Mayer M et al. (2006), Different transport mechanisms in plant and hum...

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J Gen Physiol 2006 Feb 01;1272:133-44. doi: 10.1085/jgp.200509369.

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Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters.

Mayer M , Schaaf G , Mouro I , Lopez C , Colin Y , Neumann P , Cartron JP , Ludewig U .

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The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH3 gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with Km = 3.8 mM at pHo 7.5. Uptake was strongly favored by increasing the pHo and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH4+ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pHo-independent MeA+ uptake and specific NH4+ and MeA+ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH4+, or cotransports NH3/H+, RhCG mediates electroneutral NH3 transport.

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Species referenced: Xenopus
Genes referenced: gnas rhcg

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	Figure 1. Voltage-dependent currents associated with RhCG expression are permeable to sodium and potassium. (A) Currentâvoltage relations of noninjected oocytes, (B) RhCG-expressing oocytes and (C) LeAMT1;2-expressing oocytes at pHo 7.5 (n = 4). Similar data were obtained in oocytes from three batches. (D) RhCG expression increased sodium sensitivity of an alkali metalâsensitive mutant yeast strain. Serial fivefold dilutions of 25-fold diluted saturated cultures were spotted on YNB media with added sodium (right) or without (control, left) and growth was followed for 3 (control plates) or 4 d (plates supplemented with sodium). Yeast expressing the Na+/H+ antiporter NHA1 were insensitive to sodium, but yeast expressing RhCG had increased sodium sensitivity compared with vector-transformed control.
	Figure 2. Currentâvoltage relations of endogenous ammonium-induced currents. (A) Currents from native oocytes at pHo 8.5 without ammonium (open circles), by 300 Î¼M (closed circles), 1 mM (open triangle), and 3 mM ammonium (filled triangle), respectively. (B) Ammonium-induced currents (background subtracted, from A) in a typical oocyte batch. Note that ammonium-induced currents have a reversal potential (arrow) and are outward at positive voltages. Data shown are from oocytes from a single batch, similar data were obtained in two other oocyte batches.
	Figure 3. Electroneutral uptake of methylammonium in noninjected control Xenopus oocytes is partially blocked by ammonium. (A) Currentâvoltage relations without methylammonium (open circles) and with various methylammonium concentrations, 1 mM (filled circles), 3 mM (open triangles), 10 mM (filled triangles) at pHo 7.5. Symbols are partially overlapping. (B) Concentration dependence of 14C-methylammonium uptake rate at pHo 7.5. (C) pHo dependence of 14C-methylammonium uptake rate. (D) Block of 14C-methylammonium uptake (1 mM) by ammonium.
	Figure 4. Methylammonium uptake by RhCG-expressing oocytes. (A) Linear MeA uptake by noninjected controls (circles) and RhCG (triangles) at 1 mM (open symbols) and 10 mM (filled symbols) at pHo 7.5. (B) MeA uptake (1 mM) of noninjected and RhCG-expressing oocytes in sodium-free choline (left) and sodium solutions (right). (C) Concentration dependence of RhCG at pHo 7.5. (D) Block of MeA uptake (2 mM) by ammonium, KiNH4+ = 3.8 Â± 1.2 mM at pHo 7.5. Data from four oocyte batches.
	Figure 5. MeA+ currents by LeAMT1;2. (A) MeA+-induced currents by LeAMT1;2 at pHo 7.5 (n = 4). Background currents were subtracted. Open circles, 1 mM methylammonium; filled circles, 3 mM; open triangles, 10 mM; filled triangles, 30 mM. (B) Concentration dependence of MeA+ currents at â100 mV (open circles, Km = 6.1 Â± 1.1 mM) and â140 mV (closed circles, Km = 5.6 Â± 1.0 mM) by LeAMT1;2 at pHo 7.5 (n = 4). Data are from three different batches.
	Figure 6. Distinct MeA+ currents and MeA uptake by RhCG and LeAMT1;2. (A) Currentâvoltage relations from noninjected control oocytes (A) and in RhCG-expressing oocytes (B) in the absence (open symbols) and presence (filled symbols) of methylammonium at pHo 5.5 (circles) and pHo 8.5 (triangles). Symbols are partially overlapping. (C) Currentâvoltage relations from LeAMT1;2-expressing oocytes in the absence (open circles) and presence (filled circles) of methylammonium at pHo 7.5. (D) Induced current by methylammonium (background currents subtracted) in RhCG (filled circles) and LeAMT1;2-expressing oocytes (open circles). (E) 14C-methylammonium uptake by RhCG at different pHo; background uptake of native oocytes from parallel experiments was subtracted. (F) 14C-methylammonium uptake by LeAMT1;2 at different pHo; background uptake of parallel experiments with native oocytes was subtracted. Note the different current scales in AâC. All experiments in this figure were performed with [methylammonium] = 1 mM, except for panel F: [methylammonium] = 500 Î¼M. Data were collected from six oocyte batches.
	Figure 7. Ionic currents by ammonium in H2O-injected, RhCG-expressing, and LeAMT1;2-expressing oocytes. Top, original raw current traces without (left) and with 10 mM NH4+ (right). Voltage pulses were given from 40 to â140 mV in steps of â20 mV. Note the current increase by NH4+ in LeAMT1;2 and the large RhCG-associated currents. Bottom, currentâvoltage plots of currents by ammonium in H2O-injected (n = 4), RhCG-expressing (n = 6), and LeAMT1;2-expressing (n = 5) oocytes with 10 mM NH4Cl (filled circles) and without (open circles). Data shown are from a single oocyte batch, similar data were obtained from two additional oocyte batches.
	Figure 8. Voltage dependence of ammonium-induced currents by LeAMT1;2 and RhCG. (A) Currentâvoltage relations from native oocytes. (B) RhCG-expressing oocytes. Currents were recorded in sodium-based solutions (filled circles) supplemented with 1 mM ammonium (open triangles) and with 20 mM ammonium (filled triangles). (C) Currentâvoltage relations from LeAMT1;2-expressing oocytes in sodium-based solution without ammonium (filled circles) or with 100 Î¼M ammonium (open triangles). Means Â± SD of n = 4â7 oocytes from two batches are shown. Large error bars are mainly due to different background currents. Similar results were recorded in two additional batches. (D) Induced currents (background currents subtracted) by 10 mM ammonium. Open circles, LeAMT1;2; closed circles, RhCG; open triangle, H2O injected. Arrows indicate the reversal of the induced currents. To obtain the induced current means Â± SD, the background currents were subtracted from the currents in ammonium in each individual experiment. This reduced the impact of background current magnitude on the error bars, as the resulting currents were insensitive to variable background currents. All experiments were done at pHo 7.5.
	Figure 9. Intracellular alkalinization by ammonium in H2O-injected, RhCG-expressing, and LeAMT1;2-expressing oocytes. Representative results of BCECF-loaded oocytes at pH 7.5. Oocytes were exposed to 500 Î¼M and 10 mM NH4Cl for the indicated periods. (A) H2O-injected oocyte. (B) RhCG-expressing oocyte. (C) LeAMT1;2-expressing oocyte. Measured values are indicated by black dots and are connected by thin lines. Upward deflections indicate pHi alkalinization. Similar results were obtained (n = 7, H2O-injected oocytes; n = 11, RhCG; n = 9: LeAMT1;2) in a total of five oocyte batches.
	Figure 10. Opposite effects of RhCG and LeAMT1;2 in yeast. (A) Serial 10-fold dilutions of saturated cultures of the constructs expressed in wild-type yeast. Cells were spotted on YNB media supplemented with 125 mM methylammonium. The picture was taken after 3 d at 28Â°C. Yeast expressing LeAMT1;2 were more sensitive to toxic methylammonium than vector-transformed controls, while yeast expressing RhCG were more resistant than vector transformed controls. (B and C) Serial 10-fold dilutions of saturated cultures of the constructs expressed in the ÎÎÎmep1,2,3 yeast strain that lacks three endogenous ammonium transporters. LeAMT1;2 rescued the growth on limiting ammonium (1 and 5 mM). The pictures were taken after 5 d of growth. Experiments were repeated four times.

References [+] :

Accardi, Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. 2004, Pubmed