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

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).

Figure 1. Ionic selectivity of KAAT1-expressing oocytes

A, membrane currents recorded at −80 mV holding potential in a control oocyte superfused with the indicated solutions; B, same as in A for an oocyte injected with KAAT1 cRNA. Periods of 20 s in TMA⁺ solution separated perfusion with the various alkali ion solutions.

Figure 2. Voltage-dependent currents in control and KAAT1-expressing oocytes

A, recordings of membrane currents (I_m) elicited by voltage steps from V_h=−80 mV to −180, −140, −100, −60, −20 and +20 mV, in a control (left column) and in a KAAT1-expressing oocyte (middle and right columns) bathed in the indicated solutions. B, comparison of the relaxations (step voltage to −140 mV) with and without leucine for K⁺ and Na⁺ at a higher time resolution.

Figure 3. Steady-state I-V relationships and time constants

Control (A) and KAAT1-expressing oocytes (B) in TMA⁺ (□), K⁺ (^) and Na⁺ (▵) external solutions (means ±s.e.m., n = 13). Time constants of decay of the currents relative to the ‘on’ voltage jumps as a function of voltage in control (C) and injected (D) oocytes (same symbols as in A and B).

Figure 4. Effects of leucine on steady-state I-V relationships and time constants

Data from a group of cRNA-injected oocytes in the absence (A) and presence (B) of leucine, bathed in K⁺ (□) and Na⁺ (^) solutions (means ±s.e.m., n = 5). Time constants of decay of the ‘on’ relaxation as a function of voltage in the absence (C) and presence (D) of leucine; same oocytes and same symbols as in A and B.

Figure 5

Additional currents induced by leucine in the presence of the indicated ion (means ±s.e.m., n = 5).

Figure 6. Protocol for the measurement of the effective membrane capacity

A, a small (20 mV) test step was superimposed to variable voltage levels in order to avoid saturation of the large capacitative peaks. B, sample currents elicited by the protocol. C, two traces showing the currents in response to the step from −80 mV from a control (continuous line) and from an injected (dashed line) oocyte, both in high Na⁺ solution.

Figure 7. Membrane effective capacity is ion and voltage dependent in oocytes expressing KAAT1

A, voltage dependence of C_eff in a control oocyte bathed in high Na⁺. B−D: C_eff from the same injected oocyte bathed in the indicated solutions. C_eff was calculated from the integrals of the ‘on’ (□) and ‘off’ (^) transients elicited by the protocol of Fig. 6.

Figure 8. Charge movement in Na⁺

A, I_Na - I_TMA during the 20 mV test pulses as in the protocol shown in Fig. 6A. B, integrals of the ‘on’ (□) and ‘off’ (^) transients from the traces in A, showing good correspondence with each other and a bell-shaped dependence on voltage. C, cumulative integral from B, representing the amount of charge moved at each potential.

Figure 9. Charge movement in K⁺

A, I_K - I_TMA during the 20 mV test pulses as in the protocol shown in Fig. 6A. B, integrals of the ‘on’ (□) and ‘off’ (^) transients from the traces in A, showing good correspondence with each other. C, cumulative integral from B.

Figure 10. Effect of Na⁺ concentration on charge movement

Normalized charge vs. voltage relationships for a group of oocytes bathed in 98 (□), 40 (^), 20 (▵) and 5 (▿) mM Na⁺ (TMA⁺ substitution). Data are means ±s.e.m. from 14 oocytes (two batches).

Figure 11. Calculation of forward and backward rate constants for binding of Na⁺ and K⁺ to the transporter

A, moved charge and B, time constants vs. voltage in 98 mM K⁺ (□) and 98 mM Na⁺(^); C, forward (□) and backward (^) rate constants in 98 mM Na⁺; D, energy profile of the external barrier and binding site calculated as explained in the text; the dashed part is hypothetical. Continuous lines in C are functions for α and β fitted by eye. All data are from a single oocyte.

Figure 12. Correlation between steady-state currents and Q_max

A, coupled (□) and uncoupled (^) currents at −200 mV for 10 different oocytes in 98 mM Na⁺. B, same for K⁺ (Q_max was estimated in Na⁺) in another group of 6 oocytes.

Figure 13. Comparison of charge movement and coupled current in 98 and 5 mM Na⁺

A, Q-V relationships in 98 (□) or 5 mM Na⁺ (TMA⁺ substitution, ^). B, corresponding steady-state currents in the presence of leucine.

Figure 14. Absence of charge movement in the presence of leucine

The traces shown in each column are the results of the subtraction of the currents in the presence of 1 mM leucine and the indicated ion, and the traces in TMA⁺ without leucine in the same oocyte. The ‘on’ and ‘off’ transients visible in Figs 8 and 9 disappear while the steady-state currents increase.