EFTA00611569.pdf

FEBS Leans 587 (2013) 1923-1928 ELSEVIER journal homepage: www.FEBSLetters.org Review Minireview: pH and synaptic transmission Anne Sinning'''. Christian A. Hubner' 'Institute of Human Genetics, University Hospital Jena Thechich Schiller University Jena, Kollegiengasse la 0-07743 Jena. Germany °Institute of Physiology and Pathophysiology, Ltniversary Medical Center of the Johannes Gutenberg University, Duesbergweg G 0-55128 Mainz Germany (ID CrossMarlc ARTICLE INFO Article history: Received IS April 2013 Revised 26 April 2013 Accepted 26 April 2013 Available online 10 May 2013 Edited by Alexander Cabibov. Vladimir Skulachev, Felix Wieland and Wilhelm Just Keywords: pH regulation Ion transporter Mouse model Carbonic anhydrase Synaptic transmission CAM Neuronal excitability 1. Introduction ABSTRACT Asa general rule a rise in pH increases neuronal activity, whereas it is dampened by a fall of pH. Neu- ronal activity per se also challenges pH homeostasis by the increase of metabolic acid equivalents. Moreover, the negative membrane potential of neurons promotes the intracellular accumulation of protons. Synaptic key players such as glutamate receptors or voltage-gated calcium channels show strong pH dependence and effects of pH gradients on synaptic processes are well known. How- ever. the processes and mechanisms that allow controlling the pH in synaptic structures and how these mechanisms contribute to normal synaptic function arc only beginning to be resolved. 02013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. The strong acidification of synaptic vesicles by the vacuolar - ATPase. which energizes the neurotransmitter loading of synaptic vesicles [11, is a main reason for the large fluctuations in synaptic pH. Synaptic vesicle exocytosis results in the release of protons into the synaptic cleft as well as in the incorporation of the vacu- olar H'-ATPase into the presynaptic membrane. Thus synaptic transmission causes a relatively short but strong acidification of the synaptic cleft [2-4]. The extracellular acidosis is subsequently followed by a long, yet transient increase in extrasynaptic pH 151. In the hippocampus this alkaline transient can be detected within milliseconds 16.71 and reaches magnitudes as large as 0.1-0.2 pH units 181. Mechanisms underlying this rise in pH are not fully understood but most likely presynaptic Ca2'1H'-ATPase 19.101 extracellular carbonic anhydrases [8]. and GABAA-receptor mediated bicarbonate efflux [111 are involved. Increased synaptic/neuronal activity can also cause a prolonged extracellular acidification because of the increased cell metabolism [5.12.13]. Although several studies have successfully monitored neuronal pH shifts in the brain 12.14.151. only very little is known about pH transients in neuronal microdomains because of technical limita- dons 116.171. Direct experimental data on pH fluctuations and * Corresponding author. E-mall address: christian.huebneremtiuni-Jenade (CA. Wilmer). pH regulation in intracellular synaptic compartments so far have only been obtained for motor endplates because of their signifi- cantly larger dimensions compared to central synapses [4.181. Zhang et al. used the pH-sensitive properties of the yellow fluores- cent protein to analyse the presynaptic pH in mouse motor end- plates. This study not only supports the importance of presynaptic pH regulators but further provided evidence that the release of vesicles in the peripheral nervous system is accompanied by a transient intracellular acidification. Here, the increase in pH was mainly caused by the activation of plasma membrane Ca2*/ H'-ATPase and was followed by an unexpected, longer lasting alka- linisation is due to the transient incorporation of the vacuolar Fr- ATPase into the presynaptic membrane 141. Focal injections of BCECF-AM in combination with slice imaging as used for measur- ing calcium transients in small synaptic compartments with the calcium-sensitive dye Fura [19]. genetically encoded pH indicators 1181. which also allow ratiometric imaging 120.211. may help to establish adequate and fast pH measurement in small compart- ments like central pre- and postsynaptic terminals in the future. Despite these technical limitations the occurrence of rather large, spatially and timely limited. pH fluctuations in the different synaptic compartments is generally accepted and clearly implies that pH regulatory elements are essential to maintain proper syn- aptic function. Since many synaptic elements are strongly pH dependent, limitations and alterations in synaptic pH homeostasis could potentially feed-back on neuronal activity itself. Intriguingly. 0014-5793/136.00 0 2013 Federation of European Biodiemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.dolorgf10.1016b.febslet.2013.04.04S EFTA00611569 1924 A. Sinning CA. HOWier/ FESS Letters 587 (2013) 1923-1928 it has already been shown that direct release of protons during vesicle exocytosis can act as a negative feedback on closely associ- ated calcium channels in the mammalian retina 13221. In this sys- tem. synaptic cleft acidification of retinal cells is thought to underlie surround inhibition and thereby helps to form the recep- tive field (for review see 1231). The discovery of acid-sensing ion channels (ASICs) is another example for a pH-induced feedback mechanisms 1241. At least four genes and their alternatively spliced transcripts code for subunits of such ion channels, which belong to the degenerinfepithelial Na' channel superfamily and are character- ized by a strong H'-sensitivity as well as their permeability for cat- ions. ASICs are widely expressed in the mammalian nervous system and have been shown to localize mostly to somato-dendritic regions of neurons 125261. ASICs have been implicated in many neurologi- cal disorders like e.g., ischemic stroke, epileptic seizures and pain (for review see1271). Interestingly, one study suggested that seizure termination critically depends on ASIC activation by the fall in extracellular pH in response to epileptic neuronal activity [281. 2. Effects of pH transients on presynaptic function Loading of synaptic vesicles with different neurotransmitters depends on vesicular proton gradients 1291. Hence, variations in intracellular pH could directly interfere with neurotransmitter loading. It has been shown that the glutamate uptake by astrocytes is pH sensitive and provides a mechanism which can protect neu- rons from glutamatergic excitotoxicity due to reversed glutamate uptake under ischemic conditions 1301. The function of proteins, enzymatic activity as well as protein- protein interactions are sensitive to alterations in pH and thus changes in pH can impact on the release of synaptic vesicles. which depends on the conceited action of a complex machinery of different proteins (for review see1311). In particular, the initial rise in the pre- synaptic calcium concentration mediated via voltage-gated calcium channels 1321 is pH dependent, as the opening and the conductivity of presynaptic voltage-gated calcium channels strongly depend on both extracellular and intracellular pH 1331. Protons can directly bind to sensors within the pore of the channel and thereby reduce channel conductance [34.351. shield membrane-bound charges and thus shift the channel activation voltage to more positive values 136,371. The rise in presynaptic calcium is augmented by release of calcium from intracellular stores which is mediated via inositol 1.4.5-trisphosphate and ryanodine receptors. Both receptors also show strong pH dependence138.391. Studies on spontaneous vesicle release by electrophysiological methods confirmed that lowering of intracellular pH in hippocampal neurons indeed results in a de- ceased rate of synaptic vesicle release and hence limited excitability 140,411. Further studies are necessary to investigate if presynaptic pH modulates synapse function mainly by alterations in calcium transients or if multiple effects add up. 3. Effects of pH transients on postsynaptic function NMDA receptors are strongly modulated by changes in extracel- lular pH 142,431. An increase in extracellular pH facilitates the acti- vation of NMDA receptors, whereas a decrease in extracellular pH inhibits ion channel function 142-441. The transient increase in extracellular pH elicited by high-frequency stimulation of afferents in the hippocampus has been shown to be sufficient to augment NMDA-receptor responses in vitro 1451. This is most likely also relevant in vivo both in physiological and pathophysiological conditions. In contrast, kinetics and amplitudes of AMPA- and Kainate-receptors are only marginally modulated by alteration of extracellular pH 1461. Interestingly, GABAA receptor mediated currents are enlarged by low extracellular pH. whereas a high pH rather inhibits the GABA response 147-491. GABAA receptors also conduct bicarbon- ate. As a consequence, GABAergic transmission can cause altera- tions of both intra- and extracellular pH 1111. In contrast to the direction of chloride fluxes, which can vary in dependence of the existing chloride gradients, which are set by the cation-chloride co-transporters NKCC1 and KCC2 150-521. the existing gradients always drive HCCIi out of the neurons under physiological condi- tions. Both gradients contribute to the balance between neuronal excitation and inhibition. Only little is known about the role of pH for signaling via GABA8 receptors or receptors of other neurotransmitters. In conclusion, electrical stimulation or synchronized neuronal activity results first in an initial transient alkaline shift of the extra- cellular pH that is followed by a prolonged acidosis (for review see 151). The short-lived initial increase in pH has been shown to be sufficient to augment glutamatergic excitation by activation of NMDA receptors in acute slice experiments 1451 and most likely inhibits GABAergic transmission. In contrast, under conditions of sustained stimulation [531 or pathological neuronal activity (121, the following long-lasting acidosis is predicted to diminish gluta- matergic neurotransmission and boost GABAergic which was confirmed for cultured neurons [541. This indicates that intrinsic pH transients serve as a feedback mechanism to keep the delicate balance between neuronal excit- ability and inhibition but also implies that neuronal and especially synaptic p11 has to be tightly controlled. 4. Mechanisms to regulate synaptic pH In general, cellular pH homeostasis is established by transport or buffering of acid equivalents. In neurons acid loading is largely established by Na' independent CI-/HCO3 exchangers [551. whereas Nalt exchangers [561. NC-driven CI' /HCCI; exchangers and Na'/HCO3 co-transporters [571 mediate acid extrusion. An- other family of bicarbonate transporters, which can be distin- guished from the family of SLC4 transporters 155.581, are classified as members of the SLC26 family [591. however, if at all. members of the SLC26 family of bicarbonate transporters are thought to play a minor role for neuronal pH homeostasis 1601. Neuronal pH is also affected by monocarboxylate transporters 1611 but their role in the brain under physiological conditions is limited whereas they are more important in tissues with a high en- ergy demand like in tumors 1621. Although so far no conclusive data exist that the plasma membrane calcium ATPase also plays a direct role for pH regulation, a brain-specific isoform with a pre- dominant synaptic localization has been described [631, which may contribute to synaptic pH homeostasis 19,101. Bicarbonate is a very important pH buffering system because it can be regulated by respiration. Carbonic anhydrases promote the interconversion of carbon dioxide and water to bicarbonate and protons, and thereby significantly contribute to the intra- and extracellular buffering capacity in the brain 1641. For a more general comprehensive review on cellular pH sen- sors and regulators, see 1651,151 and 1661. In the following we will mainly focus on the NalH' exchanger NHE1. the Na coupled an- ion-exchangers NCBE and NDCBE, and NC-HCO3* co-transporters. all mediating acid extrusion. 5. NHE1 The transmembrane NC-gradient is established by the Na 1K' ATPase. The Na' gradient is then used to energize the electroneu- tral exchange of one extracellular sodium for one proton by Nal H' exchangers (NHE) 1671. So far 9 different isoforms of Halle exchangers have been identified and all of these are expressed in EFTA00611570 A. Slum& CA HatinerIFEBS Letters S87(2013) i92 s "i_., 192S the central nervous system (for review see [681). NHE1iSLC9A1 is ubiquitously expressed and a multifunctional protein which does not only contribute to intracellular pH regulation but also volume regulation, cell migration, and also interacts with components of the cytoskeleton [69]. Because of the lack of suitable antibodies localization studies for Slc9a1 are limited. However, most studies suggest that Slc9a1 localizes to presynaptic nerve terminals of GABAergic neurons [54.70.711. Disruption of Slc9a1 in mice re- sulted in a severe phenotype with locomotor deficits, epileptic sei- zures. neurodegeneration, and early mortality [72.73]. Slc9a1 deficient neurons had a lower steady-state pH and a delayed recov- ery from acid loads 1741. The epileptic phenotype in Slc9a1 knock- out mice is therefore surprising, because an increase in pH is generally associated with an increase of neuronal excitability. However, disruption of Nhel results in a more complex phenotype with increased Na'-current density in hippocampal neurons [75.761 as well as increased neuronal cell death 172]. There is also indirect evidence from electrophysiological recordings with phar- macological inhibitors of Na'ffit exchange like amiloride, suggest- ing that the Neill' exchanger, most likely NHE1, localizes to inhibitory and excitatory presynaptic nerve terminals [54.70.71]. In an elegant study by Dietrich and Morad the impact of extracel- lular pH buffering on the spontaneous release of GABAergic vesi- cles in cerebellar granule cells was investigated. The results from this study suggest that Nhel activity may not only affect presynap- tic vesicle release by increasing intracellular pH but also boost GABAergic neurotransmission by increasing GABAA receptor re- sponses at the postsynapse via the extracellular pH [541. 6. Na coupled anion-exchangers From early pH recordings in the squid giant axon and in snail neurons it became evident that NC-dependent Cr/NCO; ex- change plays an essential role in the control of intracellular pH of neurons 177.781. This observation has been supported by the dem- onstration of Na'-driven Cr/NCO; exchange in different prepara- tions of hippocampal neurons [79-82]. But the molecular correlate remained unclear, until a first cDNA was cloned from drosophila [831 and from a mouse insulinoma cell line [84]. In mammals Na'-dependent Cl-/HCO; exchange is mediated by NDBCE (SLC4A8) and NCBE (SLC4A10). The initial transport characteriza- tion of NCBE/SLC4A10 as NC-dependent Cl- /HCO3 exchanger was confirmed for rat 184-861. whereas the human cDNA was rather characterized as an electroneutral /HCO; co-transporter (NBCn2) with CI- self-exchange activity 1871. Some of the controversy may be explained by the different expression systems used in the different studies like mammalian cells and Xenopus oocytes, temperature and composition of solutions. the transfectionjinjection efficiency or molecular tagging of the transport proteins. In drosophila disruption of Na'-dependent CI'/HCO3 exchange results in early lethality of the larvae [831. Surprisingly, the pheno- type of Slc4a8 knockout mice is very mild with some minor deficits in different behavioral paradigms [41 .881. whereas Slc4a10 knock- out mice experience a critical period within their first week of life with a decreased gain of body weight during postnatal develop- ment. They also display a drastic reduction of brain ventricle size [891 and visual impairment 1901. Details expression analysis in the brain revealed a significant overlap between both transporters. In the hippocampus Slc4a8 as well as Slc4a10 are expressed in pyramidal neurons 1411. The syn- aptic expression of Slc4a8 was further analyzed by ultrastructural analysis [41.911. Transmission electron microscopy of freeze-frac- tured synaptosomes of wild-type mice revealed that Slc4a8 co- localizes with different presynaptic markers like e.g., syntaxin [411 or SNAP-25 (Fig. 1). The presynaptic expression of Slc4a8 Wildtype Synaptosome Slc4a8 SNAP25 Fig. 1. Presynaplic expression of SIC4alliNIXIIL Transmission electron microscopy of a freeze-fractured synaptosomes isolated from wild-type mouse brains immu- nogold-labeled for SkOaS (large grains 10 nm) and the presynapuc marker SNAP25 (small grains 5 am). Images show the proteoplasmic side of synaptosane mem- branes. Scale bars correspond to 100 and 50 nm was nicely supported by the electrophysiological characterization of Slc4a8 knockout mice [411 and confirmed in an independent study [91]. In agreement with the classification of NDCBE as an acid extruder, Slc4a8 deficient neurons displayed a lower steady state pH and a defective pH regulation. Electrophysiological analy- sis and FM-imaging further showed a decrease in spontaneous and stimulated release of glutamatergic synaptic vesicles in knockout neurons. In accordance with a predominant presynaptic localiza- tion. there was no effect on the post-synaptic kinetics of AMPA receptor currents detected. Moreover, the release of GABAergic vesicles as evidenced from recordings of mIPSCs in acute hippo- campal brain slices did not differ between genotypes [411. In con- trast to NDCBE. NCBE has also been detected in hippocampal intemeurons and co-localizes with pre- and postsynaptic markers of GABAergic synapses in the hippocampus [891. The recovery of hippocampal principal cells from add loads was delayed in acute brain slices of Slc4a10 knockout mice, although there was no dif- ference in the steady state pH. Here, this affected network excit- ability was studied in the 4-aminopyridine model of interictal discharges in acute brain slices. Although the frequency of the interictal-like events at baseline levels did not differ between genotypes, the decreased frequency upon a propionate pulse was prolonged in the knockout [891. Interestingly, disruption of either Slc4a8 or the Slc4a10 in mice increased the seizure threshold in different seizure inducing paradigms [41,89]. In contrast, Slc9a1 and Slc4a3 knockout mice are more susceptible to seizures [72.94 7. Na' /NCO; co-transporters Na' /HCO; co-transporters also mediate net acid extrusion. The electrogenic NI/HCO3 co-transporter Slc4a4 (also called NBCe1) and the electroneutral Na'/HCCIi co-transporter Slc4a7 (also called NBCn1) are broadly expressed in the central nervous system [93.94 Slc4a7 was shown to co-localize with PSD-95, a postsynap- tic protein of glutamatergic synapses [931. Slc4a7 expression was increased upon metabolic acidosis and this up-regulation was associated with glutamate excitotoxicity [95]. Slc4a7 knockout mice have been reported to display severe sensory deficits [961. however, a detailed analysis of its role for synaptic transmission is missing to date. Recent data suggest that Slc4a4 helps to prevent the large, pro- longed, Ca2"-dependent alkaline shift upon depolarization of neu- rons 1971. As prolonged positive shifts in membrane potential. which might cause a sustained net alkaline shift, are a recurrent condition during normal brain function, depolarization activated EFTA00611571 1926 A. Sinning CA. Hubner/FEES Letters S87 (2013) 1923-1928 NCO; Slc4a301 ? Glutamatergic CA P Kos HI HI Icgel GABAergic HCO GASA.R Slc4a7 Presynapse Slc4a10 HCO3- HCOic NMDA-R NCO, SkAa10 Postsynapse Flg. 2. Model displaying different regulators involved in the control of synaptic pH. Sk4a8 localizes to glutamatergic presynapses and modulates the release of glutamate vesicles in a pH-dependent manner (411. Sk9a1 appears to play an important role for pH regulation at GABAergic nerve terminals (691. Sic4a10 is likely to be expressed on both sides of CABAergic synapses 188). Slc4a7 localizes to the postsynaptic site (941. Extracellular and probably also intracellular CM increase the buffering capacity of the different compartments 18). Whether or not 51c4a3 and other pH regulators modulate synaptic activity remains unclear. acid extrusion most likely also plays a role under physiological conditions. It was speculated that this depolarization induced alka- linization may be an adaptation to preempt untoward acidification from large intracellular Ca2' loads, while maintaining or accelerat- ing the rate of glucose utilization through the glycolytic pathway. Interestingly, a parallel Cr-dependent mechanism also contrib- uted to this depolarization induced alkalinization, but its molecu- lar correlate is yet unclear 1971. 8. Carbonic anhydrases Carbonic anhydrases (CA) catalyse the rapid interconversion of carbon dioxide and water to bicarbonate and protons and vice ver- sa. CA activity was first described in red blood cells 1981 and later became evident in many other organs. In the mammalian brain at least 10 catalytically active isoforms or CAs have been described. which differ in cellular 199,1001 and sub-cellular 1101.1021 locali- zation. Evidence on the role of carbonic anhydrases for synaptic transmission has largely been deduced from studies with pharma- cological blockers of CA. Experiments with membrane-permeant and membrane-impermeant blockers of carbonic anhydrases re- vealed that extracellular CA are involved in the regulation of the interstitial pH in the brain 18.1031. The extracellular and mem- brane-bound carbonic anhydrases CA4 and CA14 are abundantly expressed by neurons 1101,1041 and have been implicated in buf- fering extracellular alkaline shifts following neuronal activity 18.1051. Furthermore, functional coupling of CA activity of AE3- mediated bicarbonate transport was described in hippocampal neurons 11061. Intracellular CAs have been shown to be essential for synchronous firing of hippocampal neurons by enabling tonic GABAergic excitation 11071. CA inhibitors are widely used as anticovulsant drugs 11081. Hence, closer analysis of synaptic expression CM and a better understanding of their functional role could greatly impact on fu- ture clinical applications. 9. Conclusion There is ample evidence that synaptic function critically de- pends on intracellular and extracellular pH gradients and that syn- aptic activity also causes local pH gradients. 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