Vollero A et al. (2016), The D-amino acid transport by the invertebrate ...

XB-ART-51909

Physiol Rep 2016 Feb 01;44:. doi: 10.14814/phy2.12691.

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The D-amino acid transport by the invertebrate SLC6 transporters KAAT1 and CAATCH1 from Manduca sexta.

Vollero A , Imperiali FG , Cinquetti R , Margheritis E , Peres A , Bossi E .

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The ability of the SLC6 family members, the insect neutral amino acid cotransporter KAAT1(K(+)-coupled amino acid transporter 1) and its homologous CAATCH1(cation anion activated amino acid transporter/channel), to transport D-amino acids has been investigated through heterologous expression in Xenopus laevis oocytes and electrophysiological techniques. In the presence of D-isomers of leucine, serine, and proline, the msKAAT1 generates inward, transport-associated, currents with variable relative potencies, depending on the driving ion Na(+) or K(+). Higher concentrations of D-leucine (≥1 mmol/L) give rise to an anomalous response that suggests the existence of a second binding site with inhibitory action on the transport process. msCAATCH1 is also able to transport the D-amino acids tested, including D-leucine, whereas L-leucine acts as a blocker. A similar behavior is exhibited by the KAAT1 mutant S308T, confirming the relevance of the residue in this position in L-leucine binding and the different interaction of D-leucine with residues involved in transport mechanism. D-leucine and D-serine on various vertebrate orthologs B(0)AT1 (SLC6A19) elicited only a very small current and singular behavior was not observed, indicating that it is specific of the insect neutral amino acid transporters. These findings highlight the relevance of D-amino acid absorption in the insect nutrition and metabolism and may provide new evidences in the molecular transport mechanism of SLC6 family.

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	Figure 1. Transport current in the presence of Dâ and Lâ amino acids. Single oocytes (left) and mean valuesÂ Â±Â SEM for several (nÂ =Â from 5 to 25) oocytes (right). Relative efficiency of Lâ versus Dâ proline 3Â mmol/L (A), serine 3Â mmol/L (B), and leucine 0.5Â mmol/L (C) in eliciting transportâassociated current by KAAT1 when Na+ is the driving ion. In the case of proline, the Dâ enantiomer is less potent than the Lâform, whereas the opposite occurs for leucine and serine. In all cases, the Lâform is dominant over the Dâform used at the same concentration: the current amplitude upon simultaneous perfusion of both enantiomers is always close to the level generated by the Lâform alone. Currents were recorded as described in Material and Methods.
	Figure 2. Leucine doseâresponse in Na+. (A) the transport currents elicited by increasing amounts of Dâleucine show a progressive growth up to 300Â Î¼mol/L, but a further increase to 1Â mmol/L produces an abrupt change in behavior exhibiting a reduction of the maximum attained value, an inactivation process and a peculiar inward transient surge at washout (arrow). (B) the same amounts of Lâleucine induce progressively larger responses showing saturation above 100Â Î¼mol/L without any anomalous behavior.
	Figure 3. Mole fraction behavior of Lâleu and Dâleu. Mixtures containing variable proportions of Lâ and Dâleucine were applied while keeping constant the total concentration. The substantial constancy of the responses to mixtures containing Lâleucine indicates that this amino acid dominates the transport process by virtue of a higher apparent affinity. The larger current induced by Dâleucine alone is consistent with a higher turnover rate related to a lower affinity. Normalizing the transport current values to that recorded in 500Â Î¼mol/L of Lâleucine alone, the current in the presence of 350Â Î¼mol/L LâLeu+150Â Î¼mol/L DâLeu increases to 1.0769Â Â±Â 0.01566, when 250Â Î¼mol/L of both were perfused becomes 1.13986Â Â±Â 0.05827 and when 150Â Î¼mol/L LâLeu and 350Â Î¼mol/L DâLeu was tested the current is 1.20799Â Â±Â 0.03927.When 500Â Î¼mol/L Dâleucine is applied alone, it becomes from 2 to 4 times larger (mean 2.54265Â Â±Â 0.52057) data were collected from 10 oocytes of three different batches.
	Figure 4. Effects of Dâleucine at 1Â mmol/L. (A) when used at 1Â mmol/L Dâleucine produces currents with peculiar behavior. A marked decline follows the large and fast initial current during exposure to the substrate and, quite notably, an inward surge of current develops when the substrate is removed (arrows). Note that the surge is not present when the removal of Dâleucine occurs in the presence of Lâleucine (gray arrow). Furthermore, the simultaneous presence of both enantiomers causes a reduction of the current generated by either of the two when applied alone (black arrowheads). (B) subsequent application of the Dâforms of proline, leucine, and serine on the same oocyte shows the specificity of the Dâleucine response.
	Figure 5. Currentâvoltage relationship for the different conditions of recording in a representative oocyte. (A) fast voltage ramps (from â140 to +40Â mV, duration 1Â sec) before the application of substrates (a), in the presence of 1Â mmol/L Dâleucine (b), of both Dâ and Lâleucine 1Â mmol/L each (c), and upon Dâleucine washout (d). (B) IâV relationships obtained by subtracting the control current (a) from each of the others.
	Figure 6. Dâleucine in high K+ solution. When applied at 500Â Î¼mol/L in a K+âcontaining external solution, Dâleucine elicits a small current compared to Lâleucine. The addition of Dâ to Lâleucine (both at 500Â Î¼mol/L) slightly inhibits the Lâleucineâinduced current (A). The increase in Lâleucine current when applied in addition to Dâleucine (both at 500Â Î¼mol/L), does not reach the level elicited by Lâleucine alone (B). The mean valuesÂ Â±Â SEM for several (nÂ =Â from 6 to 15 from at least 3 different batches) oocytes are reported (C).Comparison of the currents generated by 1Â mmol/L Dâleucine in presence of high external Na+ or high external K+ revealed that, in the latter condition, only a very small transport current is detected (D).
	Figure 7. KAAT1, currents elicited by Dâproline and Dâserine in K+. Lâproline 3Â mmol/L elicits only small transport currents in presence of K+, whereas Dâproline at the same concentration appears to be completely ineffective. On the contrary, both Lâ and Dâserine 3Â mmol/L generate currents in K+, although with opposite relative potency compared to those in Na+ (A).The mean valuesÂ Â±Â SEM for several oocytes (nÂ =Â from 6 to 15 from at least 3 different batches) are reported (B).
	Figure 8. Action of different Lâ and Dâamino acids on the CAATCH1 transport currents in Na+. (A and B) Lâproline is more potent than the Dâform in eliciting the transport current; Lâserine and Lâleucine generate an apparent outward current, while their corresponding Dâforms produce an inward current. (A) a representative recording; (B) mean current Â Â±Â SEM from 6 to 11 oocytes from two different batches. (C) application of increasing concentrations of Dâleucine produces progressively larger inward currents, whereas the same concentrations of Lâleucine produce an apparent outward current of constant size (D), consistent with a highâaffinity block of the transporter leak current (Miszner etÂ al. 2007).
	Figure 9. Currents elicited by Lâ and Dâamino acid in KAAT1 S308T. While the currents generated by serine 3Â mmol/L, and their relative potency, are not affected by the mutation, a selective reduction of the current of the Lâform is seen for proline 3Â mmol/L. As expected, no current is generated by Lâleucine 1Â mmol/L, whereas the same concentration of Dâleucine is effective in producing a response that includes a weaker but reproducible inward current surge at wash out (arrow).

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