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

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Abstract
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+ approximately Rb+ approximately 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 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 (Qmax) 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).