When expressed in Xenopus oocytes KAAT1 increases tenfold the transport of l-leucine. Substitution of NaCl with 100 mm LiCl, RbCl or KCl allows a reduced but significant activation of l-leucine uptakes. Chloride-dependence is not strict since other pseudohalide anions such as thyocyanate are accepted. KAAT1 is highly sensitive to pH. It can transport l-leucine at pH 5.5 and 8, but the maximum uptake has been observed at pH 10, near to the physiological pH value, when amino and carboxylic groups are both deprotonated. The pH value mainly influences the V(max) in Na(+) activation curves and l-leucine kinetics. The kinetic parameters are K(mNa) = 4.6 +/- 2 mm, V(maxNa) = 14.8 +/- 1.7 pmol/oocyte/5 min for pH 8.0 and K(mNa) = 2. 8 +/- 0.7 mm, V(maxNa) = 31.3 +/- 1.9 pmol/oocyte/5 min for pH 10.0. The kinetic parameters of l-leucine uptake are: K(m) = 120.4 +/- 24. 2 microm, V(max) = 23.2 +/- 1.4 pmol/oocyte/5 min at pH 8.0 and K(m) = 81.3 +/- 24.2 microm, V(max) = 65.6 +/- 3.9 pmol/oocyte/5 min at pH 10.0. On the basis of inhibition experiments, the structural features required for KAAT1 substrates are: (i) a carboxylic group, (ii) an unsubstituted alpha-amino group, (iii) the side chain is unnecessary, if present it should be uncharged regardless of length and ramification.
[Show abstract][Hide abstract] ABSTRACT: The effects of pH on the different kinds of currents occurring at the lepidopteran amino acid cotransporter KAAT1 have been examined using heterologous expression in Xenopus oocytes and voltage clamp. Acidic pH (5.5-4.5) caused a slight depression of the uncoupled current and a complete inhibition of the coupled and transient currents, in the presence of either Na+ or K+, the two ions physiologically relevant to the transport process. Conversely, at alkaline pH (9) no statistically significant effects could be observed on uncoupled, coupled and transient currents compared to the effects at pH 7.6. These effects of pH indicate that operation of the transporter is maximal in the physiologically alkaline native environment. The dose-response curves for the inhibition of coupled and transient currents were similar, with respective pKa values of 6. 29 +/- 0.05 and 6.40 +/- 0.03 and respective Hill coefficient values (nH) of 0.93 +/- 0.07 and 1.08 +/- 0.08, suggesting that the two effects can be explained by a single proton binding to the same site in the transporter.
The Journal of Physiology 06/2000; 525 Pt 1(1):83-9. DOI:10.1111/j.1469-7793.2000.t01-1-00083.x · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phenylglyoxal (PGO), an arginine-modifying reagent, is an irreversible inhibitor of KAAT1-mediated leucine transport, expressed in Xenopus oocytes. The PGO effect was dose-dependent and 5 mm PGO determined a V(max) reduction to 24% of the control, consistent with the covalent binding to transporter arginine residues not located in the leucine binding site. The use of labelled [(14)C]PGO confirmed that the inhibitor binds KAAT1. The protein membrane domain contains seven arginine residues one of which, arginine 76, is conserved in the family of GABA transporters. Using site-directed mutagenesis we showed that only arginine 76 is crucial for KAAT1 activity and is involved in PGO binding.
[Show abstract][Hide abstract] ABSTRACT: KAAT1 is a neutral amino acid transporter activated by K+ or by Na+ (9). The protein shows significant homology with members of the Na+/Cl--dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20-30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of Vmax and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na+ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1.
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