Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative.
Na+-Dependent transmembrane transport of small neutral amino acids, such as glutamine and alanine, is mediated, among others, by the neutral amino acid transporters of the solute carrier 1 [SLC1, alanine serine cysteine transporter 1 (ASCT1), and ASCT2] and SLC38 families [sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4]. Many mechanistic aspects of amino acid transport by these systems are not well-understood. Here, we describe a new photolabile alanine derivative based on protection of alanine with the 4-methoxy-7-nitroindolinyl (MNI) caging group, which we use for pre-steady-state kinetic analysis of alanine transport by ASCT2, SNAT1, and SNAT2. MNI-alanine has favorable photochemical properties and is stable in aqueous solution. It is also inert with respect to the transport systems studied. Photolytic release of free alanine results in the generation of significant transient current components in HEK293 cells expressing the ASCT2, SNAT1, and SNAT2 proteins. In ASCT2, these currents show biphasic decay with time constants, tau, in the 1-30 ms time range. They are fully inhibited in the absence of extracellular Na+, demonstrating that Na+ binding to the transporter is necessary for induction of the alanine-mediated current. For SNAT1, these transient currents differ in their time course (tau = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (tau approximately 4-5 ms), indicating that they are associated with a fast, previously undetected, electrogenic partial reaction in the SNAT1 transport cycle. The implications of these results for the mechanisms of transmembrane transport of alanine are discussed. The new caged alanine derivative will provide a useful tool for future, more detailed studies of neutral amino acid transport.
T10 T11 T12 T13 T14 T15 T16 T17 T18 T19 T1 T20 T21 T22 T23 T24 T25 T26 T27 T28 T29 T2 T30 T31 T32 T33 T34 T35 T36 T37 T38 T39 T3 T40 T41 T42 T43 T44 T45 T46 T4 T5 T6 T7 T8 T9
CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL GENE-Y GENE-Y GENE-N GENE-Y CHEMICAL GENE-N GENE-N GENE-Y GENE-Y GENE-Y GENE-N GENE-Y GENE-Y GENE-Y GENE-Y CHEMICAL GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-Y GENE-N CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL CHEMICAL
serine cysteine sodium amino acid amino acid alanine alanine 4-methoxy-7-nitroindolinyl amino acids MNI Na+ alanine glutamine alanine alanine amino acid alanine ASCT2 SNAT1 neutral amino acid transporters SNAT1 Na+ solute carrier 1 SLC1 alanine serine cysteine transporter 1 ASCT1 ASCT2 SLC38 sodium-coupled neutral amino acid transporter 1 SNAT1 SNAT2 SNAT4 Na+ ASCT2 SNAT1 SNAT2 ASCT2 SNAT1 SNAT2 neutral amino acid transporters alanine amino acid alanine alanine amino acid alanine