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. 2015 Oct 2;290(40):24308-25.
doi: 10.1074/jbc.M115.648519. Epub 2015 Aug 3.

Molecular basis for the interaction of the mammalian amino acid transporters B0AT1 and B0AT3 with their ancillary protein collectrin

Stephen J Fairweather  1 , Angelika Bröer  1 , Nandhitha Subramanian  2 , Emrah Tumer  1 , Qi Cheng  1 , Dieter Schmoll  3 , Megan L O'Mara  2 , Stefan Bröer  4
Affiliations

Molecular basis for the interaction of the mammalian amino acid transporters B0AT1 and B0AT3 with their ancillary protein collectrin

Stephen J Fairweather et al. J Biol Chem. .

Abstract

Many solute carrier 6 (SLC6) family transporters require ancillary subunits to modify their expression and activity. The main apical membrane neutral amino acid transporters in mouse intestine and kidney, B(0)AT1 and B(0)AT3, require the ancillary protein collectrin or ACE2 for plasma membrane expression. Expression and activity of SLC6 neurotransmitter transporters are modulated by interaction with syntaxin 1A. Utilizing monocarboxylate-B(0)AT1/3 fusion constructs, we discovered that collectrin is also necessary for B(0)AT1 and B(0)AT3 catalytic function. Syntaxin 1A and syntaxin 3 inhibit the membrane expression of B(0)AT1 by competing with collectrin for access. A mutagenesis screening approach identified residues on trans-membrane domains 1α, 5, and 7 on one face of B(0)AT3 as a key region involved in interaction with collectrin. Mutant analysis established residues that were involved in collectrin-dependent functions as follows: plasma membrane expression of B(0)AT3, catalytic activation, or both. These results identify a potential binding site for collectrin and other SLC6 ancillary proteins.

Keywords: angiotensin-converting enzyme 2 (ACE2); broad neutral amino acid transporters; collectrin; intestinal epithelium; kidney; membrane transport; protein complex; protein-protein interaction; syntaxin 1A; syntaxin 3.

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Figures

FIGURE 1.
FIGURE 1.
Ancillary proteins modify substrate affinity of B0AT1 and B0AT3. X. laevis oocytes were injected with 10 ng of the indicated transporter, 2 ng of collectrin, and 10 ng of ACE2 cRNA in the indicated combinations except where otherwise stipulated. All uptake and electrophysiological measurements were made day 4–6 post-injection. A, uptake of 100 μm l-[U-14C]leucine or 100 μm l-[U-14C]alanine was measured in oocytes expressing B0AT1 or B0AT3 over 30 min. Each bar represents mean ± S.D. (n = 12, e > 3. B, B0AT1 was coexpressed with truncated (18–500del or 18–580del) or wild-type ACE2. Uptake was measured as in A (n = 10−12, e = 3). a and b above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. C, oocytes were voltage-clamped at −50 mV and then perfused with serial concentrations of l-alanine. Data were transformed according to Eadie-Hofstee and analysed by linear regression, for all regressions 0.99 > R2 > 0.88, and Pearson's r > −0.94 (n = 7). D, oocytes were injected with 6 ng of B0AT3 cRNA, 5 ng of ACE2, and 2 ng of collectrin cRNA. Uptake was measured as in A with the exception that it was challenged by 10 mm unlabeled l-amino acids as indicated. The first pair of bars from the left represents unchallenged 100 μm l-[U-14C]alanine uptake. l-[U-14C]Alanine uptake from uninjected oocytes was subtracted. Each bar represents mean ± S.D. (n = 8−10, *, p < 0.05). E, oocytes were perfused with 10 mm of all the amino acids indicated on the abscissa, and subsequent steady-state Na+ currents were recorded. All currents were normalized to the maximal current induced by 10 mm l-methionine. Each bar represents mean ± S.D. (n = 6, e = 3).
FIGURE 2.
FIGURE 2.
Collectrin is required for catalytic activation of mouse B0AT3 and B0AT1. X. laevis oocytes were injected with 10 ng of the indicated transporter or 18 ng of MCT-B0AT3 fusion cRNA and 2 ng of collectrin cRNA. All measurements were made days 4–6 post-injection of cRNA. A, schematic of B0AT3 expression conditions are as follows: B0AT3 alone cannot traffic to the plasma membrane (panel i) because it requires collectrin (panel ii). We hypothesized it would traffic without collectrin when fused to MCT1 (panel iii). B, uptake of 100 μm l-[U-14C]alanine or 100 μm l-[U-14C]lactate was carried out over 30 min. Each histogram bar represents mean ± S.D. (n = 10−15, e > 3). a–c above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. C, uptake of 100 μm l-[U-14C]lactate was carried out over 24 min. Each histogram bar represents mean ± S.D. (n = 10, e = 3). a and b above the individual bars indicates groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. D, uptake of 100 μm l-[U-14C]leucine or 100 μm l-[U-14C]lactate was carried out over 25 min. Each bar represents mean ± S.D. (n = 12−15, e = 3). a–d above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. E, correlation between l-lactate uptake and l-alanine-induced currents in the MCT-B0AT3 tandem construct. The integral of the alanine-dependent inward charge transfer (Q5 mM L-Ala) was calculated and plotted against the l-[U-14C]lactate uptake in the same oocyte (n = 10 − 13). Pearson's r = 0.92, the adjusted coefficient of determination (R2) is indicated on the graph.
FIGURE 3.
FIGURE 3.
Properties of MCT-B0AT3 activation by collectrin. X. laevis oocytes were injected with 10 ng of the indicated transporter or 18 ng of MCT-B0AT3 fusion cRNA and 2 ng of collectrin cRNA. All measurements were made 4–6 days post-injection. All oocytes were voltage-clamped at −50 mV. A, l-alanine-induced current tracings recorded from single oocytes super-fused with the serial substrate concentrations were used to determine the steady-state kinetics of MCT-B0AT3 compared with B0AT3, both in the presence of collectrin. Steady-state inward currents were recorded for both the descending (shown) and ascending (not shown) order of l-alanine concentrations. B, oocytes were perfused with serial concentrations of l-alanine as in A and averaged data for each concentration fitted to the Michaelis-Menten function. Inset, Eadie-Hofstee linear regression of the Michaelis-Menten data: adjusted R2 = 0.77 for B0AT3 + collectrin and adjusted R2 = 0.88 for MCT-B0AT3 + collectrin (n = 8−10, e = 3).
FIGURE 4.
FIGURE 4.
Surface localization of B0AT3 mutants displayed on a homology model. Transport activity of all mutants is indicated by color coding and was determined by flux assays in X. laevis oocytes as described in Fig. 1. Data were taken from Table 2. The homology model structure of mouse B0AT3 was built using the D. melanogaster dopamine transporter DAT x-ray crystal structure in the outward open confirmation (PDB 4m48) (A–C) or LeuT from A. aeolicus in an outward occluded confirmation (PDB 2A65) (D) as a template. Mouse B0AT3 is viewed parallel to the membrane. Mutated residues are shown in van der Waals representation. Wild-type activity is 100% (white); lower activity is displayed in red, and higher activity in blue. A, transmembrane helices 1α, 5, and 7 are colored yellow, orange, and green, respectively. B, homology model is rotated 180° on its vertical axis. C, surface representation of B0AT3 in the same orientation as shown in A. The pocket is flanked by TM domains 5 and 7 and partially occupied by TM1α. Only mutants located in, or peripheral to, TM1α, TM5, or TM7 are labeled. D, hypothesized interaction site between collectrin's TM domain (Met-136–Arg-171) and B0-like transporters. The model is based on the end point of a 10-ns MD simulation (see “Experimental Procedures”). The same face of mouse B0AT3 is shown as in other panels. The transporter is viewed parallel to the membrane with the POPC membrane used during MD simulation removed to see the proteins clearly. Mouse B0AT3 is visualized in gray with collectrin Met-136 to Arg-171 in dark green. The same residues are shown as in C.
FIGURE 5.
FIGURE 5.
Analysis of mutants in a MCT-B0AT3 tandem construct supports a bi-functional role for collectrin. A–E, X. laevis oocytes were injected with the indicated constructs and uptake of 100 μm l-[U-14C]alanine measured over 30 min. Each bar represents the mean ± S.D. activity of n = 10−15 (e = 3). a–d above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. B, surface biotinylation and detection of collectrin and B0AT3 was carried out as indicated under “Experimental Procedures.” Molecular mass markers (in kDa) are indicated on the left, and detected proteins are indicated on the right. No TP = no transporter. F, uptake of 100 μm l-[U-14C]lactate by wild-type and mutated MCT-B0AT3 tandem constructs. Lactate uptake for all conditions was normalized to lactate uptake of wild-type MCT-B0AT3-expressing oocytes from the same batch (i.e. either with or without collectrin). Each bar represents mean ± S.D. No difference at the p < 0.05 was detected for lactate uptake between MCT-B0AT3 WT and any MCT-B0AT3 mutant (n = 10−15, e = 3).
FIGURE 6.
FIGURE 6.
Interactions between B0AT1, collectrin, and syntaxin 1A. Uptake measurements in X. laevis were performed as outlined in Fig. 2. A, cRNA was injected as follows: transporter = 10 ng/oocyte; collectrin = 2 ng/oocyte; syntaxin-1A = 5 ng/oocyte. Uptake of 100 μm l-[U-14C]alanine was measured over 30 min. Each bar represents the mean ± S.D. (n = 10−15, e = 3). a–c (GAT1) and d–f (B0AT1) above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level. B, collectrin cRNA = 2 ng/oocyte B0AT1 cRNA = 10 ng/oocyte. Syntaxin-1A cRNA was titrated as indicated. Surface biotinylation was carried out with 15 oocytes as described under “Experimental Procedures.” Molecular mass markers (in kDa) are added to the left, and the detected proteins are indicated on the right. C, syntaxin-1A cRNA = 5 ng/oocyte B0AT1 cRNA = 10 ng/oocyte. Collectrin cRNA was titrated as indicated. 15 oocytes per sample were treated as in B. D, uptake of 100 μm l-[U-14C]leucine was carried out over 30 min. For each condition, one ancillary protein cRNA was injected at a constant amount, and the other ancillary cRNA was titrated as indicated in the legend. Each data point represents mean ± S.D. (n = 10−15, e = 3). E, cRNA was injected as follows: B0AT1 = 10 ng/oocyte; collectrin = 2 ng/oocyte; all syntaxins = 5 ng/oocyte. Uptake of 100 μm l-[U-14C]leucine was carried out over 30 min. Each bar represents mean ± S.D. (n = 15, e = 3). a–d above the individual bars indicate groupings of conditions whose differences of means are not statistically significant from each other at the p = 0.05 level.
FIGURE 7.
FIGURE 7.
Syntaxin 1A and syntaxin 3 inhibit B0AT1 activity. A, uptake of 100 μm l-[U-14C]leucine was measured over 6 min in CHO parental and CHO-SLC6A19-collectrin cells in either Hanks' buffered saline (Na+) or with NMDG replacing sodium (NMDG). Each bar represents mean transport activity ± S.D. (e = 3, n = 3, ***, p = 0.005 level). p values are calculated as difference from Na+-independent uptake condition. B, immunodetection of B0AT1 and collectrin in whole membrane preparations of CHO parental and CHO-SLC6A19-collectrin cells; 20 μg of total protein was loaded into each well. Molecular mass markers (in kDa) are indicated on the left, and detected proteins are indicated on the right. C, CHO-SLC6A19-collectrin cells were transfected with 3 μg of pcDNA3.1+ vector DNA encoding the indicated murine syntaxin gene (overexpression, left panel) or 50 pmol of siRNA against the CHO endogenous syntaxins (RNAi, right panel). 100 μm l-[U-14C]leucine uptake in CHO-SLC6A19-collectrin cells was measured over 6 min in either Hanks' buffered saline (Na+) or with NMDG replacing sodium (NMDG). The ratio of net sodium-dependent/sodium-independent uptake for each uptake condition was normalized to the pcDNA3.1+ vector-only control (overexpression) or scrambled siRNA (RNAi). Each bar represents mean ± S.E. (n = 3, e = 3, ***, p 0.005 level; **, p 0.01 level; *, p 0.05 level). p values are calculated as difference from control conditions, vector only (overexpression) or scramble (RNAi). D, levels of syntaxin 1A and syntaxin 3 mRNA transcripts were estimated by RT-PCR with 25 cycles of amplification. Closed bars represent control samples using scrambled RNAi, and open bars show the effect of specific RNAi. Samples were run on agarose gel, and quantification was made relative to clathrin mRNA expression in the same sample. Each bar represents mean ± S.E. (e = 3, ***, p 0.005 level). p values are calculated as difference from scramble conditions.
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