Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger

Ganapathy, V.; Balkovetz, Daniel F.; Ganapathy, Malliga E.; Mahesh, Virendra B.; Devoe, Lawrence D.; Leibach, F. H.

doi:10.1042/bj2450473

Cited by 23 publications

(27 citation statements)

References 14 publications

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“…The significance of His residues was also shown for the H ϩ gradient-dependent peptide transporters PepT1 and PepT2 in studies with renal and intestinal brush-border membrane vesicles, where transport activity was severely impaired following incubation with DEPC (21,29). Other examples include the Na ϩ /H ϩ exchanger (9,12), the organic cation/H ϩ antiporter (16) or the folate transporter (53). In all these transporters, one or more individual His residue(s) were identified as important factors for transport activity (1,7,63).…”

Section: Discussionmentioning

confidence: 87%

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Leuthold¹,

Hagenbuch²,

Mohebbi³

et al. 2009

American Journal of Physiology-Cell Physiology

162

176

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Organic anion transporting polypeptides (humans OATPs, rodents Oatps) are expressed in most mammalian tissues and mediate cellular uptake of a wide variety of amphipathic organic compounds such as bile salts, steroid conjugates, oligopeptides, and a large list of drugs, probably by acting as anion exchangers. In the present study we aimed to investigate the role of the extracellular pH on the transport activity of nine human and four rat OATPs/Oatps. Furthermore, we aimed to test the concept that OATP/Oatp transport activity is accompanied by extrusion of bicarbonate. By using amphibian Xenopus laevis oocytes expressing OATPs/Oatps and mammalian cell lines stably transfected with OATPs/Oatps, we could demonstrate that in all OATPs/Oatps investigated, with the exception of OATP1C1, a low extracellular pH stimulated transport activity. This stimulation was accompanied by an increased substrate affinity as evidenced by lower apparent Michaelis-Menten constant values. OATP1C1 is lacking a highly conserved histidine in the third transmembrane domain, which was shown by site-directed mutagenesis to be critically involved in the pH dependency of OATPs/Oatps. Using online intracellular pH measurements in OATP/Oatp-transfected Chinese Hamster Ovary (CHO)-K1 cells, we could demonstrate the presence of a 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid-sensitive chloride/bicarbonate exchanger in CHO-K1 cells and that OATP/Oatp-mediated substrate transport is paralleled by bicarbonate efflux. We conclude that the pH dependency of OATPs/Oatps may lead to a stimulation of substrate transport in an acidic microenvironment and that the OATP/Oatp-mediated substrate transport into cells is generally compensated or accompanied by bicarbonate efflux.

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Section: Discussionmentioning

confidence: 87%

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Leuthold¹,

Hagenbuch²,

Mohebbi³

et al. 2009

American Journal of Physiology-Cell Physiology

162

176

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“…It is known that the presence of Clin the assay medium modifies the interaction of amiloride and other inhibitors with the Na+-H+ exchanger [28,29]. Since amiloride, clonidine and cimetidine all interact with the same site on the human placental brush border membrane Na+-H+ exchanger in a mutually exclusive manner [26,30], it is very likely that Clin the assay medium exerts marked influences on the inhibitory characteristics of clonidine and cimetidine, as it does in the case of amiloride. Therefore we investigated the concentration-dependent inhibition of Na+-H+ exchanger activity in placental basal and brush border membrane preparations by clonidine and cimetidine in the presence of Cl- (Fig.…”

Section: Resultsmentioning

confidence: 99%

Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na+-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane

Kulanthaivel

Furesz²,

Moe³

et al. 1992

Biochemical Journal

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We investigated whether highly purified preparations of basal (fetal-facing) membrane isolated from normal term human placentas possess Na(+)-H+ exchanger activity. Uptake of Na+ into basal membrane vesicles was stimulated many-fold by an outwardly directed H+ gradient. This H(+)-gradient-dependent uptake was inhibitable by amiloride and its analogues. Na+ uptake in these vesicles did not occur via a Na+ channel, as it was not influenced by changes in membrane potential and, in addition, was inhibited by benzamil only at high micromolar concentrations. The results indicate that the human placental basal membrane possesses Na(+)-H+ exchanger activity. We then studied whether this exchanger is similar to or distinct from the Na(+)-H+ exchanger described in brush border (maternal-facing) membrane preparations. For this purpose, we compared the pharmacological characteristics of the basal membrane Na(+)-H+ exchanger with those of the brush border membrane Na(+)-H+ exchanger. The basal membrane exchanger was about 20-fold less sensitive to inhibition by amiloride and about 70-fold less sensitive to inhibition by dimethylamiloride than was the brush border membrane exchanger. The exchanger activity in both membrane preparations was inhibitable by clonidine and cimetidine, but the inhibition patterns with these compounds were markedly different between basal and brush border membrane preparations. These data demonstrate that the basal membrane Na(+)-H+ exchanger is distinct from the brush border membrane Na(+)-H+ exchanger. The pharmacological profiles of these exchangers indicate that the human placental brush border membrane possesses the housekeeping or non-epithelial type Na(+)-H+ exchanger (NHE-1), whereas the basal membrane possesses the epithelial or apical type Na(+)-H+ exchanger (NHE-2).

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“…Materials-Carrier-free 22 NaCl (radioactivity, 5 mCi/ml) was obtained from DuPont NEN. Amiloride, cimetidine, and ouabain were purchased from Sigma, and the amiloride derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) was obtained from Molecular Probes (Eugene, OR).…”

Section: Methodsmentioning

confidence: 99%

“…H ϩ -suicide technique) (12, 32) in order to discriminate between Na ϩ /H ϩ exchanger positive and negative transfectants. 22 Na ϩ Influx Measurements-The cells were grown to confluence in 24-well plates. NHE activity was determined by preloading the cells with H ϩ using the NH 4 Cl technique (33) and then measuring the initial rates of 22 Na ϩ influx essentially as described (12).…”

Section: Methodsmentioning

confidence: 99%

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Delineation of Transmembrane Domains of the Na+/H+ Exchanger That Confer Sensitivity to Pharmacological Antagonists

Orlowski

Kandasamy

1996

Journal of Biological Chemistry

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Plasma membrane Na+/H+ exchanger (NHE) isoforms NHE1 and NHE3 exhibit very different sensitivities to amiloride and its 5-amino-substituted analogues, benzoyl guanidinium derivatives (e.g. (3-methylsulfonyl-4-piperidinobenzoyl)guanidine methanesulfonate (HOE694)), and cimetidine. To define structural domains that confer differential sensitivity to these antagonists, unique restriction endonuclease sites were engineered into cDNAs for each isoform near the regions that encode the putative membrane-spanning domains. These new sites did not modify their pharmacological properties and allowed several chimeric Na+/H+ exchangers to be constructed by exchanging homologous segments. The modified parental (E1' and E3') and chimeric molecules were stably expressed in exchanger-deficient Chinese hamster ovary AP-1 cells and assayed for their sensitivities to amiloride, ethylisopropylamiloride, HOE694, and cimetidine. Most chimeras showed drug sensitivities corresponding to the dominant parental segment. However, interchanging a 66-amino acid segment containing the putative ninth transmembrane (M9) domain and its adjacent loops caused reciprocal alterations in the sensitivities of E1' and E3' to all antagonists. In addition, substituting the first five putative membrane-spanning domains of E3' with the corresponding region of E1' modestly reduced the transporter's sensitivity to cimetidine but not the other compounds. These data indicate that the protein segment between M8 and M10 may be a major site of interaction with these antagonists, although other regions modestly influence sensitivity to certain drugs.

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Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger

Cited by 23 publications

References 14 publications

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters

Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na+-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane

Delineation of Transmembrane Domains of the Na+/H+ Exchanger That Confer Sensitivity to Pharmacological Antagonists

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