Ion binding and permeation through the lepidopteran amino acid transporterKAAT1 expressed in Xenopus oocytes

1.The transient and steady-state currents induced by voltage jumps in Xenop us 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 control s even in the absence of amino acid substrate (uncoupled current). The sele ctivity order of this uncoupled current was Li+ > Na+ approximate to Rb+ ap proximate to K+ > Cs+; in contrast, the permeability order in noninjected o ocytes 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 (> 10 0 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 Boltzm ann equation having V-1/2 of -69 +/- 1 mV and slope factor of 26 +/- 1 mV; lowering the Na+ concentrations shifted the Q-V relationship to more negati ve potentials; the curves could be described by a generalized Hill equation with a coefficient of 1.6 suggesting two binding sites. The maximal movabl e charge (Q(max)) in Na+, 3 days after injection, was in the range 2.5-10 n C. 5. Addition of the transported substrate leucine increased the steady-state carrier current, the increase being larger in high K+ compared with high N a+ 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).