Figure 1.

Cell line and assay for high throughput screening and inhibitor characterization. (A) Isoleucine transport in CHO parental cells was entirely Na⁺‐independent. Uptake of [¹⁴C]isoleucine (100 μM) was inhibited by 10 mM phenylalanine and serine, but not by arginine. Glycine was a partial inhibitor. Transport activity is shown for the Na⁺‐independent component of total uptake (−Na), and the net Na⁺‐dependent component of total uptake (+Na(net)) (n = 5). (B) CHO parental cells stably transfected with collectrin and B⁰AT1 (CHO‐BC) showed robust net Na⁺‐dependent isoleucine transport (+Na(net)), which was undetectable in the parental cells. Na⁺‐dependent uptake of [¹⁴C]isoleucine (100 μM) was inhibited by 10 mM phenylalanine and serine, but not by arginine. Glycine was a partial inhibitor, consistent with uptake being mediated by B⁰AT1 (n = 5). Please note the similar substrate specificity of the endogenous Na⁺‐independent transporter. (C) Uptake of [¹⁴C]isoleucine (100 μM) was measured in the presence and absence of Na⁺‐ions in CHO‐BC cells. Net Na⁺‐dependent uptake of isoleucine increased proportional to time over 10 min. Na⁺‐independent uptake was rapid initially, but equilibrated quickly (n = 5). (D) A membrane potential sensitive dye was used to detect Na⁺‐amino acid cotransport by B⁰AT1. In parental CHO cells, addition of 1.5 mM isoleucine (arrow) did not change the fluorescence (n = 5), while a large increase of fluorescence (depolarisation) was observed in CHO‐BC cells (n = 10). (E) Addition of 1.5 mM leucine generated a small signal in parental cells (n = 5) and a large signal CHO‐BC cells (n = 35). (F) Cell depolarisation was not only amino acid dependent, but also Na⁺‐dependent (n = 5). (G) Surface biotinylation was used to isolate membrane proteins followed by SDS‐PAGE and immunoblotting to detect collectrin and B⁰AT1. Both proteins were only detectable in CHO‐BC cells, Na,K‐ATPase served as a loading control (n = 5). Molecular weight in kDa is indicated next to the blot. *P < 0.05, significantly different from parental cells (A); n refers to the number of independent experimental repeats for each experimental group.