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Ion binding and permeation through the lepidopteran amino acid transporter KAAT1 expressed in Xenopus oocytes
Author(s) -
Bossi Elena,
Centinaio Elena,
Castagna Michela,
Giovannardi Stefano,
Vincenti Sergio,
Sacchi V. Franca,
Peres Antonio
Publication year - 1999
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1999.729ab.x
Subject(s) - reversal potential , chemistry , xenopus , voltage clamp , amino acid , ion , steady state (chemistry) , tetramethylammonium , biophysics , analytical chemistry (journal) , membrane potential , stereochemistry , patch clamp , biochemistry , chromatography , biology , receptor , organic chemistry , gene
1 The transient and steady‐state currents induced by voltage jumps in Xenopus oocytes expressing the lepidopteran amino acid co‐transporter KAAT1 have been investigated by two‐electrode voltage clamp. 2 KAAT1‐expressing oocytes exhibited membrane currents larger than controls even in the absence of amino acid substrate (uncoupled current). The selectivity order of this uncoupled current was Li + > Na + ≈ Rb + ≈ K + > Cs + ; in contrast, the permeability order in non‐injected oocytes was Rb + > K + > Cs + > Na + > Li + . 3 KAAT1‐expressing oocytes gave rise to ‘pre‐steady‐state currents’ in the absence of amino acid. The characteristics of the charge movement differed according to the bathing ion: the curves in K + were strongly shifted (> 100 mV) towards more negative potentials compared with those in Na + , while in tetramethylammonium (TMA + ) no charge movement was detected. 4 The charge‐voltage ( Q–V ) relationship in Na + could be fitted by a Boltzmann equation having V ½ of −69 ± 1 mV and slope factor of 26 ± 1 mV; lowering the Na + concentrations shifted the Q–V relationship to more negative potentials; the curves could be described by a generalized Hill equation with a coefficient of 1.6, suggesting two binding sites. The maximal movable charge ( Q max ) in Na + , 3 days after injection, was in the range 2.5–10 nC. 5 Addition of the transported substrate leucine increased the steady‐state carrier current, the increase being larger in high K + compared with high Na + solution; in these conditions the charge movement disappeared. 6 Applying Eyring rate theory, the energy profile of the transporter in the absence of organic substrate included a very high external energy barrier (25.8 RT units) followed by a rather deep well (1.8 RT units).