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Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2014-0278 Minireview
A universal mechanism for transport and regulation of CPA sodium proton exchangers Octavian Călinescu1,2 and Klaus Fendler1,*
1
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max-von-LaueStr. 3, D-60438 Frankfurt/Main, Germany
2
Department of Biophysics, Faculty of Medicine, ‘Carol Davila’ University of Medicine and Pharmacy, RO-050474 Bucharest, Romania
*Corresponding author e-mail: [email protected]
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Universal transport mechanism for Na+/H+ exchange
Abstract Recent studies performed on a series of Na+/H+ exchangers have led us to postulate a general mechanism for Na+/H+ exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na+/H+ exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na+/H+ exchangers. Keywords: cation/proton antiporter superfamily; Na+/H+ exchangers; NhaA; NhaP1; pH regulation; transport mechanism.
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Universal transport mechanism for Na+/H+ exchange
Introduction All living organisms require a strict control of their intracellular sodium and proton concentrations, as extreme values of either can lead to cell stress and ultimately death. Among the molecular mechanisms responsible for regulating the intracellular sodium concentration and pH, a key role is played by integral membrane proteins called Na+/H+ antiporters or exchangers (Brett et al., 2005, Padan et al., 2009). Most Na+/H+ exchangers belong to the CPA1 and CPA2 subfamilies of the monovalent cation/proton antiporter superfamily, CPA (Brett et al., 2005). The CPA1 family includes exchangers that transport n protons against n Na+ ions across the membrane and are electroneutral, while the CPA2 family includes electrogenic exchangers (Brett et al., 2005). Most of the medically relevant Na+/H+ exchangers belong to the CPA1 family (NHE1-9), while only two exchangers in humans are CPA2 (NHA1-2) (Donowitz et al., 2013). Due to the inherent difficulties in purifying and reconstituting eukaryotic membrane proteins, the study of prokaryotic model systems is of great importance for the understanding of the molecular mechanisms that govern Na+/H+ exchange. By far the most studied Na+/H+ exchanger and the prototype of the whole CPA superfamily is the electrogenic E. coli NhaA (EcNhaA) CPA2 antiporter. EcNhaA owes this interest at least in part to the fact that its crystal structure was the first determined for any Na+/H+ exchanger (Hunte et al., 2005). Recently, the crystal structure of another CPA2 exchanger, NapA from Thermus thermophilus (TtNapA) was determined (Lee et al., 2013), providing another potential model for the CPA2 family. For the CPA1 class, a promising model system is the NhaP1 exchanger from Methanocaldococcus jannaschii (MjNhaP1), as it was, until recently, the only member about which structural data were available (Goswami et al., 2011, Paulino and Kuhlbrandt, 2014, Paulino et al., 2014). Recently, the crystal structure of another CPA1 exchanger, NhaP from Pyrococcus abyssi (PaNhaP), was reported (Wohlert et al., 2014), making PaNhaP another potential model for the CPA1 family. Na+/H+ exchangers are highly reversible (Mager et al., 2011) and can change their transport direction (see e.g. Kinsella and Aronson, 1982). However, most Na+/H+ exchangers function according to a preferred transport mode at physiological conditions: In general CPA1 Na+/H+ exchangers transport H+ out and Na+ into the cell while CPA2 exchangers mediate the reverse process (Leblanc et al., 1988). For example mammalian plasma membrane CPA1 exchangers (Bobulescu et al., 2005) as well as their prokaryotic CPA1 counterparts like MjNhaP1 (Thauer et al., 2008) are believed to function according to the Na+ in, H+ out mode. In 3 / 13
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Universal transport mechanism for Na+/H+ exchange contrast, CPA2 exchangers like EcNhaA (Padan et al., 2005) exchange external H+ for internal Na+. An exception to this rule may be certain CPA1 Na+/H+ exchangers, organellar NHEs, which are proposed to mediate H+ efflux out of intracellular compartments corresponding to transport of Na+ (or K+) out and H+ into the cytoplasm (Ohgaki et al., 2011). In spite of their varied stoichiometry, physiological function or selectivity for other cations, CPA Na+/H+ exchangers seem to share a few essential properties. They have a series of conserved residues that are essential for their transport function (Landau et al., 2007, Goswami et al., 2011) and they all show a pH-dependent activity (Padan et al., 2009, Goswami et al., 2011, Mager et al., 2011). Therefore, it makes perfect sense to look for a common transport mechanism that accounts for all Na+/H+ exchangers, be they electroneutral, electrogenic, eukaryotic or prokaryotic.
A minimal kinetic model for Na+/H+ exchangers The basic structural concept for our understanding of transporter function is the alternate access principle formulated nearly 50 years ago (Jardetzky, 1966). Transport is mediated by a flexible membrane-spanning macromolecule with substrate binding sites which are alternatively exposed to the cytoplasmic and the extracellular sides of the membrane. However, a complete picture only emerged ~20 years later when also the functional and thermodynamic principles of transporter mechanism were derived. A simple mechanism for antiport (Stein and Honig, 1977, Klingenberg, 1992) involves an exchanger that has a common binding site for both substrates and can switch between an inside and outside-open conformation only when this site is occupied. Thermodynamically, the energy barrier between these two conformations is high in the absence of substrate and is lowered upon substrate binding. The energy required for this conformational transition is provided by the binding energy of the substrate (Klingenberg, 1985a, Klingenberg, 1985b). Its structural and molecular background, however, remains yet to be uncovered. Based on the above described minimal exchanger mechanism we developed a kinetic model (Mager et al., 2011) using the following constraints: 1) the binding site is identical when exposed to the cytoplasmic and extracellular sides and 2) the inward and outward open forms of the carrier have the same energy. This is justified by the experimental evidence showing that the EcNhaA Na+/H+ exchanger is functionally completely symmetrical. The kinetic 4 / 13
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Universal transport mechanism for Na+/H+ exchange representation of this mechanism is the 6-state kinetic model shown in Figure 1A. Symmetry implies that the binding constants (pK and
) of the inside and outside open carrier as well
as the forward and backward rate constants of the conformational transitions are the same. Note that, although completely symmetrical, the kinetic model mediates vectorial transport of one substrate in the presence of an oppositely directed concentration gradient of the other substrate.
pH regulation as an intrinsic property of the Na+/H+ exchange mechanism The kinetic model in Figure 1A has interesting properties. It is instructive to consider the case where the pH on both sides of the membrane is identical (Figure 1B solid black line). Under these conditions the pH profile of the transporter is bell-shaped. Acidic down-regulation is due to competition of Na+ and H+ for the common extracellular binding site. In contrast, at alkaline pH down-regulation is due to depletion of the substrate H+ at the intracellular binding site. These two mechanisms of regulation are operative also at asymmetrical pH and account for the respective pH profiles. If the pH outside is alkaline and Na+ is present outside then Na+ enters while protons leave the cell and down-regulation via substrate depletion takes place when the inside pH becomes alkaline (red dashed line Figure 1B). If the pH outside is acidic and Na+ is present inside then Na+ leaves while protons enter the cell and down-regulation via substrate competition is operational as the inside pH goes down (blue dashed line Figure 1B). It is clear that depending on the conditions the pH profile of one and the same Na+/H+ exchanger can vary from alkaline down-regulation to acidic down-regulation to a bell-shaped profile and that a certain profile is not a specific property of an exchanger but critically depends on the assay conditions. The pH profiles shown in Figure 1B have important physiological implications. CPA1 Na+/H+ exchangers work in the Na+ in, H+ out mode, which corresponds to the red dashed line in Figure 1B and are therefore inherently down-regulated at alkaline pH preventing excessive akalinization of the cytoplasm. CPA2 Na+/H+ exchangers work in the H+ in, Na+ out mode, which corresponds to the blue dashed line in Figure 1B and are therefore inherently down-regulated at acidic pH preventing excessive acidification of the cytoplasm. Therefore,
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Universal transport mechanism for Na+/H+ exchange an attractive feature of the kinetic model is that Na+/H+ exchangers are down-regulated at alkaline or acidic pH by the nature of the transport mechanism itself.
Experimental validation of the kinetic model So far our discussion was based on theoretical considerations pertaining to a plausible kinetic model that has the value of being mechanistically as simple as possible. But is this mechanism correct and does the kinetic model correctly describe the properties of a real Na+/H+ exchanger? Specialized protein regions, referred to as ‘pH sensors’ or “proton modifiers” have been suggested to account for the strong pH dependence of Na+/H+ exchangers (Aronson et al., 1982, Padan et al., 2009). This concept has recently been challenged by the finding that the CPA2 exchanger EcNhaA is active at cytoplasmic pH 5 (Mager et al., 2011). This observation, being in conflict with the hypothesis of a cytoplasmic proton modifier site, suggested that alternative explanations for the pH dependence of EcNhaA had to be considered. Indeed it was shown that the substrate dependence of EcNhaA can be explained by the simple kinetic model of Figure 1A and it was proposed that its pH dependence is a unique property of its transport mechanism (Mager et al., 2011). In line with the kinetic model, a single binding site common for both Na+ and H+ was also proposed following analysis of the structure of the CPA2 exchanger TtNapA (Lee et al., 2013). Indeed, a single cation binding site is supported by molecular dynamics simulations in both TtNapA (Lee et al., 2013) and EcNhaA (Lee et al., 2014). Further electrophysiological investigation of a number of enterobacterial CPA2 NhaA Na+/H+ exchangers yielded similar results as for EcNhaA: bell-shaped pH profiles at symmetrical pH and acidic down-regulation by substrate competition as predicted by the simple kinetic model (Calinescu et al., 2014a, Lentes et al., 2014). It should also be mentioned here that we could prove (Calinescu et al., 2014a) that the previously reported “pH-independent” behavior of the NhaA exchanger from Helicobacter pylori was just a result of the investigation method and the experimental conditions used. A similar claim could also be disproved for the G338S mutant of EcNhaA (Mager et al., 2011). Both exchangers are characterized by an acidic pK (Table 1 and Mager et al., 2011) which together with the shortcomings of the applied assay (Calinescu et al., 2014a) prevented detection of acidic down-regulation in the observed pH range. It should be emphasized that the model is not only valid for CPA2 exchangers. The kinetics of the CPA1 Na+/H+ exchanger MjNhaP1 could also be described by the simple
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Universal transport mechanism for Na+/H+ exchange model and again competition of Na+ and H+ for a single binding site was detected (Calinescu et al., 2014b). The investigated Na+/H+ exchangers are summarized in Table 1 where their function and the risk they present for intracellular pH homeostasis are also given. All prokaryotic Na+/H+ exchangers given in Table 1 were analyzed with the simple kinetic model in Figure 1A. This model is completely described by only 3 parameters: pK, the rate constant ratio
and
. The parameters are also given in the table. Note that the binding
constants and the pK values determined by kinetic analysis using the kinetic model are real binding parameters, not apparent ones as obtained from the substrate concentration dependencies. The pK of the CPA1 exchanger MjNhaP1 is slightly acidic while the pK values of the CPA2 exchangers are in the alkaline range. These values reflect the physiological role (proton export or sodium export) of each respective family. The lowest pK of the CPA2 transporters is found for the Helicobacter pylori transporter, which due to its habitat has a slightly decreased intracellular pH (Krulwich et al., 2011).
Function of Na+/H+ exchangers at physiological conditions When comparing the function of the CPA1 and CPA2 Na+/H+ exchangers given in Table 1 we find that they, although described by a unified mechanism, differ in three fundamental aspects: (1) transport mode: Na+ in, H+ out (CPA1) and H+ in, Na+ out (CPA2). (2) Charge displacement during Na+ and H+ translocation, respectively: positive/positive (CPA1) and negative/zero (CPA2), respectively (Calinescu et al., 2014b). (3) pK: CPA1 more acidic than CPA2. No cooperativity was observed in the experimental pH profiles of the CPA2 exchangers in spite of the involvement of two protons. Therefore the effect of stoichiometry is mainly that of a different overall electrogenicity in the two exchanger classes. The differences summarized above lead to specific physiological properties as will be outlined in the following. For experimental reasons the electrophysiological analysis of CPA1 and CPA2 Na+/H+ exchangers was mostly performed at symmetrical pH. This is not necessarily the physiological situation but it allows the determination of all kinetic parameters (Table 1) that govern their function. Using these together with the kinetic model one can calculate exchanger activity at arbitrary cytoplasmic and extracellular pH and Na+ concentration as well as in the presence of a membrane potential (Calinescu et al., 2014b). For a comparison we
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Universal transport mechanism for Na+/H+ exchange have calculated the pH dependent activity of a representative CPA1 and a representative CPA2 Na+/H+ exchanger under typical physiological conditions (Figure 2A). Of course environmental conditions for bacteria (or archaea) vary drastically. These pH profiles are therefore only an example. However they demonstrate the principal activity of CPA1 and CPA2 transporters. The transport activity of the CPA1 transporter MjNhaP1 in the figure consists in Na+ gradient-driven H+ export while that of EcNhaA is H+-driven and membrane potential-assisted Na+ export. The pH dependent activity of the CPA1 transporter is high at low pH but vanishes at high pH. Down-regulation at alkaline pH is effected by the low concentration of substrate protons, a substrate depletion effect. CPA2 transporters on the other hand show an acidic down-regulation due to the competition of H+ and Na+ for the same intracellular binding site, a
substrate
competition
effect
well
documented
by
experimental
evidence
(Mager et al., 2011). It is interesting that the transporters show little activity at physiological intracellular pH 7.5. This makes sense because they are regulation systems meant to be active when the conditions deviate from optimal conditions and shut down when these are attained. More specifically, Na+/H+ exchanger activity vanishes in the desired pH region preventing excessive alkalinization by the activity of CPA1 transporters or excessive acidification due to CPA2 transporter activity. Clearly, using a properly tuned pK the transporters have an intrinsic mechanism of self-regulation. Therefore, there seems to be no a priori requirement for a separate system of allosteric pH regulation via a pH sensor (Padan et al., 2009) or proton modifier (Aronson et al., 1982).
The role of the membrane potential A crucial role in the tuning of Na+/H+ exchanger activity is played by the membrane potential. In EcNhaA the translocation complex (the binding site plus the translocated cation) of the Na+ translocation reaction is negatively charged (Mager et al., 2011, Calinescu et al., 2014b). Therefore, the membrane potential accelerates the Na+ translocation step and allows export of Na+ against a steep concentration gradient. Indeed, the activity of EcNhaA would be markedly different in the absence of a membrane potential (Figure 2B), with the transporter working to import Na+ instead of exporting it at normal intracellular pH 7.5 (see Figure 2B). The opposite
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Universal transport mechanism for Na+/H+ exchange effect is caused by the membrane potential in MjNhaP1. Although this CPA1 exchanger is overall electroneutral, its individual substrate translocation steps are electrogenic the translocation complexes being positively charged (see Calinescu et al., 2014b for an in-depth discussion). Here, membrane potential acts to slow down transport in both the physiological “forward” and the “reverse” directions (Figure 2B). A physiological reason for such a behavior is not immediately apparent. Possibly it is a constraint of its occlusion or translocation mechanism. A definitive answer to this question will require further investigation.
Outlook The development of a unified model for CPA Na+/H+ exchangers is a stepping stone towards a better understanding of these vital transport systems. The full picture will, however, require answering a number of questions. Chief among these is identifying the exact molecular mechanism of transport and the residues involved in substrate translocation. This might also allow identification of the molecular nature of the energetic barrier that prevents transition between the inward- and outward-open conformations of the unloaded carrier Co ↔ Ci. Another interesting aspect would be establishing the structural basis of stoichiometry and electrogenicity in CPA exchangers – what are the characteristics of the binding site that make a CPA1 transporter catalyze electroneutral 1Na+/1H+ exchange and a CPA2 transporter electrogenic 1Na+/2H+ (or different) exchange? Also, what governs selectivity of an exchanger for certain monovalent cations in favor of others? Finally, a tempting goal would be the establishment of a practical eukaryotic model system to either validate or disprove the findings gathered so far in the study of prokaryotic Na+/H+ exchangers.
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Universal transport mechanism for Na+/H+ exchange
References Aronson, P.S., Nee, J. and Suhm, M.A. (1982). Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299, 161-163. Bobulescu, I.A., Di Sole, F. and Moe, O.W. (2005). Na+/H+ exchangers: physiology and link to hypertension and organ ischemia. Curr. Opin. Nephrol. Hypertens. 14, 485-494. Brett, C.L., Donowitz, M. and Rao, R. (2005). Evolutionary origins of eukaryotic sodium/proton exchangers. Am. J. Physiol. Cell Physiol. 288, C223-C239. Calinescu, O., Danner, E., Bohm, M., Hunte, C. and Fendler, K. (2014a). Species differences in bacterial NhaA Na+/H+ exchangers. FEBS Lett. 588, 3111-3116. Calinescu, O., Paulino, C., Kuhlbrandt, W. and Fendler, K. (2014b). Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers. J. Biol. Chem. 289, 13168-13176. Donowitz, M., Ming Tse, C. and Fuster, D. (2013). SLC9/NHE gene family, a plasma membrane and organellar family of Na+/H+ exchangers. Mol. Aspects Med. 34, 236251. Goswami, P., Paulino, C., Hizlan, D., Vonck, J., Yildiz, O. and Kuhlbrandt, W. (2011). Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1. EMBO J. 30, 439-449. Hunte, C., Screpanti, E., Venturi, M., Rimon, A., Padan, E. and Michel, H. (2005). Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature 435, 1197-1202. Jardetzky, O. (1966). Simple allosteric model for membrane pumps. Nature 211, 969-970. Kinsella, J.L. and Aronson, P.S. (1982). Determination of the coupling ratio for Na+ -H+ exchange in renal microvillus membrane vesicles. Biochim. Biophys. Acta 689, 161164. Klingenberg, M. (1985a). Catalytic energy and carrier-catalyzed solute transport in biomembranes. In: Achievements and perspectives of mitochondrial research, Volume I: Bioenergetics, Quagliariello, E., Slater, E.C., Palmieri, F., Saccone, C. And Kroon, A.M. (ed.). (Amsterdam, New York, Oxford: Elsevier Science Publisher). Klingenberg, M. (1985b). Principles of carrier catalysis elucidated by comparing two similar membrane translocators from mitochondria, the ADP/ATP carrier and the uncoupling protein. Ann. N.Y. Acad. Sci. 456 279-288. Klingenberg, M. (1992). Mechanistic and energetic aspects of carrier catalysis - exemplified with mitochondrial translocators. In: A study of enzymes, Kuby, S.A. (ed.). (Boca Raton, Ann Arbor, Boston: CRC Press). Krulwich, T.A., Sachs, G. and Padan, E. (2011). Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Microbiol. 9, 330-343. Landau, M., Herz, K., Padan, E. and Ben-Tal, N. (2007). Model structure of the Na+/H+ exchanger 1 (NHE1): functional and clinical implications. J. Biol. Chem. 282, 3785437863.
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Universal transport mechanism for Na+/H+ exchange Leblanc, G., Bassilana, M. and Damiano-Forano, E. (1988). Na+/H+ exchange in bacteria and organelles. In: Na+/H+ exchange, Grinstein, S. and Piwnica-Worms, D. (ed.), (Boca Raton, Florida: CRC Press). Lee, C., Kang, H.J., Von Ballmoos, C., Newstead, S., Uzdavinys, P., Dotson, D.L., Iwata, S., Beckstein, O., Cameron, A.D. and Drew, D. (2013). A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573-577. Lee, C., Yashiro, S., Dotson, D.L., Uzdavinys, P., Iwata, S., Sansom, M.S., Von Ballmoos, C., Beckstein, O., Drew, D. and Cameron, A.D. (2014). Crystal structure of the sodiumproton antiporter NhaA dimer and new mechanistic insights. J. Gen. Physiol. 144, 529544. Lentes, C.J., Mir, S.H., Boehm, M., Ganea, C., Fendler, K. and Hunte, C. (2014). Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella typhimurium. PLoS One 9, e101575. Mager, T., Rimon, A., Padan, E. and Fendler, K. (2011). Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study. J. Biol. Chem. 286, 23570-23581. Ohgaki, R., Van, I.S.C., Matsushita, M., Hoekstra, D. and Kanazawa, H. (2011). Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions. Biochemistry 50, 443-450. Padan, E., Bibi, E., Ito, M. and Krulwich, T.A. (2005). Alkaline pH homeostasis in bacteria: new insights. Biochim. Biophys. Acta 1717, 67-88. Padan, E., Kozachkov, L., Herz, K. and Rimon, A. (2009). NhaA crystal structure: functionalstructural insights. J. Exp. Biol. 212, 1593-1603. Paulino, C. and Kuhlbrandt, W. (2014). pH- and sodium-induced changes in a sodium/proton antiporter. eLife 3, e01412. Paulino, C., Wohlert, D., Kapotova, E., Yildiz, O. and Kuhlbrandt, W. (2014). Structure and transport mechanism of the sodium/protonantiporter MjNhaP1. eLife 3, DOI: 10.7554/eLife.03583. Stein, W.D. and Honig, B. (1977). Models for active-transport of cations - steady-state analysis. Mol. Cell. Biochem. 15, 27-44. Thauer, R.K., Kaster, A.K., Seedorf, H., Buckel, W. and Hedderich, R. (2008). Methanogenic archaea: ecologically relevant differences in energy conservation. Nat. Rev. Microbiol. 6, 579-591. Wohlert, D., Yildiz, O. and Kuhlbrandt, W. (2014). Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife 3, DOI: 10.7554/eLife.03579.
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Universal transport mechanism for Na+/H+ exchange
Tables and figures Table 1
Kinetic parameters determined for different Na+/H+ exchangers using the
kinetic model of Figure 1. Organism
Exchanger
Function
Risk
pK
k2/k1
M. jannaschii*
MjNhaP1
pH regulation
alkalinization 6.8
14
0.2
H. pylori**
HpNhaA
salt exclusion
acidification
8.0
3
20
E. coli***
EcNhaA
salt exclusion
acidification
8.8
3
7
S. typhimurium**
StNhaA
salt exclusion
acidification
9.2
3
7
Kinetic parameters taken from *(Calinescu et al., 2014b), **(Calinescu et al., 2014a), ***(Mager et al., 2011). Abbreviations: EcNhaA, NhaA Na+/H+ exchanger from Escherichia coli; HpNhaA, NhaA Na+/H+ exchanger from Helicobacter pylori; MjNhaP1, NhaP1 Na+/H+ exchanger from Methanocaldococcus jannaschii; StNhaA, NhaA Na+/H+ exchanger from Salmonella typhimurium.
Figure 1
A kinetic model for Na+/H+ exchangers.
(A) Kinetic model of Na+/H+ exchange. The substrates Na+ or H+ bind to the outward open (Co) or inward open (Ci) exchanger. Substrate binding is assumed to be in rapid equilibrium described by the parameters
and
. The rate constants
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and
describe the
Universal transport mechanism for Na+/H+ exchange reorientation of the Na+- or H+-loaded carrier respectively. (B) Activity profile of a Na+/H+ exchanger calculated according to the steady-state solution of the kinetic model. The black line depicts activity under symmetrical pH conditions (see also black inset) and high Na+ outside. The red dashed line shows activity under conditions of high outside pH and Na+ activity while the blue dashed line shows activity when outside pH is low and inside Na+ is high (see respective insets).
Figure 2
pH regulation of Na+/H+ exchangers at physiological conditions.
(A) Transport activity as a function of cytoplasmic pH calculated according to the kinetic model shown in Fig. 1 and experimentally determined kinetic parameters in Table 1. Red line: MjNhaP1 Na+/H+ exchanger, pHout = 6, [Na+]out = 500 mM, [Na+]in = 5 mM, Δψ = -100 mV, displaced charge +1 elementary charge during both Na+ and H+ translocation. Blue line: EcNhaA Na+/H+ exchanger, pHout = 7, [Na+]out = 150 mM, [Na+]in = 5 mM, Δψ = -150 mV, displaced charge -1 and 0 elementary charges during Na+ and H+ translocation respectively. Forward and reverse denote the normal physiological transport direction for each exchanger (H+ export for MjNhaP1 and Na+ export for EcNhaA as also shown in insets). (B) Influence of membrane potential on transport activity. Transport activity was calculated as in (A) for either the membrane potential given in (A) (solid lines) or zero membrane potential (dashed lines). Curves in panels (A) and (B) were arbitrarily normalized to the highest activity of each respective exchanger.
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Biological Chemistry ’Just Accepted’ paper ISSN (online) 1437-4315 DOI: 10.1515/hsz-2014-0278 Minireview
A universal mechanism for transport and regulation of CPA sodium proton exchangers Octavian Călinescu1,2 and Klaus Fendler1,*
1
Department of Biophysical Chemistry, Max Planck Institute of Biophysics, Max-von-LaueStr. 3, D-60438 Frankfurt/Main, Germany
2
Department of Biophysics, Faculty of Medicine, ‘Carol Davila’ University of Medicine and Pharmacy, RO-050474 Bucharest, Romania
*Corresponding author e-mail: [email protected]
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Universal transport mechanism for Na+/H+ exchange
Abstract Recent studies performed on a series of Na+/H+ exchangers have led us to postulate a general mechanism for Na+/H+ exchange in the monovalent cation/proton antiporter superfamily. This simple mechanism employs a single binding site for which both substrates compete. The developed kinetic model is self-regulatory, ensuring down-regulation of transport activity at extreme pH, and elegantly explains the pH-dependent activity of Na+/H+ exchangers. The mechanism was experimentally verified and shown to describe both electrogenic and electroneutral exchangers. Using a small number of parameters, exchanger activity can be modeled under different conditions, providing insights into the physiological role of Na+/H+ exchangers. Keywords: cation/proton antiporter superfamily; Na+/H+ exchangers; NhaA; NhaP1; pH regulation; transport mechanism.
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Universal transport mechanism for Na+/H+ exchange
Introduction All living organisms require a strict control of their intracellular sodium and proton concentrations, as extreme values of either can lead to cell stress and ultimately death. Among the molecular mechanisms responsible for regulating the intracellular sodium concentration and pH, a key role is played by integral membrane proteins called Na+/H+ antiporters or exchangers (Brett et al., 2005, Padan et al., 2009). Most Na+/H+ exchangers belong to the CPA1 and CPA2 subfamilies of the monovalent cation/proton antiporter superfamily, CPA (Brett et al., 2005). The CPA1 family includes exchangers that transport n protons against n Na+ ions across the membrane and are electroneutral, while the CPA2 family includes electrogenic exchangers (Brett et al., 2005). Most of the medically relevant Na+/H+ exchangers belong to the CPA1 family (NHE1-9), while only two exchangers in humans are CPA2 (NHA1-2) (Donowitz et al., 2013). Due to the inherent difficulties in purifying and reconstituting eukaryotic membrane proteins, the study of prokaryotic model systems is of great importance for the understanding of the molecular mechanisms that govern Na+/H+ exchange. By far the most studied Na+/H+ exchanger and the prototype of the whole CPA superfamily is the electrogenic E. coli NhaA (EcNhaA) CPA2 antiporter. EcNhaA owes this interest at least in part to the fact that its crystal structure was the first determined for any Na+/H+ exchanger (Hunte et al., 2005). Recently, the crystal structure of another CPA2 exchanger, NapA from Thermus thermophilus (TtNapA) was determined (Lee et al., 2013), providing another potential model for the CPA2 family. For the CPA1 class, a promising model system is the NhaP1 exchanger from Methanocaldococcus jannaschii (MjNhaP1), as it was, until recently, the only member about which structural data were available (Goswami et al., 2011, Paulino and Kuhlbrandt, 2014, Paulino et al., 2014). Recently, the crystal structure of another CPA1 exchanger, NhaP from Pyrococcus abyssi (PaNhaP), was reported (Wohlert et al., 2014), making PaNhaP another potential model for the CPA1 family. Na+/H+ exchangers are highly reversible (Mager et al., 2011) and can change their transport direction (see e.g. Kinsella and Aronson, 1982). However, most Na+/H+ exchangers function according to a preferred transport mode at physiological conditions: In general CPA1 Na+/H+ exchangers transport H+ out and Na+ into the cell while CPA2 exchangers mediate the reverse process (Leblanc et al., 1988). For example mammalian plasma membrane CPA1 exchangers (Bobulescu et al., 2005) as well as their prokaryotic CPA1 counterparts like MjNhaP1 (Thauer et al., 2008) are believed to function according to the Na+ in, H+ out mode. In 3 / 13
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Universal transport mechanism for Na+/H+ exchange contrast, CPA2 exchangers like EcNhaA (Padan et al., 2005) exchange external H+ for internal Na+. An exception to this rule may be certain CPA1 Na+/H+ exchangers, organellar NHEs, which are proposed to mediate H+ efflux out of intracellular compartments corresponding to transport of Na+ (or K+) out and H+ into the cytoplasm (Ohgaki et al., 2011). In spite of their varied stoichiometry, physiological function or selectivity for other cations, CPA Na+/H+ exchangers seem to share a few essential properties. They have a series of conserved residues that are essential for their transport function (Landau et al., 2007, Goswami et al., 2011) and they all show a pH-dependent activity (Padan et al., 2009, Goswami et al., 2011, Mager et al., 2011). Therefore, it makes perfect sense to look for a common transport mechanism that accounts for all Na+/H+ exchangers, be they electroneutral, electrogenic, eukaryotic or prokaryotic.
A minimal kinetic model for Na+/H+ exchangers The basic structural concept for our understanding of transporter function is the alternate access principle formulated nearly 50 years ago (Jardetzky, 1966). Transport is mediated by a flexible membrane-spanning macromolecule with substrate binding sites which are alternatively exposed to the cytoplasmic and the extracellular sides of the membrane. However, a complete picture only emerged ~20 years later when also the functional and thermodynamic principles of transporter mechanism were derived. A simple mechanism for antiport (Stein and Honig, 1977, Klingenberg, 1992) involves an exchanger that has a common binding site for both substrates and can switch between an inside and outside-open conformation only when this site is occupied. Thermodynamically, the energy barrier between these two conformations is high in the absence of substrate and is lowered upon substrate binding. The energy required for this conformational transition is provided by the binding energy of the substrate (Klingenberg, 1985a, Klingenberg, 1985b). Its structural and molecular background, however, remains yet to be uncovered. Based on the above described minimal exchanger mechanism we developed a kinetic model (Mager et al., 2011) using the following constraints: 1) the binding site is identical when exposed to the cytoplasmic and extracellular sides and 2) the inward and outward open forms of the carrier have the same energy. This is justified by the experimental evidence showing that the EcNhaA Na+/H+ exchanger is functionally completely symmetrical. The kinetic 4 / 13
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Universal transport mechanism for Na+/H+ exchange representation of this mechanism is the 6-state kinetic model shown in Figure 1A. Symmetry implies that the binding constants (pK and
) of the inside and outside open carrier as well
as the forward and backward rate constants of the conformational transitions are the same. Note that, although completely symmetrical, the kinetic model mediates vectorial transport of one substrate in the presence of an oppositely directed concentration gradient of the other substrate.
pH regulation as an intrinsic property of the Na+/H+ exchange mechanism The kinetic model in Figure 1A has interesting properties. It is instructive to consider the case where the pH on both sides of the membrane is identical (Figure 1B solid black line). Under these conditions the pH profile of the transporter is bell-shaped. Acidic down-regulation is due to competition of Na+ and H+ for the common extracellular binding site. In contrast, at alkaline pH down-regulation is due to depletion of the substrate H+ at the intracellular binding site. These two mechanisms of regulation are operative also at asymmetrical pH and account for the respective pH profiles. If the pH outside is alkaline and Na+ is present outside then Na+ enters while protons leave the cell and down-regulation via substrate depletion takes place when the inside pH becomes alkaline (red dashed line Figure 1B). If the pH outside is acidic and Na+ is present inside then Na+ leaves while protons enter the cell and down-regulation via substrate competition is operational as the inside pH goes down (blue dashed line Figure 1B). It is clear that depending on the conditions the pH profile of one and the same Na+/H+ exchanger can vary from alkaline down-regulation to acidic down-regulation to a bell-shaped profile and that a certain profile is not a specific property of an exchanger but critically depends on the assay conditions. The pH profiles shown in Figure 1B have important physiological implications. CPA1 Na+/H+ exchangers work in the Na+ in, H+ out mode, which corresponds to the red dashed line in Figure 1B and are therefore inherently down-regulated at alkaline pH preventing excessive akalinization of the cytoplasm. CPA2 Na+/H+ exchangers work in the H+ in, Na+ out mode, which corresponds to the blue dashed line in Figure 1B and are therefore inherently down-regulated at acidic pH preventing excessive acidification of the cytoplasm. Therefore,
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Universal transport mechanism for Na+/H+ exchange an attractive feature of the kinetic model is that Na+/H+ exchangers are down-regulated at alkaline or acidic pH by the nature of the transport mechanism itself.
Experimental validation of the kinetic model So far our discussion was based on theoretical considerations pertaining to a plausible kinetic model that has the value of being mechanistically as simple as possible. But is this mechanism correct and does the kinetic model correctly describe the properties of a real Na+/H+ exchanger? Specialized protein regions, referred to as ‘pH sensors’ or “proton modifiers” have been suggested to account for the strong pH dependence of Na+/H+ exchangers (Aronson et al., 1982, Padan et al., 2009). This concept has recently been challenged by the finding that the CPA2 exchanger EcNhaA is active at cytoplasmic pH 5 (Mager et al., 2011). This observation, being in conflict with the hypothesis of a cytoplasmic proton modifier site, suggested that alternative explanations for the pH dependence of EcNhaA had to be considered. Indeed it was shown that the substrate dependence of EcNhaA can be explained by the simple kinetic model of Figure 1A and it was proposed that its pH dependence is a unique property of its transport mechanism (Mager et al., 2011). In line with the kinetic model, a single binding site common for both Na+ and H+ was also proposed following analysis of the structure of the CPA2 exchanger TtNapA (Lee et al., 2013). Indeed, a single cation binding site is supported by molecular dynamics simulations in both TtNapA (Lee et al., 2013) and EcNhaA (Lee et al., 2014). Further electrophysiological investigation of a number of enterobacterial CPA2 NhaA Na+/H+ exchangers yielded similar results as for EcNhaA: bell-shaped pH profiles at symmetrical pH and acidic down-regulation by substrate competition as predicted by the simple kinetic model (Calinescu et al., 2014a, Lentes et al., 2014). It should also be mentioned here that we could prove (Calinescu et al., 2014a) that the previously reported “pH-independent” behavior of the NhaA exchanger from Helicobacter pylori was just a result of the investigation method and the experimental conditions used. A similar claim could also be disproved for the G338S mutant of EcNhaA (Mager et al., 2011). Both exchangers are characterized by an acidic pK (Table 1 and Mager et al., 2011) which together with the shortcomings of the applied assay (Calinescu et al., 2014a) prevented detection of acidic down-regulation in the observed pH range. It should be emphasized that the model is not only valid for CPA2 exchangers. The kinetics of the CPA1 Na+/H+ exchanger MjNhaP1 could also be described by the simple
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Universal transport mechanism for Na+/H+ exchange model and again competition of Na+ and H+ for a single binding site was detected (Calinescu et al., 2014b). The investigated Na+/H+ exchangers are summarized in Table 1 where their function and the risk they present for intracellular pH homeostasis are also given. All prokaryotic Na+/H+ exchangers given in Table 1 were analyzed with the simple kinetic model in Figure 1A. This model is completely described by only 3 parameters: pK, the rate constant ratio
and
. The parameters are also given in the table. Note that the binding
constants and the pK values determined by kinetic analysis using the kinetic model are real binding parameters, not apparent ones as obtained from the substrate concentration dependencies. The pK of the CPA1 exchanger MjNhaP1 is slightly acidic while the pK values of the CPA2 exchangers are in the alkaline range. These values reflect the physiological role (proton export or sodium export) of each respective family. The lowest pK of the CPA2 transporters is found for the Helicobacter pylori transporter, which due to its habitat has a slightly decreased intracellular pH (Krulwich et al., 2011).
Function of Na+/H+ exchangers at physiological conditions When comparing the function of the CPA1 and CPA2 Na+/H+ exchangers given in Table 1 we find that they, although described by a unified mechanism, differ in three fundamental aspects: (1) transport mode: Na+ in, H+ out (CPA1) and H+ in, Na+ out (CPA2). (2) Charge displacement during Na+ and H+ translocation, respectively: positive/positive (CPA1) and negative/zero (CPA2), respectively (Calinescu et al., 2014b). (3) pK: CPA1 more acidic than CPA2. No cooperativity was observed in the experimental pH profiles of the CPA2 exchangers in spite of the involvement of two protons. Therefore the effect of stoichiometry is mainly that of a different overall electrogenicity in the two exchanger classes. The differences summarized above lead to specific physiological properties as will be outlined in the following. For experimental reasons the electrophysiological analysis of CPA1 and CPA2 Na+/H+ exchangers was mostly performed at symmetrical pH. This is not necessarily the physiological situation but it allows the determination of all kinetic parameters (Table 1) that govern their function. Using these together with the kinetic model one can calculate exchanger activity at arbitrary cytoplasmic and extracellular pH and Na+ concentration as well as in the presence of a membrane potential (Calinescu et al., 2014b). For a comparison we
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Universal transport mechanism for Na+/H+ exchange have calculated the pH dependent activity of a representative CPA1 and a representative CPA2 Na+/H+ exchanger under typical physiological conditions (Figure 2A). Of course environmental conditions for bacteria (or archaea) vary drastically. These pH profiles are therefore only an example. However they demonstrate the principal activity of CPA1 and CPA2 transporters. The transport activity of the CPA1 transporter MjNhaP1 in the figure consists in Na+ gradient-driven H+ export while that of EcNhaA is H+-driven and membrane potential-assisted Na+ export. The pH dependent activity of the CPA1 transporter is high at low pH but vanishes at high pH. Down-regulation at alkaline pH is effected by the low concentration of substrate protons, a substrate depletion effect. CPA2 transporters on the other hand show an acidic down-regulation due to the competition of H+ and Na+ for the same intracellular binding site, a
substrate
competition
effect
well
documented
by
experimental
evidence
(Mager et al., 2011). It is interesting that the transporters show little activity at physiological intracellular pH 7.5. This makes sense because they are regulation systems meant to be active when the conditions deviate from optimal conditions and shut down when these are attained. More specifically, Na+/H+ exchanger activity vanishes in the desired pH region preventing excessive alkalinization by the activity of CPA1 transporters or excessive acidification due to CPA2 transporter activity. Clearly, using a properly tuned pK the transporters have an intrinsic mechanism of self-regulation. Therefore, there seems to be no a priori requirement for a separate system of allosteric pH regulation via a pH sensor (Padan et al., 2009) or proton modifier (Aronson et al., 1982).
The role of the membrane potential A crucial role in the tuning of Na+/H+ exchanger activity is played by the membrane potential. In EcNhaA the translocation complex (the binding site plus the translocated cation) of the Na+ translocation reaction is negatively charged (Mager et al., 2011, Calinescu et al., 2014b). Therefore, the membrane potential accelerates the Na+ translocation step and allows export of Na+ against a steep concentration gradient. Indeed, the activity of EcNhaA would be markedly different in the absence of a membrane potential (Figure 2B), with the transporter working to import Na+ instead of exporting it at normal intracellular pH 7.5 (see Figure 2B). The opposite
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Universal transport mechanism for Na+/H+ exchange effect is caused by the membrane potential in MjNhaP1. Although this CPA1 exchanger is overall electroneutral, its individual substrate translocation steps are electrogenic the translocation complexes being positively charged (see Calinescu et al., 2014b for an in-depth discussion). Here, membrane potential acts to slow down transport in both the physiological “forward” and the “reverse” directions (Figure 2B). A physiological reason for such a behavior is not immediately apparent. Possibly it is a constraint of its occlusion or translocation mechanism. A definitive answer to this question will require further investigation.
Outlook The development of a unified model for CPA Na+/H+ exchangers is a stepping stone towards a better understanding of these vital transport systems. The full picture will, however, require answering a number of questions. Chief among these is identifying the exact molecular mechanism of transport and the residues involved in substrate translocation. This might also allow identification of the molecular nature of the energetic barrier that prevents transition between the inward- and outward-open conformations of the unloaded carrier Co ↔ Ci. Another interesting aspect would be establishing the structural basis of stoichiometry and electrogenicity in CPA exchangers – what are the characteristics of the binding site that make a CPA1 transporter catalyze electroneutral 1Na+/1H+ exchange and a CPA2 transporter electrogenic 1Na+/2H+ (or different) exchange? Also, what governs selectivity of an exchanger for certain monovalent cations in favor of others? Finally, a tempting goal would be the establishment of a practical eukaryotic model system to either validate or disprove the findings gathered so far in the study of prokaryotic Na+/H+ exchangers.
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Universal transport mechanism for Na+/H+ exchange
References Aronson, P.S., Nee, J. and Suhm, M.A. (1982). Modifier role of internal H+ in activating the Na+-H+ exchanger in renal microvillus membrane vesicles. Nature 299, 161-163. Bobulescu, I.A., Di Sole, F. and Moe, O.W. (2005). Na+/H+ exchangers: physiology and link to hypertension and organ ischemia. Curr. Opin. Nephrol. Hypertens. 14, 485-494. Brett, C.L., Donowitz, M. and Rao, R. (2005). Evolutionary origins of eukaryotic sodium/proton exchangers. Am. J. Physiol. Cell Physiol. 288, C223-C239. Calinescu, O., Danner, E., Bohm, M., Hunte, C. and Fendler, K. (2014a). Species differences in bacterial NhaA Na+/H+ exchangers. FEBS Lett. 588, 3111-3116. Calinescu, O., Paulino, C., Kuhlbrandt, W. and Fendler, K. (2014b). Keeping it simple, transport mechanism and pH regulation in Na+/H+ exchangers. J. Biol. Chem. 289, 13168-13176. Donowitz, M., Ming Tse, C. and Fuster, D. (2013). SLC9/NHE gene family, a plasma membrane and organellar family of Na+/H+ exchangers. Mol. Aspects Med. 34, 236251. Goswami, P., Paulino, C., Hizlan, D., Vonck, J., Yildiz, O. and Kuhlbrandt, W. (2011). Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1. EMBO J. 30, 439-449. Hunte, C., Screpanti, E., Venturi, M., Rimon, A., Padan, E. and Michel, H. (2005). Structure of a Na+/H+ antiporter and insights into mechanism of action and regulation by pH. Nature 435, 1197-1202. Jardetzky, O. (1966). Simple allosteric model for membrane pumps. Nature 211, 969-970. Kinsella, J.L. and Aronson, P.S. (1982). Determination of the coupling ratio for Na+ -H+ exchange in renal microvillus membrane vesicles. Biochim. Biophys. Acta 689, 161164. Klingenberg, M. (1985a). Catalytic energy and carrier-catalyzed solute transport in biomembranes. In: Achievements and perspectives of mitochondrial research, Volume I: Bioenergetics, Quagliariello, E., Slater, E.C., Palmieri, F., Saccone, C. And Kroon, A.M. (ed.). (Amsterdam, New York, Oxford: Elsevier Science Publisher). Klingenberg, M. (1985b). Principles of carrier catalysis elucidated by comparing two similar membrane translocators from mitochondria, the ADP/ATP carrier and the uncoupling protein. Ann. N.Y. Acad. Sci. 456 279-288. Klingenberg, M. (1992). Mechanistic and energetic aspects of carrier catalysis - exemplified with mitochondrial translocators. In: A study of enzymes, Kuby, S.A. (ed.). (Boca Raton, Ann Arbor, Boston: CRC Press). Krulwich, T.A., Sachs, G. and Padan, E. (2011). Molecular aspects of bacterial pH sensing and homeostasis. Nat. Rev. Microbiol. 9, 330-343. Landau, M., Herz, K., Padan, E. and Ben-Tal, N. (2007). Model structure of the Na+/H+ exchanger 1 (NHE1): functional and clinical implications. J. Biol. Chem. 282, 3785437863.
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Universal transport mechanism for Na+/H+ exchange Leblanc, G., Bassilana, M. and Damiano-Forano, E. (1988). Na+/H+ exchange in bacteria and organelles. In: Na+/H+ exchange, Grinstein, S. and Piwnica-Worms, D. (ed.), (Boca Raton, Florida: CRC Press). Lee, C., Kang, H.J., Von Ballmoos, C., Newstead, S., Uzdavinys, P., Dotson, D.L., Iwata, S., Beckstein, O., Cameron, A.D. and Drew, D. (2013). A two-domain elevator mechanism for sodium/proton antiport. Nature 501, 573-577. Lee, C., Yashiro, S., Dotson, D.L., Uzdavinys, P., Iwata, S., Sansom, M.S., Von Ballmoos, C., Beckstein, O., Drew, D. and Cameron, A.D. (2014). Crystal structure of the sodiumproton antiporter NhaA dimer and new mechanistic insights. J. Gen. Physiol. 144, 529544. Lentes, C.J., Mir, S.H., Boehm, M., Ganea, C., Fendler, K. and Hunte, C. (2014). Molecular characterization of the Na+/H+-antiporter NhaA from Salmonella typhimurium. PLoS One 9, e101575. Mager, T., Rimon, A., Padan, E. and Fendler, K. (2011). Transport mechanism and pH regulation of the Na+/H+ antiporter NhaA from Escherichia coli: an electrophysiological study. J. Biol. Chem. 286, 23570-23581. Ohgaki, R., Van, I.S.C., Matsushita, M., Hoekstra, D. and Kanazawa, H. (2011). Organellar Na+/H+ exchangers: novel players in organelle pH regulation and their emerging functions. Biochemistry 50, 443-450. Padan, E., Bibi, E., Ito, M. and Krulwich, T.A. (2005). Alkaline pH homeostasis in bacteria: new insights. Biochim. Biophys. Acta 1717, 67-88. Padan, E., Kozachkov, L., Herz, K. and Rimon, A. (2009). NhaA crystal structure: functionalstructural insights. J. Exp. Biol. 212, 1593-1603. Paulino, C. and Kuhlbrandt, W. (2014). pH- and sodium-induced changes in a sodium/proton antiporter. eLife 3, e01412. Paulino, C., Wohlert, D., Kapotova, E., Yildiz, O. and Kuhlbrandt, W. (2014). Structure and transport mechanism of the sodium/protonantiporter MjNhaP1. eLife 3, DOI: 10.7554/eLife.03583. Stein, W.D. and Honig, B. (1977). Models for active-transport of cations - steady-state analysis. Mol. Cell. Biochem. 15, 27-44. Thauer, R.K., Kaster, A.K., Seedorf, H., Buckel, W. and Hedderich, R. (2008). Methanogenic archaea: ecologically relevant differences in energy conservation. Nat. Rev. Microbiol. 6, 579-591. Wohlert, D., Yildiz, O. and Kuhlbrandt, W. (2014). Structure and substrate ion binding in the sodium/proton antiporter PaNhaP. eLife 3, DOI: 10.7554/eLife.03579.
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Universal transport mechanism for Na+/H+ exchange
Tables and figures Table 1
Kinetic parameters determined for different Na+/H+ exchangers using the
kinetic model of Figure 1. Organism
Exchanger
Function
Risk
pK
k2/k1
M. jannaschii*
MjNhaP1
pH regulation
alkalinization 6.8
14
0.2
H. pylori**
HpNhaA
salt exclusion
acidification
8.0
3
20
E. coli***
EcNhaA
salt exclusion
acidification
8.8
3
7
S. typhimurium**
StNhaA
salt exclusion
acidification
9.2
3
7
Kinetic parameters taken from *(Calinescu et al., 2014b), **(Calinescu et al., 2014a), ***(Mager et al., 2011). Abbreviations: EcNhaA, NhaA Na+/H+ exchanger from Escherichia coli; HpNhaA, NhaA Na+/H+ exchanger from Helicobacter pylori; MjNhaP1, NhaP1 Na+/H+ exchanger from Methanocaldococcus jannaschii; StNhaA, NhaA Na+/H+ exchanger from Salmonella typhimurium.
Figure 1
A kinetic model for Na+/H+ exchangers.
(A) Kinetic model of Na+/H+ exchange. The substrates Na+ or H+ bind to the outward open (Co) or inward open (Ci) exchanger. Substrate binding is assumed to be in rapid equilibrium described by the parameters
and
. The rate constants
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and
describe the
Universal transport mechanism for Na+/H+ exchange reorientation of the Na+- or H+-loaded carrier respectively. (B) Activity profile of a Na+/H+ exchanger calculated according to the steady-state solution of the kinetic model. The black line depicts activity under symmetrical pH conditions (see also black inset) and high Na+ outside. The red dashed line shows activity under conditions of high outside pH and Na+ activity while the blue dashed line shows activity when outside pH is low and inside Na+ is high (see respective insets).
Figure 2
pH regulation of Na+/H+ exchangers at physiological conditions.
(A) Transport activity as a function of cytoplasmic pH calculated according to the kinetic model shown in Fig. 1 and experimentally determined kinetic parameters in Table 1. Red line: MjNhaP1 Na+/H+ exchanger, pHout = 6, [Na+]out = 500 mM, [Na+]in = 5 mM, Δψ = -100 mV, displaced charge +1 elementary charge during both Na+ and H+ translocation. Blue line: EcNhaA Na+/H+ exchanger, pHout = 7, [Na+]out = 150 mM, [Na+]in = 5 mM, Δψ = -150 mV, displaced charge -1 and 0 elementary charges during Na+ and H+ translocation respectively. Forward and reverse denote the normal physiological transport direction for each exchanger (H+ export for MjNhaP1 and Na+ export for EcNhaA as also shown in insets). (B) Influence of membrane potential on transport activity. Transport activity was calculated as in (A) for either the membrane potential given in (A) (solid lines) or zero membrane potential (dashed lines). Curves in panels (A) and (B) were arbitrarily normalized to the highest activity of each respective exchanger.
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