Ion binding and permeation through the lepidopteran amino acid transporter KAAT1 expressed in Xenopus oocytes

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. 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 + . 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. 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. 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. 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).