellar mechano-sensitive channels are only characterized in yeast cells (Nakayamaand Iida [25] in this SI). Finally, sperm ion channels are coveredto exemplify how electrophysiological studies in non-intracellularorganelles can be instrumental (Miller et al. [26] in this SI).

Common themes emerge upon a collective reading ofthese reviews. First, improved electrophysiological methods andfluorescence-based functional assays have led to functional iden-tification of new organellar channels. Recent examples includelysosomal TRPML1 [21], lysosomal two-pore TPC channels [20],and mitochondrial MCU channels [16]. Second, improved biochem-ical and system-based methods have led to the discovery of newintracellular channels/transporters, such as mitochondrial MCUchannels [14–16] and metal transporters in chloroplasts [22]. Third,advances in the understanding of organellar channels/transportershave led to the identification of novel targets for therapeutics.

nellar channels and transporters, Cell Calcium (2015),

Examples of new “druggable targets” in this series are lysosomalchannels for lysosome storage diseases (LSDs) and mitochondrialchannels for cancer [17,18,21]. Together, these studies providedan updated “toolkit” for tackling the difficult study of intracellular

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hannels and transporters. Due to space limitation, many of theecently discovered organellar channels and transporters are notovered in this SI. We first outline common challenges, then dis-uss the progress in each subfield/organelle, with the focus on theitochondria, chloroplasts, lysosomes, and plant vacuoles.

. Common challenges in studying organellar channels

There are common challenges in studying channels from dif-erent intracellular organelles. Unlike plasma membrane channels,hose working environment has been unambiguously defined, the

asic information for most organelles has yet to be established,ncluding luminal ionic composition, organellar membrane poten-ial, and lipid composition of the organellar membranes.

Luminal ionic composition varies greatly in different subcell-lar contexts, adding a layer of difficulty in the task of properlyharacterizing the function of organellar ion channels. The mostelevant luminal ions are Ca2+ and K+. While [Ca2+]lumen is high forhe ER and lysosomes, and low for mitochondria, [K+]lumen is highn the ER, nucleus, and Golgi, but relatively low in mitochondriand lysosomes [2,27]. Importantly, in small-sized organelles likendosomes and lysosomes, the luminal concentration of one ionust be viewed in the context of other ions and ion-dependent

hannels/transporters. Due to the enrichment of various ion co-ransporters and exchangers in organelles [28], an increase in theermeability of one ion may alter the concentration gradientsf others. Hence, unlike their plasma membranes counterparts,rganellar ion transporters may have a direct and acute influencen the functions of organellar channels.

What is the membrane potential (� , defined aslumen − Vcytosol for comparison) for each organelle? Resting is around 0 mV for the ER and nucleus, very negative (−150 to180 mV) for mitochondria, and slightly positive (+20 to 30 mV)

or the Golgi apparatus, phagosomes, and lysosomes [1,2,28]. Forlant vacuoles, a membrane potential around +30 mV is assumed,owever, it remains unknown whether it can fluctuate in responseo changes in environmental conditions [29]. For the chloroplastnvelope membrane, a value of approximately −110 mV has beeneported [30]. The ionic permeabilities that set � at rest or upontimulation remain to be determined.

What are the identified channels and transporters in therganelles? Many ion channels and transporters are reportedlyresent in the organelles based on molecular expression analy-is, pharmacological manipulation, or functional characterization.owever, only few of them are supported by strong data in all threespects. In addition, while some channels are targeted specificallynd exclusively to one organelle, others are present in multiple cel-ular compartments. Hence, for channels present in both plasma

embrane and organelles, it is necessary to set up the criteria toefine organellar versus plasma membrane channels. On the otherand, the fact that phamarcological properties of such channels,

ocated either intracellularly or at the plasma membrane, appearo be the same in many cases renders assigning a definite role tontracellular channels in a given process a difficult task.

. The endocytic compartments

.1. Regulation of lysosomal function and trafficking byysosomal ion fluxes

Lysosomes, acidic vesicles that are filled with Ca2+ and

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ydrolases, mediate the degradation of both endocytic andutophagic cargos [31]. Subsequently, the digested metabolites areransported out of the lysosome via specific exporters or throughesicular membrane trafficking [32,33]. Lysosomal channels and

PRESSxxx (2015) xxx–xxx 3

transporters mediate ion fluxes across perimeter membranes inorder to regulate lysosomal ion homeostasis, membrane potential,catabolite export, and membrane trafficking [28]. Deregulation oflysosomal channels may underlie the pathogenesis of many lyso-some storage diseases (LSDs) and possibly some metabolic diseases[34].

There exist large concentration gradients for Ca2+ (∼5000 fold),H+ (∼1000 fold), Na+ (∼10 fold), and K+ (∼10 fold) [35,36]. Theproton gradient (pHlumen ∼ 4.6) is established and maintained byV-ATPase [31]. Cl− influx regulates lysosomal acidification by pro-viding counter ions for H+ pumping [37,38]. [Ca2+]lumen is ∼0.5 mMfor lysosomes [28], higher than the low micromolar ranges forearly and late endosomes [39]. Ca2+ efflux from lysosomes isimportant for signal transduction [28]. Lysosomal Ca2+ is alsoknown to regulate multiple steps in lysosomal trafficking, includ-ing fusion of lysosomes with autophagosomes, late endosomes[32,40], lysosomal exocytosis [36,41], retrograde trafficking to theGolgi apparatus, and lysosome reformation from the autolyso-somes [42] or endolysosome hybrids [32]. The high [Na+]lumen andlow [K+]lumen may help set the � , which, like H+ flux, may indi-rectly affect lysosomal Ca2+ release [28,43]. With the exception ofV-ATPase, most lysosomal ion transporters have yet to be identified.The Ca2+ gradient is thought to be established by a putative Ca2+–H+

exchanger in the mammalian cells [28]. The molecular identity ofthis high affinity Ca2+ transporter is still unknown. In contrast, inthe yeast and plant vacuoles, both Ca2+–H+ exchanger and Ca2+-ATPase are required for the maintenance of the vacuolar Ca2+ store[44].

Although the importance of lysosomal ionic flux has been longappreciated, the ion channels responsible for lysosomal Na+, K+,Ca2+, Cl−, and H+ fluxes are only beginning to be discovered.

3.2. Endolysosomal patch-clamping

The traditional way to study endosomal and lysosomal chan-nels is to reconstitute them into a planar lipid bilayer [45].However, bilayer studies require a high degree of purity inmembrane and protein preparation, and typically do not yieldlarge macroscopic currents. To characterize endosomal and lyso-somal channels in their native membranes, the biggest hurdleis their relatively small size (<0.5 �m in diameter), suboptimalfor patch-clamping studies [11,12]. Recently, this barrier hasbeen overcome by advances in cell biology. Large early endo-somes (>3 �m in diameter) can be formed by expressing mutantforms of trafficking proteins [11]. Alternatively, late endosomesand lysosomes can be selectively enlarged using small moleculevacuole-enlargement reagents, such as vacuolin-1 [12,13,27].Four different configurations can be made for endolysosomalelectrophysiology: endolysosome-attached, whole-endolysosome,luminal-side-out, and cytoplasmic-side-out [11,12]. Geneticallyencoded ion indicators that are targeted to endolysosomes maybe employed to study the flux of ions. However, the whole-endolysosome technique represents the most powerful method tostudy ion channels in the endosomes, lysosomes, and other relatedintracellular vesicles, including phagosomes and melanosomes[13,27,46].

3.3. Lysosomal conductances

As H+ and Ca2+ are 1000–5000 times more abundant in thelysosome lumen than in the cytosol, lysosomal Ca2+ and H+-permeant channels must be tightly regulated. Meanwhile, the

nellar channels and transporters, Cell Calcium (2015),

high Na+ and K+ gradients across lysosomal membranes suggestthe existence of selective Na+ and K+ channels in the lysosome.Using a lysosome patch-clamp technique [12], multiple lysosomalconductances have been functionally characterized, including INa,

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Ca, IFe and ICl [12,13,27,47–49]. IK and IH have not been fully charac-erized. In addition, endogenous INAADP has been reported in mousebroblasts [50]. Among these conductances, TPCs have been molec-larly identified to encode INa [27] and TRPMLs to encode ICa [12]see Fig. 1). CLC-7 is presumed to encode ICl [38] (see Fig. 1).

.4. Lysosomal Ca2+ channels

Mucolipin TRPs (TRPML1-3) are the principle Ca2+ release chan-els in lysosomes. TRPML1 is a key regulator of most lysosomalrafficking processes [40,51], and human mutations of TRPML1ause lysosomal storage and Type IV Mucolipidosis (ML-IV) [52,53]also see [21]). Using whole-endolysosome patch-clamp technique,RPML1 is demonstrated to be a late endosome and lysosome-ocalized, Ca2+ and Fe2+/Zn2+ dually permeable channel activatedy an endolysosome-localized phosphoinositide, i.e. PI(3,5)P2 [21].he regulation of TRPML1 by PI(3,5)P2 provides an example ofompartment-specific regulation of organellar channels.

The cell biological roles of TRPML1 were uncovered with theid of membrane-permeable synthetic agonists [54,55]. Usingucolipin Synthetic Agonist 1 (ML-SA1), which robustly acti-

ates TRPML1 at low micromolar concentrations [55], TRPML1s found to be a primary regulator of lysosomal exocytosis [46].RPML1-mediated lysosomal exocytosis is required for the phago-ytic uptake of large particles in macrophages [46] and repair oflasma membrane damage in skeletal muscle [56]. Loss-of-functionutations in TRPML1 cause ML-IV, a LSD manifested by men-

al retardation, muscular dystrophy, and constitutive achlorhydria53,56]. In addition, TRPML1’s role may also be extended to otherSDs [55], in which TRPML1-mediated lysosomal Ca2+ release andysosomal trafficking are partially blocked [55].

Two-pore channels (TPC s) are also thought to be lysosomala2+ channels [20]. TPCs are localized in the lysosomes, and over-xpression of TPCs increases NAADP-activated Ca2+-release [57].n whole-endolysosome recordings, INAADP was increased in TPC-verexpressing cells, but abolished in TPC2 KO cells [50,58]. TPC KOice exhibit susceptibility to liver disease and impaired starvation

ndurance [20].P2X4 channels are recently identified to be ATP-activated Ca2+-

ermeable channels in the lysosomes of Cos1 cells [59]. P2X4roteins are localized in the lysosome, and overexpression of P2X4esults in large non-selective cationic currents activated by luminalTP and alkalization [59]. The physiological significance of lysoso-al P2X4 channels remains to be established.

.5. Lysosomal Na+ channels

Whole-endolysosome TPC currents are highly selective for Na+

ver K+ or Ca2+ [27,49]. TPC channels are regulated by PI(3,5)P227], membrane voltage [49], and cytoplasmic Mg2+/ATP [48,58].iven TPC’s high permeability to Na+, regulation of TPC currentsay provide mechanisms to rapidly change lysosomal � · Under

onditions when PI(3,5)P2 levels are high but ATP levels are low,ndolysosomes that lack TPCs have a less depolarized (luminal-ess-positive) � [48].

.6. Endosomal and phagosomes

Several CLC (CLC3-7) proteins are localized in the early and latendosomes [38]. Although the biological functions of CLCs in endo-omes have been clearly established, their channel or transporter

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roperties are characterized mostly at the plasma membrane19]. Therefore, endosomal patch-clamping is needed to charac-erize CLCs in their native settings. Several other proteins are alsoresent in the early endosomes, including TRPML3, TPC1, and also

PRESSxxx (2015) xxx–xxx

possibly TRPV2 [11,51,60]. However, their roles in early endosomalfunctions are unclear.

Whole-phagosome patch-clamping techniques have beenrecently developed in macrophages [46]. This technique shouldbe employed to study phagosomal conductances, including thoseare already known to exist – for example, the voltage-gated pro-ton conductance mediated by Hv1 [61]. Whether there exist anyautophagosome-specific conductances is not known.

3.7. Cell-type-specific compartments

Whole-endolysosome patch-clamp methods can be employedto study ion channels in lysosome-related-organelles. For example,the albinism-causing OCA2 proteins are reported to encode a Cl−

channel in melanosomes that are important for pigmentation [62].

4. Endoplasmic reticulum

No K+ concentration gradient is thought to exist across the ERmembrane, and the ER � is around 0 mV [2]. The only major con-centration gradient across the ER membrane is for Ca2+, suggestingthat a major function of ER ion transport is Ca2+ signaling. Free[Ca2+]lumen in the ER is 0.3–0.7 mM, which is established and main-tained by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA)[63]. Although there are no concentration gradients, the flux ofother ions such as H+ and K+ under certain conditions may regulateCa2+ release and uptake [9].

4.1. Nuclear patch-clamping

Because the outer membrane of ER is continuous withthe nuclear membrane, studying ER channels has been madepossible by developing a nuclear patch-clamping method [8]. Sev-eral configurations can be achieved, including nucleus-attached,luminal-side-out, cytoplasmic-side-out, and nucleoplasmic-side-out [8]. Whole-nucleus configuration would be tremendouslyhelpful in studying macroscopic currents, but has not been reportedyet.

4.2. ER Ca2+ channels

There are two major ER Ca2+ channels in mammalian cells(see Fig. 1). Localized on the ER and nuclear membranes, theubiquitously expressed IP3-Rs (IP3-R1-3) are large conductanceCa2+-permeant channels. IP3Rs are activated by the second mes-senger InsP3, which is generated upon activation of PLC-coupledreceptors on the plasma membrane by extracellular agonists [8].

RyRs (RyR1-3) are the second class of ER Ca2+ channels thatare activated upon opening of DHPRs in the sarcolemmal mem-branes to amplify the Ca2+ signals [64]. Alternatively, RyRs canbe activated directly by Ca2+ in cardiac muscle cells and neurons[64]. While endogenous RyRs are studied mostly by reconstitutioninto the lipid bilayer, overexpressed RyRs are studied using nuclearpatch-clamping [65,66].

Several non-selective cation channels, including TRPP2, TRPV1,TRPM8, presenilins, mitsugumin23, and pannexin channels are alsofound in the ER/SR membranes of various cell types and are pro-posed to be the ER Ca2+ leak channels [2]. Whereas confirmationfrom nuclear patch-clamping is still lacking, Ca2+ imaging studieshave demonstrated the roles of these proteins in passive depletionof the ER reservoir [67].

nellar channels and transporters, Cell Calcium (2015),

4.3. ER K+ channels

ER � is negligible, thus cation influx is not driven by a lumi-nal negative potential. However, several studies point to functional

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xpression of different K+ channels and a K+–H+ exchanger in theR membrane (Fig. 1). Most of these K+ channels/transporters areot ER-specific, and are located in the plasma membrane and other

ntracellular organelles such as mitochondria. These include theTP-sensitive K+ channel (in PM and mitochondria), the small and

arge-conductance Ca2+-activated K+ channels (present in PM anditochondria), and the mitochondrial K+–H+ exchanger KHE (pre-

umably formed by LETM1 present in the ER and mitochondria)67,68] (see Fig. 1). The monovalent cation-permeable trimericntracellular channels (TRIC channels) are expressed in the ER/SRs well as in the nucleus of myocytes [67]. Two proteins located inhe nuclei of plants, Castor and Pollux, have been shown to form+-permeable channels (see Fig. 1) when reconstituted in the pla-ar lipid bilayer [69]. Both proteins are required for the initiationf nuclear Ca2+ spiking [69].

The prevailing view is that ER K+ channels, along with ERl− channels of the CLC family (see Fig. 1) might ensure rapidounter-ion fluxes across the ER/SR to compensate for the chargeovements associated with Ca2+ release and re-uptake processes

9,67]. During the Ca2+ uptake phase, SERCA extrudes protons fromhe ER, which can re-enter the lumen via the KHE [9]. In turn, thisould lead to an asymmetry in K+ concentration, which would

e re-adjusted following entry of K+ into the lumen via the afore-entioned K+ channels. K+ re-entry also facilitates H+ entry and K+

xport via KHE, fostering the activity of SERCA2 [9]. In addition, thebserved voltage sensitivity of the ER/SR K+ channels suggests thathese channels might “clamp” � close to zero mV [67]. Finally, ER+ channels may also control the volume of the ER lumen, therebyodifying functional properties of this organelle [9].

.4. Yeast mechano-sensitive channels

Yeast ER membranes may express mechano-sensitive chan-els that are activated by hypo-osmolarity [25]. Mechanosensitivehannels are also expressed in yeast vacuoles, in which TRPY1 isctivated by membrane stretch and Ca2+ [70]. As membrane cur-ature is expected to generate force during membrane fusion andssion processes of organelles, mechano-sensitive channels maylay important roles in membrane trafficking. However, mechano-ensitive channels in the intracellular organelles of mammalianells have not been reported.

. Golgi apparatus

Because the Golgi apparatus receives input from ER-derivedesicles, many of the ER channels and transporters are also localizedn the Golgi apparatus, including IP3Rs and SERCA pumps. How-ver, there are also specific Ca2+ transporters in the Golgi apparatus,ncluding SPCA pumps [71]. Development of direct patch-clamp

ethods on isolated Golgi apparatus may promote functional char-cterization of Golgi-specific channels.

. Mitochondria

The existence of ion-conducting pathways in mitochondria haseen long known from classical bioenergetics studies. The chan-el activities in mitochondria have been observed during the

ast 30 years either by patch-clamping isolated mitochondria anditoplasts devoid of their outer membrane, or by incorporatingitochondrial membrane vesicles or purified native/recombinant

roteins into planar lipid bilayers [68].

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Due to the highly negative membrane potential in mitochon-ria (−150 mV to −180 mV), a strong driving force exists for

on movement through ion channels in the inner mitochondrialembrane (IMM). Since oxidative phosphorylation requires an

PRESSxxx (2015) xxx–xxx 5

electrochemical gradient across the IMM, ion channels in this mem-brane are expected to play an important role in the regulation ofenergy metabolism. Indeed, the channels operating in the IMMare highly regulated in order to avoid imbalances in energy trans-duction and consequent processes, e.g. increased production ofreactive oxygen species. In fact, as illustrated by some reviewsin this SI [17,18], disturbance of mitochondrial ion homeostasisand/or membrane potential by affecting channel activity leadsto severe mitochondrial dysfunction with consequent metabolicchanges and/or cell death.

6.1. Mitochondrial conductances

The mitochondrial channels characterized over the past threedecades include the voltage-dependent anion channel (VDAC) inthe outer membrane (see Fig. 1). In the inner membrane, thelist includes KATP, Ca2+-activated large, intermediate and small-conductance K+ channels, Kv1.3, the TWIK-related acid-sensitiveK+ channel-3 (TASK-3), the nonselective permeability transitionpore MPTP, chloride channels, the magnesium-permeable Mrs-2, the calcium uniporter MCU, and uncoupling UCP proteins (seeFig. 1) (for recent reviews see e.g. [68]). Interestingly, the sin-gle channel conductances range from a few pS (MCU), to the nSrange (MPTP). Despite the successful introduction of large-scaleproteomics into the mitochondrial channel research, molecularidentification of these channels is still incomplete. Mitochondrialchannels are encoded by the nucleus and in most cases do not har-bor clear targeting sequences. In addition, their low abundance andhigh hydrophobicity render proteomic identification extremelydifficult. Nevertheless, the Mitocarta compendium, an inventoryof more than 1000 proteins with proven mitochondrial location[72], significantly moved the field ahead, by allowing identifica-tion of some channel modulators and/or components. It must bementioned that in addition to the channels observed by electro-physiology of mitochondrial preparations, some proteins, knownto give rise to channel activities in other membranes (e.g. the vac-uolating toxin VacA, a nicotinic acetylcholine receptor, a glutamatereceptor family member) have been discovered to reside in mito-chondrial membranes as well [68]. However, whatever channelsthey form in the IMM need to be determined.

6.2. Mitochondrial Ca2+ channels

Ca2+ uptake across the IMM is performed by the mitochondrialCa2+ uniporter (MCU) and possibly by mitochondrial ryanodinereceptors (mitoRyR). On the other hand, Ca2+ efflux is mediatedby both Na+-dependent (mitoNCX) and Na+-independent Ca2+

transporters (see Fig. 1). Mitochondrial calcium homeostasis hasreceived particular attention due to its regulatory roles in the aer-obic metabolism and cellular signaling under both physiologicaland pathological conditions [68]. The long-sought calcium uni-porter, characterized in bioenergetic studies, was identified as ahighly calcium-selective ion channel observed in mitoplasts ina seminal work [73]. Recent discovery of a variety of moleculesimpacting mitochondrial calcium uptake supports the emergingview that the uniporter is a protein complex rather than a sin-gle protein. However, the exact components of this complex, aswell as of the factors determining its assembly, are highly debatedand represent a hot topic in the field. A 40 kDa protein namedMCU, when expressed in recombinant form in vitro, is able to formcalcium-selective ion channels [14,15]. However, some character-istic features of the mitochondrial Ca2+ uptake machinery (e.g. the

nellar channels and transporters, Cell Calcium (2015),

observation that mitochondrial Ca2+ uptake varies greatly amongdifferent cells and tissues and that the channel displays low activityat resting state but an increased activity after cellular stimulation)are due to the important contribution of several modulators of the

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hannel-forming protein. Indeed, the uniporter is likely a com-lex composed of an inner-membrane channel (MCU and MCUb,

dominant-negative subunit) and regulatory subunits (MICU1,ICU2, MCUR1, and EMRE) (for recent reviews see e.g. [68,74]).

n particular, both MICU1 and MICU2 are regulated by calciumhrough their EF-hand domains, thus accounting for the sig-

oidal response of MCU to [Ca2+]cytosol in situ and allowing tighthysiological control. At low [Ca2+]cytosol, the dominant effect ofICU2 largely shuts down MCU activity; at higher [Ca2+]cytosol,

he stimulatory effect of MICU1 allows the prompt response ofitochondria to Ca2+ signals generated in the cytoplasm. In a

ecent study the whole-mitoplast calcium current was found toe different in mitochondria isolated from different types of tis-ues [75]. The study of the expression of MCU complex membersy quantitative proteomics in different mouse tissues reveal sig-ificant differences in the various tissues in the MCU/MICU1 asell as MICU1/MICU2 ratio [16], pointing to the possibility of

issue-dependent activity/composition of the uniporter complex.osttranslational modifications, e.g. by the calmodulin-dependentinase II [76], might also account for the differences in MCU activity.

.3. Mitochondrial K+ channels

As mentioned above, IMM K+ channels recorded by patch-lamp include calcium-dependent K+ channels (KCa), Kv1.3, andASK-3 (see Fig. 1). Although not all channels are recorded inll tissues, most of these channels have wide tissue-expressionrofiles. With the exception of KATP that is thought to differrom its plasma membrane counterpart, the K+ channels foundn the IMM display biophysical, biochemical, and pharmacologi-al characteristics resembling those of the correspondent plasmaembrane channels, leading to the assumption that the pro-

ein entities are the same. Therefore, the generation of geneticodels (cells or animals) exclusively lacking the IMM channels

s a challenging task. In some cases, for instance, mitoKATP, definitive molecular identification has not been achieved.itoKATP has received much attention since its activity has been

inked to ischemic preconditioning, ischemic postconditioning,nd cytoprotection in general. The confounding non-specificityf available pharmacological agents and antibodies has ham-ered efforts to identify this long-sought channel at a molecular

evel (see e.g. [77]). Recently, a short form of the renal outeredullary K+ ROMK channel (ROMK2 or Kir1.1b) has emerged

s a possible candidate [78], but this identification is still underebate.

The mechanisms underlying dual/multiple targeting is stillnclear for most mitochondrial channels, as is the case forhe ER channels (see above). One exception is BKCa, which isocated on the plasma membrane, Golgi, ER, and mitochon-ria [79]. A recent study found that mitoBKCa in the heart isncoded by a splice variant of the KCNMA1 gene that encodeslasma membrane BKCa. A 50-aa splice insert is essential for itsrafficking to the mitochondria [80]. K+ channel subcellular tar-eting may also depend on intrinsic characteristics of the proteinuch as the length and/or amino-acid sequence of transmem-rane segments, as elegantly demonstrated for a viral K+ channel24].

Modulation of IMM K+ channels causes changes in ROS produc-ion and oxidative phosphorylation capacity, suggesting a role inne-tuning the oxidative and metabolic state of the cell [81]. For

Please cite this article in press as: H. Xu, et al., Orgahttp://dx.doi.org/10.1016/j.ceca.2015.02.006

xample, inhibition of mitoKv1.3 by membrane-permeable block-rs results in increased ROS production and selective induction ofpoptosis in cancer cells in vivo, whereas membrane-impermeablev1.3 inhibitors are without effect [17].

PRESSxxx (2015) xxx–xxx

6.4. Mitochondrial permeability transition pore

Another mitochondrial channel that has a crucial, well-documented influence on mitochondrial function is the perme-ability transition pore (MPTP; [18,68]). Persistent MPTP openingleads to the loss of � and mitochondrial integrity, ultimatelycausing cell death. MPTP has been shown to correspond to ahigh-conductance channel recorded by patch-clamp in the IMM.Recently the ATP synthase has been proposed to be a crucial com-ponent of MPTP by several groups [82,83]. According to one study,MPTP may form at the interface between two adjacent FO domainsof the ATP synthase in a dimer [83]. However, in another study,the pore-forming part is the c-subunit ring of the FO of the F1FOATP synthase [82]. Although no consensus has been reached on theexact way of MPTP formation by the ATP synthase complexes, thesefindings open new perspectives for several pathologies that areinfluenced by MPTP activation. The signaling pathways leading tothe transition from an energy-conserving to an energy-dissipatingdevice are of great importance in the context of cell survival. Indeed,MPTP modulation can be exploited e.g. by cancer cells to increasetheir chemo-resistance [18]. Hopefully, better understanding of thepore structure and function will help design MPTP-active com-pounds to treat cancer and degenerative diseases.

7. Chloroplasts

Chloroplasts have a double-membrane envelope as well asinternal membrane structure called thylakoids, where photosyn-thesis and ATP production take place (Fig. 1). The outer envelopemembrane is considered to be permeable to most ions and metabo-lites. In contrast, the inner envelope membrane and the thylakoidsharbor numerous selective ion and metabolite transport path-ways, allowing regulation of optimal metabolic activities andof signaling within this organelle [22,84,85]. A multidisciplinaryapproach exploiting modern genetics, plant physiology, biophysics,biochemistry and proteomics represents one of the new fron-tiers in chloroplast research. Indeed, recent results pinpoint ionhomeostasis within the chloroplasts as the master regulator of pho-tosynthesis, as illustrated by the paper of Finazzi et al. [22] in thisSI.

7.1. Chloroplast conductances

Several different solute transporters and chloride, potassium,and divalent cation-selective ion channels have been identified,either directly in chloroplast membranes using the patch-clamptechnique or after reconstitution of purified envelope membranesor thylakoid vesicles into the planar lipid bilayer (for reviewssee Refs. [84,86]). Unfortunately, not all techniques are suitablefor chloroplasts of the model plant Arabidopsis, for which manyknock-out mutant lines are available. While patch clamping ofpea chloroplast is technically demanding but feasible [87], to ourknowledge, this technique has not been successfully applied toArabidopsis chloroplasts.

As a result, many cases of molecular entities giving riseto chloroplast conductances are unknown. However, excellentmass spectrometry studies became available on chloroplast sub-membranes as well [88] (also see [22] and [23]), leading to thediscovery of many transporter proteins within this organelle.Intriguingly, only few bona fide channels were revealed by thistechnique. Luckily, even though activity is still not experimentally

nellar channels and transporters, Cell Calcium (2015),

proven for many new candidates emerging from the high-throughput approaches, sequence analysis and homology searcheswould allow predictions of their functions, as in the case of the plantcounterpart of the MCU [1]. Thus, knock-out plants might be used

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o unravel their importance for the metabolism/ion homeostasisf chloroplasts. In addition, when looking for ion channels withossible chloroplast location, the cyanobacterial origin of theserganelles can be informative and evolutionary conserved proteinsight become good candidates (for recent review see Ref. [89]).In the few cases in which molecular identification of chloro-

last channels was successfully achieved, precious information wasbtained on the physiological roles of these channels by usingnock-out Arabidopsis plants. For example, small mechanosensitivehannel-like (MscS-like) Arabidopsis homolog AtMSL3, was showno rescue the osmotic-shock sensitivity of a bacterial mutant lack-ng MS-channel activity [90]. Localized in the envelope, AtMSL3 haseen shown to control plastid size and shape, to protect plastidsrom hypo-osmotic stress, and serve as component of the chloro-last division machinery [90]. Two-pore potassium channel TPK3,

ocated in the thylakoid membrane, was shown to be a cruciallayer in the optimization of photosynthesis, by influencing theroton motive force (see [22] and [91]).

A plethora of ion transporters are also present in the chloro-last, allowing the transport of metals, inorganic anions, calcium,nd potassium (see Table 1 in Ref. [23]). The function of these trans-orters ranges from osmoprotection and protection from oxidativetress to ammonium assimilation. Likewise, many metaboliteransporters are expressed in the chloroplast, participating in pho-orespiration and nitrogen/sulfur metabolism (see [85]).

. Plant vacuoles

Vacuoles are the plant counterparts to lysosomes. Therefore, likeysosomes, plant vacuoles accommodate high levels of hydrolyticctivities, and store high concentrations of Ca2+ and Na+ [92].nlike lysosomes, plant vacuoles are large organelles, since theyccupy 80–90% of the size of an adult plant cell with a diame-er of 20–40 �m. This large size has made vacuoles interesting for

any electrophysiologists since the beginning of the patch-clampra [29]. The vacuole fulfills many diverse roles, such as the tempo-ary storage of solutes or potentially toxic compounds. A plethora ofransporters and channels are characterized on the vacuolar mem-rane (also called tonoplast). Many of them have been molecularly

dentified during the last 10–15 years. Here we will focus on trans-orters and channels involved in Ca2+, Na+, K+, and anion transport.

more detailed and exhaustive overview is given in some recenteviews [29,93].

.1. Vacuolar Ca2+ transporters and channels

The vacuole is the major Ca2+ store and it is generally assumedhat Ca2+ released for signal transduction is mainly from the vac-ole. This assumption is made based on the experiments performedy Alesandre et al. [94] showing that InsP3 releases Ca2+ pre-ominantly from the vacuole. Later experiments by Lemtiri-Chlieh95] and Munnik et al. [96] indicated that in plants inositol-exakisphosphate (InsP6) plays a much more important role in

ntracellular signal transduction than InsP3. However, the channelseleasing Ca2+ have remained unidentified. TPC1 proteins may acts vacuolar Ca2+ channels (see Fig. 1), with conflicting evidence inavor and against its Ca2+ conductivity [92]. The propagation of thealt-stress induced Ca2+ waves is dependent on TPC1 in Arabidopsis97], suggesting that TPC1 may indeed act as a Ca2+ channel underertain in vivo conditions.

Ca2+ is taken up into the vacuole likely by two P-type Ca2+ pumps

Please cite this article in press as: H. Xu, et al., Orgahttp://dx.doi.org/10.1016/j.ceca.2015.02.006

s well as a small gene family encoding calcium-proton exchangersCAXs; see Fig. 1), which exhibit a high sequence homology to theireast counterparts residing also on the vacuolar membrane [98].nterestingly, different CAX proteins exhibit slight differences in

PRESSxxx (2015) xxx–xxx 7

their substrate recognition, with a subset of them transporting notonly Ca2+, but also heavy metals such as Cd and Mn [29,99].

8.2. Vacuolar Na+/K+ channels and transporters

The vacuole lumen is iso-osmotic to the cytosol, since the vac-uolar membrane is permeable for water [29,92]. Plants store a largeamount of inorganic ions as osmolites within the vacuole [29,92].This is energetically favorable compared with the production oforganic compounds such as glucose or sucrose. Sodium and potas-sium are taken up into the vacuole by K+/Na+ proton antiporters ofthe NHX family [100] (see Fig. 1). The first NHX was identified asa Na+/H+ antiporter [101]. During the first years after this discov-ery, it was thought that NHXs act as Na+/H+ antiporters to detoxifysodium [101]. Later studies demonstrated that vacuolar NHXs, aswell as NHXs from the secretory system, mediate also potassiumuptake into the vacuole [102].

Several Na+/K+ channels have been shown to reside in the vac-uolar membrane. The aforementioned vacuolar TPC1 is also likelyto be involved in sodium export, mainly in response to signalingcues, since this channel is activated by Ca2+. Furthermore, four outof five members of the TPK family (see Fig. 1) are localized in thetonoplast in Arabidopsis [103]. The most well characterized TPK isAtTPK1, whose activation is Ca2+-dependent. The channel activityof AtTPK1 is also stimulated by its interaction with 14-3-3 proteins,but suppressed at high [pH]Lumen > 6.8 [104].

8.3. Vacuolar anion channels and transporters

Two types of transporters and channels have been described forinorganic anions. In addition, a homologue of the renal carboxylatetransporters [105] has been shown to act as a malate transporterin Arabidopsis and citrate exporter in Citrus [29]. The class of ABCCtransporters that is presumed to be localized in the vacuolar mem-brane can transport organic anions [106].

ALMTs have been described first as plasma membrane-localized,aluminum-activated malate exporters [107]. Later, it was shownthat members of a ALMT subfamily reside in the tonoplast [107].Vacuolar ALMTs are permeable to malate, but it is likely that theirphysiological role is to act as malate-activated chloride channels[108]. ALMTs are specific to plants and have so far not been foundin other organisms.

In contrast to animal CLCs, plant CLCs reside exclusively in inter-nal membranes [109]. Two members have been characterized indetail [109]. While CLCc acts as a chloride-proton exchanger, thebest characterized plant CLC, CLCa, is a nitrate proton antiporter(see Fig. 1). CLCa is required to drive nitrate accumulation in thevacuole. It was shown that a very steep nitrate gradient is main-tained in plants accumulating nitrate as a nitrogen reserve [110].Interestingly, the phosphorylation status of CLCa set the direction ofnitrate flux. Hence CLCa is also required, at least partially to unloadnitrate [110].

In conclusion, a large number of transporters and channels havebeen identified in the plant vacuolar membrane. What we lackis more insight into the regulation of this network and how allthese transporters and channels interact with each other in orderto maintain a cytosolic ion homeostasis.

9. Future directions

Despite the rapid progress made in the research of organellarchannels and transporters, many questions related to subcellular

nellar channels and transporters, Cell Calcium (2015),

targeting, regulation, structure–function relationships, and phys-iological roles of intracellular channels and transporters remain.Furthermore, identification of possible ion channel/transportermodulators (e.g. kinases and lipids) in some organelles has begun,

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pening the exciting possibility of studying organelle-specific reg-lation of intracellular channels/transporters. The recent exciting

nsights in organellar channels and transporters will undoubtedlyrovide further motivation for the scientific community in pursuitf this goal.

Importantly, techniques developed to study one organelle couldpark ideas and provide methods to study other organelles. Forxample, the plant studies have led directly to the moleculardentification of new ligand-activated channels in the intracellu-ar membranes of animal cells. This molecular “cross-pollination”xemplifies the importance of establishing a discussion forum inhe area of intracellular transport systems: both plant and animalommunities must meet and challenge each other with new resultsnd thoughts. Likewise, bringing together scientists working onifferent organelles will result in innovative ideas and research.or example, it will prove fruitful to repurpose the research andechniques developed in studying lysosomes and mitochondria forhe studies of other organelles including autophagosomes, synapticesicles, and lysosome-related organelles. For example, whole-elanosome recording has been achieved recently to discover new

nion channels in the melanosome [62].Recent studies reveal that different intracellular organelles (i.e.

he ER, mitochondria, and lysosomes) cross-talk with each othero form intracellular networks to regulate basic cell biologicalrocesses such as Ca2+ signaling, ion and lipid exchange, signalransduction, autophagy, and metabolism. In fact, membrane-ontact-sites (MCSs) are crucial for ion transport and lipid exchangeetween organelles, e.g. ER and mitochondria [111]. The rolesf channels/transporters in MCSs, which are difficult-to-study atresent, are just beginning to be discovered. For example, theoltage-dependent anion channels (VDACs) in mitochondria wereecently shown to interact directly with IP3 receptors in the ER, andhis interaction is crucial for cellular metabolism and ATP produc-ion [111]. Likewise, ER and endosomes may also cross-talk withach other to regulate Ca2+ signaling [112]. In the future we will seeore studies on inter-organellar communication and regulation.

cknowledgements

We apologize to colleagues whose works are not cited due topace limitations. We thank Matteo Simonetti for his assistancen making the figure. The work in the authors’ laboratories isupported by NIH grants NS062792 and AR060837 to H.X.; AIRCG15544, PRIN 2010CSJX4F 005 grants to I.S.; grants of the Swissational Foundation and EU to E.M. We appreciate helpful com-ents from other members of the Xu, Szabo, and Martinoia’s

aboratories.

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