Abstract
CAATCH1 (cation-amino acid transporter/channel) is a recently cloned insect epithelial membrane protein related to mammalian Na(+)-, Cl(-)-coupled neurotransmitter transporters (Feldman, D. H., Harvey, W. R., and Stevens, B. R. (2000) J. Biol. Chem. 275, 24518-24526). In the present study we analyze the relationship between CAATCH1-mediated amino acid transport and ion fluxes by utilizing the Xenopus oocyte expression system in conjunction with electrophysiology and radiotracer uptake. Simultaneous flux measurements reveal that electrical currents and amino acid transport are thermodynamically uncoupled. This observation is supported by measuring significant uptake even in the absence of external alkali cations. Remarkably, CAATCH1-associated Na(+) or K(+) currents are large and do not saturate with voltage nor with cation concentration. These currents reverse in Nernstian fashion, thereby conferring channel activity in CAATCH1. Upon step-changes in the membrane potential, CAATCH1-expressing oocytes exhibit transient currents. Detailed analyses of these transients in the absence and presence of amino acids reveal direct ligand-protein interaction, demonstrating that binding by different amino acids (e.g. proline, threonine, methionine) differentially affects the state probability of CAATCH1 but has no effect on the maximal charge movement (Q(max)). Together these data suggest that CAATCH1 is a multifunction membrane protein that mediates thermodynamically uncoupled amino acid uptake but functions predominantly as an amino acid-gated alkali cation channel.
Highlights
CAATCH1 is a 70kDa membrane protein recently cloned from Manduca sexta midgut [1]
Unlike L-proline, which increased the magnitude of the inward currents, significant uptake was observed for L-threonine and L-methionine, even though these amino acids inhibited (L-methionine) or slightly increased (L-threonine) the currents at a comparable test potential under voltage-clamped conditions (Ϫ30 mV, see Fig. 2, A and B)
In the present study we exploit the Xenopus oocyte expression system in conjunction with electrophysiological and flux measurements to analyze the relationship between CAATCH1associated amino acid transport and electrical currents
Summary
CAATCH1 (cation-amino acid transporter/channel) is a 70kDa membrane protein recently cloned from Manduca sexta midgut [1]. Unlike L-proline, which increased the magnitude of the inward currents, significant uptake was observed for L-threonine and L-methionine, even though these amino acids inhibited (L-methionine) or slightly increased (L-threonine) the currents at a comparable test potential under voltage-clamped conditions (Ϫ30 mV, see Fig. 2, A and B).
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