Conserved tyrosine-147 plays a critical role in the ligand-gated current of the epithelial cation/amino acid transporter/channel CAATCH1.
CAATCH1 functions both as an amino-acid-gated cation channel and as a cation-dependent, proline-preferring, nutrient amino acid transporter in which the two functions are thermodynamically uncoupled. This study focuses on the ionic channel aspect, in which a Tyr(147) (wild type) to Phe(147) (Y147F) site-directed mutation was investigated by steady-state electrophysiological measurements in the Xenopus laevis oocyte expression system. This tyrosine residue is conserved within the third transmembrane domain in members of the Na(+):neurotransmitter transporter family (SNF), where it plays a role in binding pharmacological ligands such as cocaine to the serotonin (SERT), dopamine (DAT) and norepinephrine (NET) transporters. Epithelial CAATCH1 is a member of the SNF family. The results show that amino acid ligand-gating selectivity and current magnitudes in Na(+)- and K(+)-containing media are differentially altered in CAATCH1 Y147F compared with the wild type. In the absence of amino acid ligands, the channel conductance of Na(+), K(+) and Li(+) that is observed in the wild type was reduced to virtually zero in Y147F. In the wild type, proline binding increased conductance strongly in Na(+)-containing medium and moderately in K(+)-containing medium, whereas in Y147F proline failed to elicit any cation currents beyond those of N-methyl-D-glucamine- or water-injected oocytes. In the wild type, methionine binding strongly inhibited inward Na(+) currents, whereas in Y147F it strongly stimulated inward currents in both Na(+) and K(+)-containing media. Indeed, in Na(+)-containing medium, the relative potency ranking for inward current inhibition in the wild type (Met>Leu>Gly>Phe>Thr) was similar to the ranking of ligand-permissive gating of large inward currents in Y147F. In Na(+)-containing medium, current/voltage relationships elicited by ligands in the wild type were complex and reversing, whereas in Y147F they were linear and inwardly rectifying. In K(+)-containing medium, current/voltage relationships remained non-linear in Y147F. Both wild-type and Y147F currents were Cl(-)-independent. Together, these data demonstrate a critical role for Tyr(147) in ligand-binding selectivity and modulation of the ionic channel conductance in CAATCH1. The results support the argument that inhibition of the CAATCH1 conductance by free methionine shares some properties in common with ligand inhibition of DAT, SERT, NET and the gamma-aminobutyric acid transporter (GAT1).
The Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32652, USA.
Bruce R
BR
Stevens
Daniel H
DH
Feldman
Zhilin
Z
Liu
William R
WR
Harvey
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017931:DNA Primers; D006801:Humans; D007150:Immunohistochemistry; D019476:Insect Proteins; D015640:Ion Channel Gating / Q000502:physiology*; D008024:Ligands; D018402:Manduca; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000502:physiology*
C413023:CAATCH1 protein, insect; D002352:Carrier Proteins; D017931:DNA Primers; D019476:Insect Proteins; D008024:Ligands; D008565:Membrane Proteins; D014443:Tyrosine