The C-terminal domain of the neutral amino acid transporter SNAT2 regulates transport activity through voltage-dependent processes

Abstract

SNAT (sodium-coupled neutral amino acid transporter) 2 belongs to the SLC38 (solute carrier 38) family of solute transporters. Transport of one amino acid molecule into the cell is driven by the co-transport of one Na(+) ion. The functional significance of the C-terminus of SNAT2, which is predicted to be located in the extracellular space, is currently unknown. In the present paper, we removed 13 amino acid residues from the SNAT2 C-terminus and studied the effect of this deletion on transporter function. The truncation abolished amino acid transport currents at negative membrane potentials (<0 mV), as well as substrate uptake. However, transport currents were observed at positive membrane potentials demonstrating that transport was accelerated while the driving force decreased. Membrane expression levels were normal in the truncated transporter. SNAT2(Del C-ter) (13 residues deleted from the C-terminus) showed 3-fold higher apparent affinity for alanine, and 2-fold higher Na(+) affinity compared with wild-type SNAT2, suggesting that the C-terminus is not required for high-affinity substrate and Na(+) interaction with SNAT2. The pH sensitivity of amino acid transport was retained partially after the truncation. In contrast with the truncation after TM (transmembrane domain) 11, the deletion of TM11 resulted in an inactive transporter, most probably due to a defect in cell surface expression. Taken together, the results demonstrate that the C-terminal domain of SNAT2 is an important voltage regulator that is required for a normal amino acid translocation process at physiological membrane potentials. However, the C-terminus appears not to be involved in the regulation of membrane expression.

Figure 1. Deletion of the C-terminus of SNAT2 inhibits transport current and amino acid uptake

(A) Typical transport currents induced by application of 10 mM alanine to non-transfected (control), SNAT2_{Del C-ter} and SNAT2_WT-expressing cells. The bath solution contained 140 mM NaMes, and the pipette solution contained 140 mM KMes at 0 mV transmembrane potential. (B) Average alanine-induced transport currents (gray bars, left axis) and MeAIB uptake (black bars, right axis) in HEK293 cells transiently transfected with vecter pBK-CMV(Δ(1098–1300)), SNAT2_{Del C-ter}, and SNAT2_WT cDNA. The inset shows the predicted SNAT2 trans-membrane topology and the truncation site.

Figure 2

Confocal microscopy images testing for cellular localization of truncated SNAT2-AcGFP fusion constructs. (A) SNAT2_WT, (B) SNAT2_{Del C-ter}, and (C) SNAT2_{Del TM11}. Panel (D) shows control, non-transfected cells, and (E) is the bright-field control image, showing that cells were present. The arrows indicate regions of intense cell boundary fluorescence. AcGFP was attached in frame to the N-terminus of SNAT2.

Figure 3. The C-terminal deletion does not interfere with substrate binding

Apparent affinities for the substrate L-alanine of SNAT2_WT (A) and SNAT2_{Del C-ter} (B) were determined by recording substrate-inhibited anion leak currents as a function of [alanine] at 0 mV in the presence of 140 mM intracellular KSCN (SNAT2_{Del C-ter}) and 140 mM extracelluar NaSCN (SNAT2_WT).

Figure 4. The deletion of the C-terminus affects the voltage dependence of substrate transport

I-V relationships of L-alanine-induced transport currents at a concentration of 10 mM for SNAT2_{Del C-ter} (A) and SNAT2_WT (B). C, average current-voltage relationships of alanine-induced transport currents in nontransfected cells (control, open circles), SNAT2_{Del C-ter} (solid squares), and SNAT2_WT (solid triangles).

Figure 5. Deletion of the C-terminus decreases, but does not eliminate anion leak current

Voltage dependence of alanine-sensitive currents mediated by SNAT2_{Del C-ter} (A) and SNAT2_WT (B), in the presence of 140 mM intracelluare KSCN and 140 mM extracellular NaMes. 10 mM L-alanine was applied at t = 0 s, as indicated by the bar. C, current-voltage relationships of 10 mM L-alanine-sensitive currents in nontransfected cells (open circles), SNAT2_{Del C-ter} (up triangles), and SNAT2_WT (squares) expressing cells in the presence of 140 mM intracellular KSCN.

Figure 6. SNAT2_{Del C-ter} retains normal apparent affinity for Na⁺

Whole-cell current recordings were performed with a KSCN-based pipette solution (140 mM) and at 0 mV trans-membrane potential. (A), Comparison of typical leak anion currents induced by the application of 140 mM extracellular Na⁺ among non-transfected cells (control), SNAT2_{Del C-ter}, and SNAT_WT -expressing cells, and indicated by the gray bars. (B), Statistical analysis of average Na⁺-induced currents as the ones shown in (A). (C) and (D) show leak anion currents as a function of extracellular [Na⁺] for SNAT2_{Del C-ter} (C) and SNAT_WT (D), respectively (solid squares, after subtracting the unspecific currents, open circles, determined from non-transfected cells). The open squares in (C) and (D) are results from the original experiments before subtraction of the unspecific leak currents (open circles) determined in non-transfected control cells.

Figure 7. Deletion of TM11 results in a non-functional transporter

(A) Transport currents induced by nontransfected cells, SNAT2_WT- and SNAT2_{Del TM11}-expressing cells at 10 mM of alanine and MeAIB uptake by vector (pBK-CMV (Δ[1098–1300])), SNAT2_{Del C-ter} and SNAT2_WT in transiently transfected HEK293 cells. Transport of [¹⁴C]MeAIB (40 μM) was measured at 1 min in NaMes-containing buffer. (B) Voltage-dependence of transport currents induced by SNAT2_{Del TM11} in the presence of 10 mM of alanine in nontranfected cells (open circles), SNAT2_WT- (squares) and SNAT2_{Del TM11}-expressing cells (triangles). (C) Average current-voltage relationships of anion leak current in the presence of 10 mM of alanine among nontransfected cells (contol, open circles), SNAT2_WT- (squares) and SNAT2_{Del TM11}- expressing cells (triangles). (D) Anion leak currents induced by 140 mM of extracellular Na⁺. The open triangles (SNAT2_WT) and open circles (SNAT2_{Del TM11}) are the currents from original experiments before subtraction of the unspecific leak currents (open squares, control, nontransfected cells). The solid circles are the anion leak currents induced by SNAT2_{Del TM11} after subtracting the unspecific currents (open circles). The inset shows the truncation site.