Ehud Ohana

Identification of the Zn2+ Binding Site and Mode of Operation of a Mammalian Zn2+ Transporter

Vesicular zinc transporters (ZnTs) play a critical role in regulating Zn2+ homeostasis in various cellular compartments and are linked to major diseases ranging from Alzheimer disease to diabetes. Despite their importance, the intracellular localization of ZnTs poses a major challenge for establishing the mechanisms by which they function and the identity of their ion binding sites. Here, we combine fluorescence-based functional analysis and structural modeling aimed at elucidating these functional aspects. Expression of ZnT5 was followed by both accelerated removal of Zn2+ from the cytoplasm and its increased vesicular sequestration. Further, activity of this zinc transport was coupled to alkalinization of the trans-Golgi network. Finally, structural modeling of ZnT5, based on the x-ray structure of the bacterial metal transporter YiiP, identified four residues that can potentially form the zinc binding site on ZnT5. Consistent with this model, replacement of these residues, Asp599 and His451, with alanine was sufficient to block Zn2+ transport. These findings indicate, for the first time, that Zn2+ transport mediated by a mammalian ZnT is catalyzed by H+/Zn2+ exchange and identify the zinc binding site of ZnT proteins essential for zinc transport.

IRBIT coordinates epithelial fluid and HCO3– secretion by stimulating the transporters pNBC1 and CFTR in the murine pancreatic duct

Fluid and HCO3- secretion are vital functions of secretory epithelia. In most epithelia, this entails HCO3- entry at the basolateral membrane, mediated by the Na+-HCO3- cotransporter, pNBC1, and exit at the luminal membrane, mediated by a CFTR-SLC26 transporters complex. Here we report that the protein IRBIT (inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3), a previously identified activator of pNBC1, activates both the basolateral pNBC1 and the luminal CFTR to coordinate fluid and HCO3- secretion by the pancreatic duct. We used video microscopy and ion selective microelectrodes to measure fluid secretion and Cl- and HCO3- concentrations in cultured murine sealed intralobular pancreatic ducts. Short interference RNA-mediated knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, luminal Cl- absorption and HCO3- secretion, and the associated fluid secretion. Single-channel measurements suggested that IRBIT regulated CFTR by reducing channel mean close time. Furthermore, expression of IRBIT constructs in HEK cells revealed that activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, suggesting that IRBIT activates these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO3- secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis.

Diverse transport modes by the solute carrier 26 family of anion transporters

The solute carrier 26 (SLC26) transporters are anion transporters with diverse substrate specificity. Several members are ubiquitous while others show limited tissue distribution. They are expressed in many epithelia and to the extent known, play a central role in anion secretion and absorption. Members of the family are primarily Cl− transporters, although some members transport mainly SO42−, Cl−, HCO3− oru2003 I−. A defining feature of the family is their functional diversity. Slc26a1 and Slc26a2 function as specific SO42− transporters while Slc26a4 functions as an electroneutral Cl−/I−/HCO3− exchanger. Slc26a3 and Slc26a6 function as coupled electrogenic Cl−/HCO3− exchangers or as bona fide anion channels. SLC26A7 and SLC26A9 function exclusively as Cl− channels. This short review discusses the functional diversity of the SLC26 transporters.

Silencing of ZnT-1 expression enhances heavy metal influx and toxicity

ZnT-1 reduces intracellular zinc accumulation and confers resistance against cadmium toxicity by a mechanism which is still unresolved. A functional link between the L-type calcium channels (LTCC) and ZnT-1 has been suggested, indicating that ZnT-1 may regulate ion permeation through this pathway. In the present study, immunohistochemical analysis revealed a striking overlap of the expression pattern of LTCC and ZnT-1 in cardiac tissue and brain. Using siRNA to silence ZnT-1 expression, we then assessed the role of ZnT-1 in regulating cation permeation through the L-type Ca2+ channels in cells that are vulnerable to heavy metal permeation. Transfection of cortical neurons with ZnT-1 siRNA resulted in about 70% reduction of ZnT-1 expression and increased Ca2+ influx via LTCC by approximately fourfold. Moreover, ZnT-1 siRNA transfected neurons showed ∼30% increase in synaptic release, monitored using the FM1-43 dye. An increased cation influx rate, through the LTCC, was also recorded for Zn2+ and Cd2+ in cells treated with the ZnT-1 siRNA. Furthermore, Cd2+-induced neuronal death increased by approximately twofold after transfection with ZnT-1 siRNA. In addition, ZnT-1 siRNA transfection of the ovarian granulosa cell line, POGRS1, resulted in a twofold increase in Cd2+ influx rate via the LTCC. Finally, a robust nimodipine-sensitive Cd2+ influx was observed using a low extracellular Cd2+ concentration (5xa0μM) in neurons and testicular slice cultures, attesting to the relevance of the LTCC pathway to heavy metal toxicity. Taken together, our results indicate that endogenously-expressed ZnT-1, by modulating LTCC, has a dual role: regulating calcium influx, and attenuating Cd2+ and Zn2+ permeation and toxicity in neurons and other cell types.

Solute Carrier Family 26 Member a2 (Slc26a2) Protein Functions as an Electroneutral SO42−/OH−/Cl− Exchanger Regulated by Extracellular Cl−

Background: Slc26a2 is an SO42− transporter, mutations in which cause diastrophic dysplasia. How Slc26a2 transports SO42− is unknown. Results: We found that Slc26a2 exchanges SO42− for 2OH− or 2Cl− and is regulated by a promiscuous extracellular anion site. Conclusion: Slc26a2 functions as SO42−/2OH− or SO42−/2Cl− exchanger, regulated by extracellular Cl−o. Significance: The findings should help in understanding aberrant SLC26A2 function in diastrophic dysplasia. Slc26a2 is a ubiquitously expressed SO42− transporter with high expression levels in cartilage and several epithelia. Mutations in SLC26A2 are associated with diastrophic dysplasia. The mechanism by which Slc26a2 transports SO42− and the ion gradients that mediate SO42− uptake are poorly understood. We report here that Slc26a2 functions as an SO42−/2OH−, SO42−/2Cl−, and SO42−/OH−/Cl− exchanger, depending on the Cl− and OH− gradients. At inward Cl− and outward pH gradients (high Cl−o and low pHo) Slc26a2 functions primarily as an SO42−o/2OH−i exchanger. At low Cl−o and high pHo Slc26a2 functions increasingly as an SO42−o/2Cl−i exchanger. The reverse is observed for SO42−i/2OH−o and SO42−i/2Cl−o exchange. Slc26a2 also exchanges Cl− for I−, Br−, and NO3− and Cl−o competes with SO42− on the transport site. Interestingly, Slc26a2 is regulated by an extracellular anion site, required to activate SO42−i/2OH−o exchange. Slc26a2 can transport oxalate in exchange for OH− and/or Cl− with properties similar to SO42− transport. Modeling of the Slc26a2 transmembrane domain (TMD) structure identified a conserved extracellular sequence 367GFXXP371 between TMD7 and TMD8 close to the conserved Glu417 in the permeation pathway. Mutation of Glu417 eliminated transport by Slc26a2, whereas mutation of Phe368 increased the affinity for SO42−o 8-fold while reducing the affinity for Cl−o 2 fold, but without affecting regulation by Cl−o. These findings clarify the mechanism of net SO42− transport and describe a novel regulation of Slc26a2 by an extracellular anion binding site and should help in further understanding aberrant SLC26A2 function in diastrophic dysplasia.

Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters.

Significance Cl− is the major cellular anion that controls the intracellular activity of many ions, the membrane potential, and transepithelial fluid and electrolyte secretion. How cells sense intracellular Cl− (Cl−in) to coordinate all Cl−-dependent activities is not known. We report a molecular mechanism for Cl−in sensing that involves interaction of Cl− with GXXXP-containing sites and show how these sites are used to regulate the activity of several Na+-HCO3− cotransporters. Although these transporters do not transport Cl−, they sense Cl−in in a manner specific for each transporter that is suitable for the transporter physiological activity. Our data has fundamental implications for the role of Cl− in cellular ion homeostasis and fluid and electrolyte secretion. Cl− is a major anion in mammalian cells involved in transport processes that determines the intracellular activity of many ions and plasma membrane potential. Surprisingly, a role of intracellular Cl− (Cl−in) as a signaling ion has not been previously evaluated. Here we report that Cl−in functions as a regulator of cellular Na+ and HCO3− concentrations and transepithelial transport through modulating the activity of several electrogenic Na+-HCO3− transporters. We describe the molecular mechanism(s) of this regulation by physiological Cl−in concentrations highlighting the role of GXXXP motifs in Cl− sensing. Regulation of the ubiquitous Na+-HCO3− co-transport (NBC)e1-B is mediated by two GXXXP-containing sites; regulation of NBCe2-C is dependent on a single GXXXP motif; and regulation of NBCe1-A depends on a cryptic GXXXP motif. In the basal state NBCe1-B is inhibited by high Cl−in interacting at a low affinity GXXXP-containing site. IP3 receptor binding protein released with IP3 (IRBIT) activation of NBCe1-B unmasks a second high affinity Cl−in interacting GXXXP-dependent site. By contrast, NBCe2-C, which does not interact with IRBIT, has a single high affinity N-terminal GXXP-containing Cl−in interacting site. NBCe1-A is unaffected by Cl−in between 5 and 140 mM. However, deletion of NBCe1-A residues 29–41 unmasks a cryptic GXXXP-containing site homologous with the NBCe1-B low affinity site that is involved in inhibition of NBCe1-A by Cl−in. These findings reveal a cellular Cl−in sensing mechanism that plays an important role in the regulation of Na+ and HCO3− transport, with critical implications for the role of Cl− in cellular ion homeostasis and epithelial fluid and electrolyte secretion.

Identification of residues that control Li(+) versus Na(+) dependent Ca(2+) exchange at the transport site of the mitochondrial NCLX.

BACKGROUNDnThe Na+/Ca2+/Li+ exchanger (NCLX) is a member of the Na+/Ca2+ exchanger family. NCLX is unique in its capacity to transport both Na+ and Li+, unlike other members, which are Na+ selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li+ or Na+ selective Ca2+ transport and map their putative location on NCLX cation transport site.nnnMETHODnWe combined molecular modeling to map transport site of NCLX with euryarchaeal H+/Ca2+ exchanger, CAX_Af, and fluorescence analysis to monitor Li+ versus Na+ dependent mitochondrial Ca2+ efflux of transport site mutants of NCLX in permeabilized cells.nnnRESULTnMutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca2+ exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li+/Ca2+ exchange activity but a reduced Na+/Ca2+ exchange activity. On the other hand, D471A showed dramatically reduced Li+/Ca2+ exchange, but Na+/Ca2+ exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca2+ binding residues was required to completely abolish both Na+/Ca2+ and Li+/Ca2+ exchange activities.nnnCONCLUSIONSnWe identified distinct Na+ and Li+ selective residues in the NCLX transport site. We propose that functional segregation in Li+ and Na+ sites reflects the functional properties of NCLX required for Ca2+ exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane.nnnGENERAL SIGNIFICANCEnThe results of this study provide functional insights into the unique Li+ and Na+ selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

A missense mutation in SLC26A3 is associated with human male subfertility and impaired activation of CFTR

Chloride absorption and bicarbonate excretion through exchange by the solute carrier family 26 member 3 (SLC26A3) and cystic fibrosis transmembrane conductance regulator (CFTR) are crucial for many tissues including sperm and epithelia of the male reproductive tract. Homozygous SLC26A3 mutations cause congenital chloride diarrhea with male subfertility, while homozygous CFTR mutations cause cystic fibrosis with male infertility. Some homozygous or heterozygous CFTR mutations only manifest as male infertility. Accordingly, we studied the influence of SLC26A3 on idiopathic infertility by sequencing exons of SLC26A3 in 283 infertile and 211 control men. A heterozygous mutation c.2062u2009Gu2009>u2009C (p.Asp688His) appeared in nine (3.2%) infertile men, and additionally, in two (0.9%) control men, whose samples revealed a sperm motility defect. The p.Asp688His mutation is localized in the CFTR-interacting STAS domain of SLC26A3 and enriched in Finland, showing a significant association with male infertility in comparison with 6,572 Finnish (Pu2009<u20090.05) and over 120,000 global alleles (Pu2009<u20090.0001) (ExAC database). Functional studies showed that while SLC26A3 is a strong activator of CFTR-dependent anion transport, SLC26A3-p.Asp688His mutant retains normal Cl−/HCO3− exchange activity but suppresses CFTR, despite unaffected domain binding and expression. These results suggest a novel mechanism for human male infertility─impaired anion transport by the coupled SLC26A3 and CFTR.