Substrate Selectivity and pH Dependence of KAAT1 Expressed in Xenopus laevis Oocytes
Istituto di Fisiologia Generale e di Chimica Biologica, Facoltà di Farmacia, Università di Milano, Via Trentacoste 2, 20134 Milano, Italy. Journal of Membrane Biology
(Impact Factor: 2.46).
05/2000; 174(3):213-24. DOI: 10.1007/s002320001046
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.
Available from: Elena Bossi
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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
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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.
Insect Molecular Biology 09/2002; 11(4):283-9. DOI:10.1046/j.1365-2583.2002.00327.x · 2.59 Impact Factor
Journal of Experimental Biology 01/2003; 206(2):245-254. DOI:10.1242/jeb.00065 · 2.90 Impact Factor
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