The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1

Abstract

System L is the major Na(+)-independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineral corticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two-electrode voltage-clamp technique. Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na(+)-independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non-rectifying and non-selective, conducting Li(+) > Cs(+) = Na(+) > K(+). After replacement of Na(+) by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2(+), TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3(+), Gd3(+) and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone-regulated protein kinase sgk-1. The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge-constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected. It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

Figure 1. Characteristics of isoleucine transport in 4F2hc- and LAT1-expressing oocytes

Oocytes were injected with 5 ng 4F2hc cRNA, 5 ng LAT1 cRNA or with 10 ng of a mixture of both cRNAs. Non-injected oocytes were used as controls. A, after 1 day, uptake of ¹⁴C-labelled isoleucine (0.1 mM) was determined in Na⁺-free transport buffer. The mean (±s.d.) uptake activity of seven oocytes is given for each injection. n.i., non-injected. B, uptake of ¹⁴C-labelled isoleucine (0.1 mM) was determined in Na⁺-free transport buffer in the presence of a hundredfold excess of unlabelled amino acid. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment. The uptake activity of non-injected oocytes has been subtracted. MeAIB, methyl-aminoisobutyric acid; BCH, 2-amino-[2,2,1]heptane-2-carboxylic acid; other amino acids indicated by the single letter code; -, no addition. C, efflux of preloaded ¹⁴C-labelled isoleucine in 4F2hc/LAT1-expressing oocytes. Seven oocytes were preloaded with 100 μM [¹⁴C]isoleucine for 10 min. Subsequently, oocytes were washed four times with ice-cold amino acid free incubation buffer and finally suspended in 1 ml transport buffer with (•) or without (○) added isoleucine (1 mM). Samples (100 μl) were removed at the indicated times and radioactivity was determined. The released radioactivity was integrated over time and the values were corrected for the number of oocytes. D, pH dependence of Na⁺-independent [¹⁴C]isoleucine uptake in 4F2hc/LAT1-injected oocytes. Uptake of labelled isoleucine (0.1 mM) was determined in Na⁺-free transport buffer of different pH. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of two experiments shown). The uptake activity of non-injected oocytes has been subtracted.

Figure 2. Expression of 4F2hc/LAT1 induces a cation conductance (I_cat)

Original tracing showing the inhibition of I_cat on replacement of all cations by NMDG. The oocyte was held at a potential of -50 mV (n = 27).

Figure 3. Cation selectivity and pharmacology of I_cat

A, cation selectivity of I_cat in 4F2hc/LAT1-expressing Xenopus oocytes. Voltage ramps were performed from a holding potential of -40 mV to -90 and +40 mV and cation-induced currents were measured as the difference between the current in the presence of the respective cation and the current in the absence of all cations (NMDG). B, cation currents in control oocytes. C, concentration-dependent inhibition of I_cat in 4F2hc/LAT1-expressing Xenopus oocytes by LaCl₃ (IC₅₀ = 20.6 ± 14.5 μM) and GdCl₃ (IC₅₀ = 40.9 ± 6.3 μM) and of Na⁺-independent isoleucine transport by GdCl₃ (IC₅₀ = 2.4 ± 0.4 μM). The IC₅₀ value was calculated by fitting the curves to the data using the equation: I =I_maxC^n_H/(C^n_H+ IC₅₀), where n_H and C are the Hill coefficient and the inhibitor concentration, respectively. I_max is the maximal extrapolated inhibition and IC₅₀ is the concentration needed for half-maximal inhibition. The experiment was performed twice with five oocytes (or seven in the flux studies).

Figure 4. Identification of single cation-selective channels in inside-out excised patches

A, original current traces recorded at different voltages (as indicated) with KCl bath and ND96 pipette solutions. B, I-V relations recorded under control conditions (as in A; ▪) and with 70 mM sodium gluconate/26 mM NaCl pipette solution (▴). Channel amplitudes represent means ±s.e.m. of five inside-out patches. C and D, inhibition of non-selective cation channels by NMDG, with transition from KCl bath solution to NMDGCl solution (C) and transition from NMDGCl to KCl solution (D). The original current traces shown were recorded at +100 mV (upper traces) and -100 mV (lower traces). Four different batches of oocytes were tested.

Figure 5. Amino acid transport and cation channel activity in wild-type and mutated 4F2hc/LAT1 heterodimers

Oocytes were injected with 10 ng of the following cRNA mixtures: h4F2hc/rLAT1, h4F2hc(C109S)/rLAT1 or h4F2hc/rLAT1(C165S); non-injected oocytes were used as controls. After an 8-30 h expression period groups of seven oocytes were removed to determine [¹⁴C]isoleucine uptake (A), as described in the legend to Fig. 1. I_cat was determined simultaneously in oocytes of the same batch (B). Due to the differences in the activity between different experiments, the uptake activity of wild-type (wt) was set to 100 %; absolute transport activities ranged between 20 and 50 pmol (10 min)⁻¹ oocyte⁻¹. The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of three experiments shown). The uptake activity of non-injected oocytes has been subtracted.

Figure 6. Effect of HgCl₂ on amino acid transport and cation channel activity in wild-type and mutated 4F2hc/LAT1 heterodimers

Oocytes were injected with 10 ng of cRNA mixtures of r4F2hc/rLAT1 (•), h4F2hc/rLAT1 (▪) or h4F2hc(C109S)/rLAT1 (□) or were uninjected (control). A, after 1 day expression groups of seven oocytes were removed to determine [¹⁴C]isoleucine uptake in the presence of increasing concentrations of HgCl₂. B, in parallel experiments oocytes were treated for 15 min with 1 μM HgCl₂ and I_cat was determined before (-) and after (+) incubation, in oocytes expressing h4F2hc/rLAT1 (), h4F2hc(C109S)/rLAT1 (□) or in uninjected control oocytes (▪). The mean (±s.d.) uptake activity of seven oocytes is given for each experiment (one of three experiments shown). The uptake activity of non-injected oocytes has been subtracted.

Figure 7. Correlation between the increase in isoleucine uptake activity and I_cat

Oocytes were injected with 10 ng of a mixture of 4F2hc and LAT1 cRNAs or were not injected (control). At the times indicated, groups of seven oocytes were removed to determine [¹⁴C]isoleucine uptake (○) as described in the legend to Fig. 1. I_cat (•) was determined simultaneously in oocytes of the same batch. The mean (±s.d.) uptake activity of seven oocytes is given for isoleucine uptake (one of two different experiments shown). The uptake activity of non-injected oocytes has been subtracted.