Giovanola M et al. (2018), Threonine 67 is a key component in the coupling...

XB-ART-54592

Biochim Biophys Acta Biomembr 2018 May 01;18605:1179-1186. doi: 10.1016/j.bbamem.2018.01.020.

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Threonine 67 is a key component in the coupling of the NSS amino acid transporter KAAT1.

Giovanola M , Vollero A , Cinquetti R , Bossi E , Forrest LR , Di Cairano ES , Castagna M .

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	Graphical abstract
	Fig. 1. A, B, C, Structure of regions corresponding to Thr 67 in KAAT1, LeuT and hSERT. The predicted binding site region in the homology model of msKAAT1 (A) is compared to the sites in LeuT (PDB code 2A65) and hSERT (PDB code 5I6X, with Na2 from PDB entry 5171). Thr 67 is shown using purple sticks, and other notable binding site residues are shown as sticks using the colors of the helices to which they belong: TM1 (red), TM6 (green), and TM8 (cyan), shown also in cartoon representation. Bound amino acid leucine and paroxetine are shown as gray sticks and paroxetine is semi-transparent, for clarity. Sodium (orange) and chloride ions, where present (green), are shown as spheres. Probable hydrogen-bonds mentioned in the main text are indicated with dashed black lines. For all groups, oxygen and nitrogen atoms are colored red and blue, respectively. A few binding site residues have been omitted for clarity. D, Alignment of transmembrane domain I of some NSS members highlighting Thr 67 of KAAT1 and the corresponding residues in the other proteins.
	Fig. 2. Surface expression of FLAG-tagged KAAT1 WT and mutants. Oocytes expressing the wild-type or the indicated mutants of KAAT1-FLAG were secondarily labelled with peroxidase-conjugated goat anti-mouse (IgG-HRP) after exposure to mouse anti FLAG. The chemiluminescence was detected from 38 to 80 oocytes from 3 different batches (NI:nâ¯=â¯85;WT:nâ¯=â¯54; A66G:nâ¯=â¯55; T67G:nâ¯=â¯38; T67Y:nâ¯=â¯50; S68A:nâ¯=â¯38). The data were normalized to the mean value of the wild-type KAAT1 FLAG of each batch. (*) Statistically significant, Pâ¯<â¯0.05, when compared to all the other transporter expressing condition.
	Fig. 3. Ion dependence of 0.1â¯mM leucine uptake induced by KAAT1 WT and mutants. Data are meansâ¯Â±â¯S.E. of three independent experiments with n ranging from 30 to 40, and are expressed as percentage of the WT induced uptake. (A): Uptake measured in the presence of 100â¯mM NaCl. Insert: Na+-dependence of KAAT1 induced uptake. In the absence of Na+, osmolarity was maintained substituting NaCl with choline chloride. (B) Uptake measured in the presence of 150â¯mM KCl. Insert: uptake measured in non-injected oocytes or in oocytes injected with KAAT1 WT cRNA. Data are meansâ¯Â±â¯SE of a representative experiment with n ranging from 10 to4. (), () Statistically significant, Pâ¯<â¯0.01 when compared with WT or other mutants in the same condition; () Statistically significant, Pâ¯<â¯0.001 when compared with non-injected oocytes.
	Fig. 4. Transport associated currents. Means of the transport currents recorded at â140â¯mV and at â60â¯mV in the presence of threonine (3â¯mM) or leucine (1â¯mM). Values are meansâ¯Â±â¯s.e.m. from several oocytes (nâ¯=â¯from 4 to 25) heterologously expressing KAAT1 WT or the indicated mutants, and with Na+ as driving ion (sodium buffer solution pHâ¯7.5). In the inset the currents generated by T67Y are enlarged, to highlight the reduced currents recorded in threonine. For S68A currents were measured also in the presence of proline (â307.45â¯Â±â¯91.07â¯nA at â140â¯mV and â67.60â¯Â±â¯20.77â¯nA at â60â¯mV; data not reported in the histograms).
	Fig. 5. Current-Voltage relationships of normalized currents. Sodium (left) or potassium buffer solution (right) at pHâ¯7.6 in the presence of threonine and leucine (proline only for S68A). Data were obtained by subtracting the traces recorded in the absence of the indicated substrate from those recorded in its presence, and normalized to the mean value in threonine at â140â¯mV for each batch before averaging. Values are meansâ¯Â±â¯SE from several oocytes (nâ¯=â¯from 4 to 20) in each tested group. Currents were recorded as described in Materials and Methods.
	Fig. 6. Chloride dependence of the transport activity of KAAT1 WT and mutants. (A) Uptake of 0.1â¯mM leucine in the absence of chloride was measured substituting 100â¯mM NaCl with 100â¯mM sodium gluconate. Data are expressed as percentage of the uptake measured in control conditions (i.e. in the presence of 100â¯mM NaCl) and are meansâ¯Â±â¯S.E of three independent experiments with n ranging from 30 to 40. (*) Statistically significant, Pâ¯<â¯0.001 when compared with WT or other mutants. (B) Transport-associated current recorded at â60â¯mV in the presence of leucine, threonine, or proline (only for S68A), in the absence of chloride (substituted by sodium gluconate), expressed as percentage of the currents measured in the control condition (presence of chloride). Data are the meansâ¯Â±â¯S.E from 3 different experiments with n ranging from 3 to 15, according to the different condition.
	Fig. 7. Effects of 10â¯mM PGO (phenylglyoxal) treatment. Leucine uptake was measured before and after treatment with 10â¯mM PGO conducted in the presence of 100â¯mM NaCl or 100â¯mM sodium gluconate. Data are expressed as percentage of the uptake measured in control conditions (i.e. in the absence of PGO) and are meansâ¯Â±â¯S.E of three independent experiments with n ranging from 30 to 40. () Statistically significant, Pâ¯<â¯0.03 when compared with gluconate condition; (*) Statistically significant, Pâ¯<â¯0.01 when compared with WT treated in the presence of chloride.
	Fig. 8. Effects of D-leucine application on L-leucine uptake. A) 0.1â¯mM [3H]-L-leucine uptake induced by KAAT1 WT and the T67Y mutant was measured in the absence or in the presence of 4â¯mM unlabeled D-leucine. Data are meansâ¯Â±â¯S.E. of three independent experiments with n ranging from 30 to 40 and are expressed as percentage of control uptake. (*) Statistically significant, Pâ¯<â¯0.001 when compared with WT. B) Transport current in the presence of D- and L- leucine reported as raw mean valuesâ¯Â±â¯SEM for WT (nâ¯=â¯25) and for T67Y (nâ¯=â¯7). The currents recorded in the presence of D-leucine are larger than the currents recorded in L-leucine for the wild type, while the opposite is true for the T67Y mutant. The inset is an enlargement of the data for T67Y (0.05 level one-way ANOVA).

References [+] :

Abramson, Structure and function of Na(+)-symporters with inverted repeats. 2009, Pubmed