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.

Show Gene links Show Anatomy links

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 .

???displayArticle.abstract???
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.

???displayArticle.pubmedLink??? 26884475
???displayArticle.pmcLink??? PMC4759042
???displayArticle.link??? Physiol Rep

???attribute.lit??? ???displayArticles.show???

	Figure 1. Transport current in the presence of D‐ and L‐ amino acids. Single oocytes (left) and mean values ± SEM for several (n = from 5 to 25) oocytes (right). Relative efficiency of L‐ versus D‐ proline 3 mmol/L (A), serine 3 mmol/L (B), and leucine 0.5 mmol/L (C) in eliciting transport‐associated current by KAAT1 when Na+ is the driving ion. In the case of proline, the D‐ enantiomer is less potent than the L‐form, whereas the opposite occurs for leucine and serine. In all cases, the L‐form is dominant over the D‐form used at the same concentration: the current amplitude upon simultaneous perfusion of both enantiomers is always close to the level generated by the L‐form alone. Currents were recorded as described in Material and Methods.
	Figure 2. Leucine dose‐response in Na+. (A) the transport currents elicited by increasing amounts of D‐leucine show a progressive growth up to 300 μmol/L, but a further increase to 1 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 L‐leucine induce progressively larger responses showing saturation above 100 μmol/L without any anomalous behavior.
	Figure 3. Mole fraction behavior of L‐leu and D‐leu. Mixtures containing variable proportions of L‐ and D‐leucine were applied while keeping constant the total concentration. The substantial constancy of the responses to mixtures containing L‐leucine indicates that this amino acid dominates the transport process by virtue of a higher apparent affinity. The larger current induced by D‐leucine alone is consistent with a higher turnover rate related to a lower affinity. Normalizing the transport current values to that recorded in 500 μmol/L of L‐leucine alone, the current in the presence of 350 μmol/L L‐Leu+150 μmol/L D‐Leu increases to 1.0769 ± 0.01566, when 250 μmol/L of both were perfused becomes 1.13986 ± 0.05827 and when 150 μmol/L L‐Leu and 350 μmol/L D‐Leu was tested the current is 1.20799 ± 0.03927.When 500 μmol/L D‐leucine is applied alone, it becomes from 2 to 4 times larger (mean 2.54265 ± 0.52057) data were collected from 10 oocytes of three different batches.
	Figure 4. Effects of D‐leucine at 1 mmol/L. (A) when used at 1 mmol/L D‐leucine 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 D‐leucine occurs in the presence of L‐leucine (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 D‐forms of proline, leucine, and serine on the same oocyte shows the specificity of the D‐leucine response.
	Figure 5. Current–voltage relationship for the different conditions of recording in a representative oocyte. (A) fast voltage ramps (from −140 to +40 mV, duration 1 sec) before the application of substrates (a), in the presence of 1 mmol/L D‐leucine (b), of both D‐ and L‐leucine 1 mmol/L each (c), and upon D‐leucine washout (d). (B) I–V relationships obtained by subtracting the control current (a) from each of the others.
	Figure 6. D‐leucine in high K+ solution. When applied at 500 μmol/L in a K+‐containing external solution, D‐leucine elicits a small current compared to L‐leucine. The addition of D‐ to L‐leucine (both at 500 μmol/L) slightly inhibits the L‐leucine‐induced current (A). The increase in L‐leucine current when applied in addition to D‐leucine (both at 500 μmol/L), does not reach the level elicited by L‐leucine alone (B). The mean values ± SEM for several (n = from 6 to 15 from at least 3 different batches) oocytes are reported (C).Comparison of the currents generated by 1 mmol/L D‐leucine 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 D‐proline and D‐serine in K+. L‐proline 3 mmol/L elicits only small transport currents in presence of K+, whereas D‐proline at the same concentration appears to be completely ineffective. On the contrary, both L‐ and D‐serine 3 mmol/L generate currents in K+, although with opposite relative potency compared to those in Na+ (A).The mean values ± SEM for several oocytes (n = from 6 to 15 from at least 3 different batches) are reported (B).
	Figure 8. Action of different L‐ and D‐amino acids on the CAATCH1 transport currents in Na+. (A and B) L‐proline is more potent than the D‐form in eliciting the transport current; L‐serine and L‐leucine generate an apparent outward current, while their corresponding D‐forms produce an inward current. (A) a representative recording; (B) mean current ± SEM from 6 to 11 oocytes from two different batches. (C) application of increasing concentrations of D‐leucine produces progressively larger inward currents, whereas the same concentrations of L‐leucine produce an apparent outward current of constant size (D), consistent with a high‐affinity block of the transporter leak current (Miszner et al. 2007).
	Figure 9. Currents elicited by L‐ and D‐amino acid in KAAT1 S308T. While the currents generated by serine 3 mmol/L, and their relative potency, are not affected by the mutation, a selective reduction of the current of the L‐form is seen for proline 3 mmol/L. As expected, no current is generated by L‐leucine 1 mmol/L, whereas the same concentration of D‐leucine is effective in producing a response that includes a weaker but reproducible inward current surge at wash out (arrow).

References [+] :

Bauer, Preferred stereoselective brain uptake of d-serine--a modulator of glutamatergic neurotransmission. 2005, Pubmed