Functional Characterization of Two Novel Mammalian Electrogenic Proton-dependent Amino Acid Cotransporters

Abstract

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H ؉ -symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H ؉ -symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as ␥-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.

Substrate specificity and transport mode of the proton‐dependent amino acid transporter mPAT2

“…They have an exceptional position among the mammalian amino acid transporters, as they act as electrogenic amino acid/proton symporters. Transport is dependent on the extracellular pH, but is independent of sodium, chloride, and potassium ions [1,2,4,7]. Moreover, PAT1 and PAT2 mediated amino acid influx leads to a pronounced intracellular acidification [2,9] by cotransport of the zwitterionic amino acid substrates and protons with a stoichiometric coupling of 1 : 1 as shown for the model substrate L-proline [2].…”

“…Moreover, PAT1 and PAT2 mediated amino acid influx leads to a pronounced intracellular acidification [2,9] by cotransport of the zwitterionic amino acid substrates and protons with a stoichiometric coupling of 1 : 1 as shown for the model substrate L-proline [2]. Substrates of both transporters are the small apolar amino acids glycine, L-alanine, and L-proline [1,2,[4][5][6][7][8]. Besides these common functional properties, distinct differences became obvious within substrate recognition and electrophysiological characteristics of the two transporters.…”

“…Whereas substrate affinity is dependent on extracellular pH, proton binding affinity to PAT2 is substrate-independent, favouring a sequential binding of proton followed by substrate. Maximal transport currents are substrate-dependent which suggests that the translocation of the loaded carrier to the internal side is the rate-limiting step.Keywords: electrophysiology; functional characterization; proton symporter; substrate recognition; transport mode.The proton/amino acid transporter family PAT (SLC36) has been identified from human, mouse and rat origin during the last three years [1][2][3][4][5][6][7]. The PAT family is comprised of four members (PAT1-PAT4, SLC36A1-4); the PAT proteins consist of around 470-500 amino acids and are thought to represent integral membrane proteins with a > 60% similarity to each other [8].…”

“…The proton/amino acid transporter family PAT (SLC36) has been identified from human, mouse and rat origin during the last three years [1][2][3][4][5][6][7]. The PAT family is comprised of four members (PAT1-PAT4, SLC36A1-4); the PAT proteins consist of around 470-500 amino acids and are thought to represent integral membrane proteins with a > 60% similarity to each other [8].…”

“…PAT1 recognizes, besides L-a amino acids, a number of additional substrates, e.g. b-alanine, c-aminobutyrate (GABA), betaine, D-Ser, and D-Ala [1,2,5,6,9], whereas for the PAT2 transporter only sarcosine has been identified as an additional high affinity substrate beside the normal L-a amino acid substrates [2,10].Recent studies on PAT1 suggest it to play a dual role in mammalian cells [8]. Based on its localization in lysosomal membranes in neurons [1,6] it is considered to act as an export system for amino acids out of lysosomes after their intralysosomal release from proteolytic processes.…”