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Clathrin-mediated Endocytosis and Recycling of the Neuron-specific Na+/H+ Exchanger NHE5 Isoform

REGULATION BY PHOSPHATIDYLINOSITOL 3′-KINASE AND THE ACTIN CYTOSKELETON*
  • Katalin Szászi
    Footnotes
    Affiliations
    Programme in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada
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  • Anders Paulsen
    Footnotes
    Affiliations
    Programme in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada
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  • Elöd Z. Szabó
    Affiliations
    Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the
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  • Masayuki Numata
    Affiliations
    Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the
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  • Sergio Grinstein
    Footnotes
    Affiliations
    Programme in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada
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  • John Orlowski
    Correspondence
    Supported by an Investigator Award from the CIHR. To whom correspondence should be addressed: Dept. of Physiology, McGill University, McIntyre Medical Science Bldg., 3655 Promenade Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-8335; Fax: 514-398-7452; E-mail:
    Affiliations
    Department of Physiology, McGill University, Montreal, Quebec H3G 1Y6, Canada and the
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  • Author Footnotes
    * This work was supported by the Canadian Institutes of Health Research (CIHR).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Supported by a CIHR fellowship.
    ¶ These authors contributed equally to this work.
    ** Cross-appointed to the Department of Biochemistry of the University of Toronto. Supported by a Distinguished Investigator Award from the CIHR and the current holder of the Pitblado Chair in Cell Biology.
Open AccessPublished:August 29, 2002DOI:https://doi.org/10.1074/jbc.M206629200
      Mammalian Na+/H+exchangers (NHEs) are a family of integral membrane proteins that play central roles in sodium, acid-base, and cell volume homeostasis. The recently cloned NHE5 isoform is expressed predominantly in brain, but its functional and cellular properties are poorly understood. To facilitate its characterization, an epitope-tagged construct of NHE5 was ectopically expressed in nonneuronal and neuronal cells. In NHE-deficient Chinese hamster ovary AP-1 cells, NHE5 localized at the plasmalemma, but a significant fraction accumulated intracellularly in vesicles that concentrated in a juxtanuclear region. Similarly, in nerve growth factor-differentiated neuroendocrine PC12 cells and primary hippocampal neurons, immunolabeling of NHE5 was detected in endomembrane vesicles in the perinuclear region of the cell body but also along the processes. More detailed characterization in AP-1 cells using organelle-specific markers showed that NHE5 co-localized with internalized transferrin, a marker of recycling endosomes. Transient transfection of a dominant negative mutant of dynamin-1, which inhibits clathrin-mediated endocytosis, blocked uptake of transferrin as well as internalization of NHE5. Likewise, wortmannin inhibition of phosphatidylinositol 3′-kinase, a lipid kinase implicated in endosomal traffic, induced coalescence of vesicles containing NHE5 and caused a pronounced inhibition of plasmalemmal Na+/H+ exchange. By contrast, disruption of the F-actin cytoskeleton with cytochalasin D increased cell surface NHE5 activity and abundance. These observations demonstrate that NHE5 is localized to the recycling endosomal pathway and is dynamically regulated by phosphatidylinositol 3′-kinase and by the state of F-actin assembly.
      NHE
      Na+/H+ exchanger
      BCECF
      2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein
      GFP
      enhanced green fluorescent protein
      HA
      hemagglutinin
      PBS
      phosphate-buffered saline
      pHi
      cytosolic pH
      r-Tfn: rhodamine-labeled transferrin
      PI3-K, phosphatidylinositol 3-kinase
      CHO
      Chinese hamster ovary
      FITC
      fluorescein isothiocyanate
      NGF
      nerve growth factor
      Subtle fluctuations in intra- or extracellular pH of neurons can significantly modulate membrane excitability and are thought to fulfill a regulatory role in central nervous system function (
      • Krishtal O.A.
      • Osipchuk Y.V.
      • Shelest T.N.
      • Smirnoff S.V.
      ,
      • Ransom B.R.
      ,
      • Takahashi K.I.
      • Copenhagen D.R.
      ,
      • Deitmer J.W.
      • Rose C.R.
      ,
      • Putnam R.W.
      ). On the other hand, extreme disturbances in acid-base homeostasis are associated with the progression of certain neuropathies, such as acute ischemic stroke (
      • Bondarenko A.
      • Chesler M.
      ,
      • Li P.A.
      • Siesjo B.K.
      ,
      • Horikawa N.
      • Nishioka M.
      • Itoh N.
      • Kuribayashi Y.
      • Matsui K.
      • Ohashi N.
      ), glial swelling (
      • Kempski O.
      • Staub F.
      • Jansen M.
      • Baethmann A.
      ), and cerebral edema (
      • Kempski O.
      ). Hence, fine pH control of the neural milieu is a vital biological process. Maintenance of acid-base homeostasis in the central nervous system is complex and involves the coordinated activities of several distinct plasma membrane ion transporters, including Na+-HCO 3 cotransporters, Na+-dependent and -independent Cl/HCO 3 exchangers, and Na+/H+ exchangers (for an overview, see Ref.
      • Putnam R.W.
      ). This diversity is further enriched by the presence of multiple isoforms for some of these transporters (
      • Havenga M.J.E.
      • Bosman G.J.C.G.M.
      • Appelhans H.
      • De Grip W.J.
      ,
      • Kobayashi S.
      • Morgans C.W.
      • Casey J.R.
      • Kopito R.R.
      ,
      • Ma E.
      • Haddad G.G.
      ,
      • Douglas R.M.
      • Schmitt B.M.
      • Xia Y.
      • Bevensee M.O.
      • Biemesderfer D.
      • Boron W.F.
      • Haddad G.G.
      ). However, their specific regulatory properties and contributions to neural physiology are not well understood.
      In mammals, seven distinct Na+/H+ exchangers (NHEs)1 have been described to date; five (NHE1 to -5) are resident primarily in the plasma membrane, whereas two (NHE6 and -7) localize predominantly to intracellular organelles, and all are present in brain (
      • Ma E.
      • Haddad G.G.
      ,
      • Douglas R.M.
      • Schmitt B.M.
      • Xia Y.
      • Bevensee M.O.
      • Biemesderfer D.
      • Boron W.F.
      • Haddad G.G.
      ,
      • Orlowski J.
      • Grinstein S.
      ,
      • Miyazaki E.
      • Sakaguchi M.
      • Wakabayashi S.
      • Shigekawa M.
      • Mihara K.
      ,
      • Numata M.
      • Orlowski J.
      ). NHE1 is ubiquitously expressed and is noted for its high sensitivity to inhibitory drugs such as amiloride. It is chiefly responsible for restoration of steady-state pHi following cytosolic acidification and for maintenance of cell volume. Recent findings indicate that it is also crucial for neural function and viability. Mice with null mutations of Nhe1 exhibit locomotor abnormalities, epileptic-like seizures, and considerable mortality (67%) prior to weaning (
      • Cox G.A.
      • Lutz C.M.
      • Yang C.L.
      • Biemesderfer D.
      • Bronson R.T., Fu, A.
      • Aronson P.S.
      • Noebels J.L.
      • Frankel W.N.
      ,
      • Bell S.M.
      • Schreiner C.M.
      • Schultheis P.J.
      • Miller M.L.
      • Evans R.L.
      • Vorhees C.V.
      • Shull G.E.
      • Scott W.J.
      ). Moreover, hippocampal CA1 neurons isolated from these animals display enhanced membrane excitability and increased Na+ current density, implicating a functional association between NHE1 and voltage-sensitive Na+ channels that seemingly contributes to neural dysfunction when disrupted (
      • Gu X.Q.
      • Yao H.
      • Haddad G.G.
      ). However, the precise molecular nature of this relationship is unknown.
      By comparison, NHE2 to -4 are predominantly expressed in epithelia of the kidney and gastrointestinal tract (
      • Orlowski J.
      • Grinstein S.
      ) but are also detected at low levels in discrete regions of the brain (
      • Ma E.
      • Haddad G.G.
      ). The best studied of these is the amiloride-resistant isoform, NHE3. In epithelia, NHE3 resides along the apical membrane as well as in recycling endosomes and fulfills an important role in luminal Na+ and HCO 3 (re)absorption; the latter effected by H+ extrusion (
      • Biemesderfer D.
      • Rutherford P.A.
      • Nagy T.
      • Pizzonia J.H.
      • Abu-Alfa A.K.
      • Aronson P.S.
      ,
      • Chow C.W.
      • Khurana S.
      • Woodside M.
      • Grinstein S.
      • Orlowski J.
      ,
      • Wang T.
      • Hropot M.
      • Aronson P.S.
      • Giebisch G.
      ,
      • Schultheis P.J.
      • Clarke L.L.
      • Meneton P.
      • Miller M.L.
      • Soleimani M.
      • Gawenis L.R.
      • Riddle T.M.
      • Duffy J.J.
      • Doetschman T.
      • Wang T.
      • Giebisch G.
      • Aronson P.S.
      • Lorenz J.N.
      • Shull G.E.
      ). Its activity is also subject to acute regulation by several protein and lipid kinases, including protein kinase A (
      • Azarani A.
      • Goltzman D.
      • Orlowski J.
      ,
      • Kurashima K., Yu, F.H.
      • Cabado A.G.
      • Szabó E.Z.
      • Grinstein S.
      • Orlowski J.
      ,
      • Zizak M.
      • Lamprecht G.
      • Steplock D.
      • Tariq N.
      • Shenolikar S.
      • Donowitz M.
      • Yun C.H.C.
      • Weinman E.J.
      ,
      • Szaszi K.
      • Kurashima K.
      • Kaibuchi K.
      • Grinstein S.
      • Orlowski J.
      ,
      • Hu M.C.
      • Fan L.Z.
      • Crowder L.A.
      • Karim-Jimenez Z.
      • Murer H.
      • Moe O.W.
      ), protein kinase C (
      • Azarani A.
      • Goltzman D.
      • Orlowski J.
      ,
      • Yip J.W., Ko, W.H.
      • Viberti G.
      • Huganir R.L.
      • Donowitz M.
      • Tse C.M.
      ,
      • Janecki A.J.
      • Montrose M.H.
      • Zimniak P.
      • Zweibaum A.
      • Tse C.M.
      • Khurana S.
      • Donowitz M.
      ), Rho-associated kinase (
      • Szászi K.
      • Kurashima K.
      • Kapus A.
      • Paulsen A.
      • Kaibuchi K.
      • Grinstein S.
      • Orlowski J.
      ), and phosphatidylinositol 3′-kinase (PI3-K) (
      • Khurana S.
      • Nath S.K.
      • Levine S.A.
      • Bowser J.M.
      • Tse C.M.
      • Cohen M.E.
      • Donowitz M.
      ,
      • Kurashima K.
      • Szabó E.Z.
      • Lukacs G.
      • Orlowski J.
      • Grinstein S.
      ). These kinases act on NHE3 by direct phosphorylation or through intermediary effectors and alter one or more of its biological parameters including proton affinity, cell surface abundance, and/or association with the actin cytoskeleton. In brain, NHE3 is present primarily in cerebellar Purkinje and glial cells (
      • Ma E.
      • Haddad G.G.
      ) but is also detected in chemosensitive neurons of the ventrolateral medulla oblongata, where it has been proposed to play a role in the regulation of breathing rhythm (
      • Wiemann M.
      • Schwark J.R.
      • Bonnet U.
      • Jansen H.W.
      • Grinstein S.
      • Baker R.E.
      • Lang H.J.C.
      • Wirth K.
      • Bingmann D.
      ,
      • Wiemann M.
      • Bingmann D.
      ,
      • Kiwull-Schone H.
      • Wiemann M.
      • Frede S.
      • Bingmann D.
      • Wirth K.J.
      • Heinelt U.
      • Lang H.J.
      • Kiwull P.
      ).
      NHE5 is distinguished from the other isoforms by its almost exclusive expression in the central nervous system. This isoform is found in multiple regions of the brain including the dentate gyrus, cerebral cortex, hippocampus, amygdala, caudate nucleus, hypothalamus, subthalamic nucleus, and thalamus but not in glia-enriched structures such as the corpus callosum, suggesting that NHE5 might be neuron-specific (
      • Baird N.R.
      • Orlowski J.
      • Szabó E.Z.
      • Zaun H.C.
      • Schultheis P.J.
      • Menon A.G.
      • Shull G.E.
      ,
      • Attaphitaya S.
      • Park K.
      • Melvin J.E.
      ). Structurally, it is most closely related to NHE3 (∼50% amino acid identity) and shares similar pharmacological (
      • Attaphitaya S.
      • Park K.
      • Melvin J.E.
      ,
      • Szabó E.Z.
      • Numata M.
      • Shull G.E.
      • Orlowski J.
      ) and regulatory (
      • Attaphitaya S.
      • Nehrke K.
      • Melvin J.E.
      ) properties. These observations have led to the suggestion that NHE5 may be the amiloride-resistant Na+/H+ exchanger reported in hippocampal neurons (
      • Raley-Susman K.M.
      • Cragoe Jr., E.J.
      • Sapolsky R.M.
      • Kopito R.R.
      ). Aside from these few reports, however, little else is known about the functional properties of NHE5. Information regarding its subcellular distribution and regulation are hampered by the absence of isoform-specific antibodies and by the difficulties of isolating, maintaining, and transfecting neurons in primary culture. Additionally, the presence of multiple NHE isoforms in neurons further complicates interpretation of functional measurements.
      To gain further insight into the sorting and regulation of NHE5, we have stably expressed an epitope-tagged version of human NHE5 cDNA in a subline (AP-1) of Chinese hamster ovary (CHO) cells that lacks endogenous plasmalemmal NHE activity, thereby facilitating transport measurements. The usefulness of this cell line for studying membrane proteins normally resident in polarized cells is bolstered by studies showing that undifferentiated cells, such as CHO cells, contain both regulated and constitutive secretory as well as endocytic vesicles analogous to those found in more specialized cell types such as epithelia and neurons (
      • Cameron P.L.
      • Sudhof T.C.
      • Jahn R.
      • De Camilli P.
      ,
      • Yoshimori T.
      • Keller P.
      • Roth M.G.
      • Simons K.
      ,
      • Chavez R.A.
      • Miller S.G.
      • Moore H.P.
      ,
      • Wilson J.M.
      • Colton T.L.
      ,
      • Coorssen J.R.
      • Schmitt H.
      • Almers W.
      ,
      • Morimoto T.
      • Popov S.
      • Buckley K.M.
      • Poo M.M.
      ,
      • D'Souza S.
      • Garcia-Cabado A., Yu, F.
      • Teter K.
      • Lukacs G.
      • Skorecki K.
      • Moore H.P.
      • Orlowski J.
      • Grinstein S.
      ). In this report, we show that NHE5, like NHE3, is present both in the plasma membrane and in clathrin-associated recycling endosomes that are regulated by PI3-K when expressed in AP-1 cells. Pharmacological disruption of the actin cytoskeleton leads to increased rates of transport and cell surface-exposed NHE5, possibly by blocking internalization. Interestingly, this latter finding is in marked contrast to that observed for NHE3, which is inhibited under comparable conditions (
      • Kurashima K.
      • D'Souza S.
      • Szászi K.
      • Ramjeesingh R.
      • Orlowski J.
      • Grinstein S.
      ). Although characterized less extensively, a comparable subcellular distribution of NHE5 was also observed in transiently transfected neuroendocrine PC12 cells and hippocampal neurons in primary culture.

      Acknowledgments

      We thank Drs. Damian Wheeler and Ellis Cooper for providing the primary cultures of rat hippocampal neurons and for valuable discussions.

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