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Conserved tyrosine-147 plays a critical role in the ligand-gated current of the epithelial cation/amino acid transporter/channel CAATCH1

Bruce R. Stevens¹, Daniel H. Feldman^1,*, Zhilin Liu² and William R. Harvey^2,

¹ The Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32652, USA
² The Whitney Laboratory, University of Florida, 9505 Ocean Shore Boulevard, St Augustine, FL 32080, USA
^* Present address: Shriners Hospital for Children of Northern California, Sacramento, CA 98517, USA

View larger version (61K): [in a new window]   Fig. 1. CAATCH1 (TC 2.A.22.2.4 transporter superfamily classification), Na+:neurotransmitter transporter family members (SNF; TC 2.A.22) all possess a conserved tyrosine residue (filled circles). (A) Location of Tyr147 in the third transmembrane domain of CAATCH1. The predicted secondary structure is adapted from Feldman et al. (2000). (B) Alignment of third transmembrane domain residues of CAATCH1 with some representative members of the SNF family showing a universally conserved tyrosine residue representing Tyr147 in CAATCH1 (http://ca.expasy.org/cgi-bin/blast.pl?sequence=Q9U5A9 ).

View larger version (104K): [in a new window]   Fig. 2. Immunohistochemistry of CAATCH1 wild type (WT) and mutant Y147F expressed in Xenopus laevis oocytes. (A) Confocal scanning micrographs of the outer membrane surface of oocytes expressing CAATCH1 (WT or Y147F) and a water-injected control. (B) Cross sections of intact oocytes from A were obtained by confocal microscopy scans focused using depths of field of 2 µm.

View larger version (23K): [in a new window]   Fig. 3. Voltage-dependence of CAATCH1 steady-state currents in the absence of free amino acid ligands. Net total currents measured in representative oocytes expressing WT or Y147F were obtained without amino acids in the presence of 100 mmol l-1 Na+ (open squares), K+ (filled diamonds), Li+ (filled triangles) or NMG+ (open triangles). (A) Total WT currents (adapted from Feldman et al., 2000). (B) Net cation-specific WT-dependent currents obtained by subtracting the NMG+ currents in A at each voltage. (C) Total Y147F currents. (D) Net cation-specific Y147F-dependent currents obtained by subtracting the NMG+ currents in C at each voltage. Vm, membrane potential; I, current. The experiment was repeated 3 times.

View larger version (31K): [in a new window]   Fig. 4. Representative current/voltage (I/V) relationships elicited by free amino acids in 100 mmol l-1 Na+/0 mmol l-1 K+ medium. The I data represent total currents obtained by subtracting control I/V data from the I/V data in the presence of 0.5 mmol l-1 amino acids for (A) CAATCH1 WT and (B) CAATCH1 Y147F. For A, in the case of methionine, leucine, phenylalanine and glycine, the blockade of inward current is represented by inverted I/V data as a result of the subtraction protocol representing the algebraic inverse of the inhibited current. Vm, membrane potential.

View larger version (29K): [in a new window]   Fig. 5. Representative current/voltage (I/V) relationships elicited by free amino acids in 100 mmoll-1 Na+ medium. The I data represent total currents obtained by substracting control I/V data from the I/V data in the presence of 0.5 mmoll-1 amino acids for (A) CAATCH1 WT and (B) CAATCH1 Y147F. In B, data were obtained using the same oocyte as Fig. 3C,D, demonstrating that currents elicited by Gly, Pro and Phe were similar to those in the presence of NMG+ alone. Vm, membrane potential.

View larger version (17K): [in a new window]   Fig. 6. Net change in currents (I) elicited by 0.5 mmoll-1 free amino acids in 100 mmoll-1 (A) Na+- or (B) K+-containing medium obtained at a holding potential of -60 mV. Negative values represent blockade of net inward current, as described in Materials and methods. Values were normalized to the CAATCH1 WT preferences (Feldman et al., 2000; Quick and Stevens, 2001) for gating amino acids in Na+-containing (Pro) or K+-containing (Thr) medium. KAAT1 WT values were also measured for comparison using the same electrophysiological apparatus. Values are means + S.E.M., N=3.

View larger version (42K): [in a new window]   Fig. 7. Cl--independence of amino-acid-elicited inward currents of WT or Y147F in 100 mmoll-1 Na+- or K+-containing medium. In all media, Cl- salts were replaced by gluconate salts. There was no significant differences (P0.05) between currents in Cl-- and gluconate-containing media. Values are means + S.E.M., N=3.

View larger version (37K): [in a new window]   Fig. 8. Schematic representation of amino acid ligand-gating of cationic channel conductance in CAATCH1 (Tyr147) and the Y147F (Phe147) mutant. (A) Endogenous WT currents in the absence of ligands. (B) No Y147F current in the absence of ligands. (C) Increased WT currents gated by extracellular L-proline. (D) No Y147F current gated by extracellular L-proline. (E) WT Na+ inward current blocked by extracellular L-methionine. (F) Y147F currents enhanced by extracellular L-methionine.

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