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K⁺ amino acid transporter KAAT1 mutant Y147F has increased transport activity and altered substrate selectivity

Zhilin Liu^1,3, Bruce R. Stevens², Daniel H. Feldman^2,*, Matthias A. Hediger³ and William R. Harvey^1,2,

¹ The Whitney Laboratory, University of Florida, St Augustine, FL 32080, USA
² Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610, USA
³ Harvard Institutes of Medicine, Harvard Medical School, Boston, MA 02115, USA
^* Present address: Research Department, Shriners Hospital for Children of Northern California, Sacramento, CA 95817, USA.

View larger version (66K): [in a new window]   Fig. 1. Predicted secondary structure of KAAT1 polypeptide, showing location of mutations employed in the present study (blue, mutation; red, location) and conserved tyrosine 147 (yellow Y in lower box) among related transporters.

View larger version (62K): [in a new window]   Fig. 2. Targeting of expressed proteins to Xenopus oocyte plasma membrane 3 days after injection of anti-sense RNA from (A) wild-type (WT) (B) Y147F mutant or (C) water control.

View larger version (12K): [in a new window]   Fig. 3. Uptake of [3H]L-leucine by KAAT1 wild-type (WT) and several mutants (Y147F, R76E and W75L). Accumulated radioactivity was measured in oocytes after 30 min exposure to Na+ media containing 0.2 mmol l-1 [3H]L-leucine. Oocytes had been injected 2-3 days earlier with cRNA encoding W75L, R76E, Y147F, WT or water control.

View larger version (18K): [in a new window]   Fig. 4. Typical amino acid selectivity spectrum for KAAT1 Y147F mutant and wild type (WT; insert) in K+ and Na+ media. Data represent net inward currents evoked by each amino acid in the presence of 100 mmol l-1 cation.

View larger version (16K): [in a new window]   Fig. 5. [3H]L-leucine uptake by KAAT1 (A) Y147F mutant and (B) wild type (WT) in K+ media.

View larger version (12K): [in a new window]   Fig. 6. Typical activation of L-leucine-associated net inward currents as a function of leucine concentration in 100 mmoll-1 K+-containing medium. (A) KAAT1 Y147F mutant and (B) wild type (WT).

View larger version (11K): [in a new window]   Fig. 7. Typical activation of L-leucine-associated net inward current as a function of K+ concentration in 500 µmoll-1 leucine-containing medium. (A) KAAT1 Y147F mutant and (B) wild type (WT).

View larger version (14K): [in a new window]   Fig. 8. Typical Li+-, K+- and Na+-induced currents in mutant Y147F and wild type (WT).

View larger version (19K): [in a new window]   Fig. 9. KAAT1 wild type (WT) uptake at pH 7.5 and pH 10. Upper panel: bars A and B, 0.2 mmoll-1 [3H]-L-leucine at pH 7.5 and pH 10, respectively; C and D, 0.2 mmoll-1 [3H]-L-lysine uptake at pH 7.5 and pH 10, respectively; E, 0.2 mmoll-1 [3H]-L-leucine uptake with 10 mmoll-1 lysine; F, 0.2 mmoll-1 [3H]-L-lysine with 10.0 mmoll-1 leucine; G and H, 10 mmoll-1 [3H]-L-leucine and 10 mmoll-1 [3H]-L-lysine uptake, respectively, by water-injected oocytes. Lower panel: currents measured in KAAT1 cRNA-injected oocytes in Na+-free K+ medium with lysine added (0.2 mmoll-1) and then removed.

View larger version (32K): [in a new window]   Fig. 10. Comparison of cation and amino-acid-associated net inward currents and transport occurring in Na+ medium by Xenopus oocytes injected with full-length cRNA from wild type (WT) and mutants of nutrient and neurotransmitter transporters. (A) In GAT1, WT Na+ and Cl- activate the uptake of -aminobutyric acid (GABA) but not -amino acids, and a water channel is present. The transport is curtailed completely in the equivalent mutant Y140F. (B) In KAAT1, WT Na+ and Cl- activate the uptake of leucine. The co-transport of the amino acids and ions is enhanced more than sevenfold in the mutant Y147F. (C) In CAATCH1 WT, there is no Cl- activation and Na+ fluxes are thermodynamically uncoupled from the transport of proline and other neutral amino acids. In CAATCH1 Y147F mutant, methionine, but not proline, activates the Na+ channel (Stevens et al., 2002). AA, amino acid.