Palaniappan Kulanthaivel

Tyrosine residues are essential for the activity of the human placental taurine transporter

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.

Solubilization and functional reconstitution of the human placental taurine transporter

The taurine transporter from purified human placental brush-border membranes was solubilized and reconstituted into proteoliposomes in a functional form. Solubilization was done with 2.5% cholate in the presence of 4 M urea. The proteins in the solubilizate were precipitated with 6% poly(ethylene glycol) and the precipitated proteins were reconstituted into proteoliposomes with an asolectin/protein ratio of 10:1. Under these experimental conditions, the taurine transport activity in the proteoliposomes was maximal. SDS-PAGE analysis of proteins, however, revealed that the proteoliposomes still contained a majority of the proteins originally present in the brush-border membranes. Uptake of taurine in the reconstituted proteoliposomes was obligatorily dependent on the presence of Na+ as well as Cl-. Substitution of Na+ with other monovalent cations such as K+ and Li+ reduced the taurine transport activity drastically. Similarly, substitution of Cl- with other monovalent anions such as SCN-, F-, I- and NO3- could support the transport activity only to a maximum of 30% of the control activity. In the presence of Cl-, the uptake rate was sigmoidally related to Na+ concentration, resulting in a Na+/taurine coupling ratio of 2:1. The apparent dissociation constant for Na+ was about 195 mM. In the presence of Na+, the uptake rate was hyperbolically related to Cl- concentration, indicating a Cl-/taurine coupling ratio of 1:1. The apparent dissociation constant for Cl- was about 205 mM. The NaCl-dependent taurine uptake was stimulated by an inside-negative membrane potential, showing that the uptake process was electrogenic. The uptake system was specific for beta-amino acids. The affinity of the system for taurine was high with an apparent dissociation constant of 2.7 +/- 0.1 microM. It is concluded that the taurine transporter can be dislodged from the placental brush-border membranes and reconstituted in a catalytically active form in proteoliposomes with no significant change in its characteristics.

Inactivation of taurine transporter by calcium in purified human placental brush border membrane vesicles

We investigated the effects of calcium on the activity of the taurine transporter in purified human placental brush border membrane vesicles. Treatment of the membrane vesicles with calcium markedly inhibited taurine uptake. The magnitude of inhibition was dependent on the calcium concentration and the treatment time. Free ionized Ca2+ was responsible for this effect because EGTA, a Ca2+ chelator, totally abolished the calcium-induced effect. Uptake of succinate, which occurs via a Na(+)-dependent process as does the uptake of taurine, was reduced only to a small extent by the calcium treatment. This result indicates that the effect of Ca2+ on taurine uptake was not due to an accelerated dissipation of the Na+ gradient as a result of an increased Na+ permeability of the membrane. Preloading the vesicles with phospholipase inhibitors such as neomycin and quinacrine significantly protected the taurine transporter from the Ca2+ effect, raising the possibility that Ca(2+)-activated phospholipases may mediate the Ca2+ effect. Kinetic analysis revealed that Ca2+ decreased the affinity of the transporter for taurine as well as the translocation rate of the taurine-loaded transporter complex.

An essential role for vicinal dithiol groups in the catalytic activity of the human placental Na+-H+ exchanger

We examined the effects of phenylarsine oxide, a reagent specific for vicinal dithiol groups, on the catalytic activities, Na+ influx and H+ efflux, of the human placental Na(+)-H+ exchanger. Treatment of the placental brush-border membrane vesicles with the reagent markedly inhibited both the activities. The inhibition was partially reversible by dithiols. The effect of phenylarsine oxide was to reduce the maximal velocity of the exchanger without influencing its affinity for Na+. The exchanger was partially protected from this inhibition by amiloride but not by cimetidine even though both these compounds interacted with the Na(+)-binding site. The data demonstrate that vicinal dithiol groups are essential for the catalytic function of the placental Na(+)-H+ exchanger and that the critical dithiol groups are located at a site distinct from the Na(+)-binding site.

Expression of the cocaine-sensitive norepinephrine transporter in the human placental syncytiotrophoblast

Maternal-facing brush border membrane vesicles isolated from normal term human placentas were found to accumulate norepinephrine in a concentrative manner in the presence of an inwardly directed NaCl gradient. Both Na+ and Cl- were obligatory for maximal uptake. The NaCl-dependent norepinephrine uptake was further stimulated by the presence of K+ or an acidic pH in the intravesicular medium. The uptake process was electrogenic, being stimulated by an inside-negative membrane potential, and this characteristic was observed in the absence as well as in the presence of K+ inside the vesicles. Kinetic analyses revealed that one Na+ and one Cl- were involved per transport of one norepinephrine molecule. The apparent Michaelis-Menten constant for norepinephrine was 104 +/- 5nM. The uptake process exhibited higher affinity for dopamine than for norepinephrine but had low affinity for serotonin and histamine. The uptake of norepinephrine was inhibited very effectively by nomifensine, desipramine, imipramine, and cocaine, but much less effectively by bupropion and GBR 12909. Northern blot analysis with the cDNA of the human (SK-N-SH cell) norepinephrine transporter as the probe revealed that the human placenta contained two mRNAs, 5.8 and 3.6 kb in size, which hybridized to the probe. The JAR human placental choriocarcinoma cells were found unable to accumulate norepinephrine in a NaCl-dependent manner. These cells were also found not to contain mRNAs which hybridized to the norepinephrine cDNA probe in northern blot. It is concluded that the human placental syncytiotrophoblast expresses a cocaine-sensitive norepinephrine transporter and that these findings may be directly relevant and important to the clinical complications of maternal cocaine abuse during pregnancy.