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The Journal of Experimental Biology 206, 245-254 (2003)
doi: 10.1242/jeb.00065

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.

Author for correspondence (e-mail: wharvey{at}whitney.ufl.edu)

Accepted 10 October 2002

KAAT1, a K⁺-coupled, neutral amino acid transporter from larval insect midgut, differs from other members of the Na⁺:neurotransmitter transporter family (SNF) in two important ways: (1) it transports nutrient L-, -amino acids, rather than neurotransmitters such as -aminobutyric acid (GABA), and (2) it accepts K⁺ as well as Na⁺ as a co-substrate. To determine whether the restoration of KAAT1 residues to their GABA transporter GAT1 cation-binding equivalents might abolish its K⁺ but not its Na⁺ recognition site, we constructed a multiple mutant in which nine divergent KAAT1 residues were mutated back to the conserved form of the superfamily. To investigate the amino-acid-binding site, we constructed several single mutants that had been identified in GAT1. Wild-type (WT) or mutant cRNA was injected into Xenopus oocytes and the effects of external amino acids and ions upon labeled leucine uptake and substrate-induced currents were examined.

The multiple mutant exhibited no amino-acid-induced currents, indicating that one or more of the mutated residues are crucial for function. W75L and R76E mutations in the first transmembrane helix of KAAT1 led to results equivalent to those observed in the corresponding mutants of GAT1; namely, substrate (leucine) uptake and substrate-evoked net inward current were severely curtailed. The KAAT1 A523S mutant, which corresponds to a serotonin transporter mutant that is thought to render Li⁺ equivalent to Na⁺ as a co-transported ion, functioned no differently to WT.

The effects of mutation Y147F in the third transmembrane helix of KAAT1 were dramatically different from the equivalent mutation, Y140F, in GAT1. Although kinetic characteristics, expression levels and plasma membrane localization were all similar in Y147F and WT, the Y147F mutant exhibited a sevenfold increase in labeled leucine uptake by Xenopus oocytes in Na⁺ buffer. This increase is in sharp contrast to the complete loss of uptake activity in the GAT1 Y140F mutant. KAAT1 Y147F also differed from WT in cation selectivity and substrate spectrum, as revealed by amino-acid-induced net inward currents that were measured with a two-electrode voltage clamp.

Amino-acid-independent currents induced by Li⁺ and Na⁺ chloride salts were observed in both WT and the Y147F mutant. The Li⁺-induced current was 30% higher in Y147F than in WT, whereas no substrate-independent K⁺-induced currents above control levels were detected either in WT or Y147F. These results suggest that transport of K⁺, the physiological co-substrate in insect midgut, is tightly coupled to that of amino acids in KAAT1, in contrast to the independence of cation and amino acid transport in the closely related cation amino acid transporter channel, CAATCH1.

Key words: CAATCH1, KAAT1, GAT1, potassium, sodium, amino acid, transporter, leakage current

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