Pre-steady-state currents in neutral amino acid transporters induced by photolysis of a new caged alanine derivative.

Neutral amino acids are transported across cell membranes by a variety of transport systems [reviewed in (1)]. Some of these systems, which actively transport neutral amino acids against a concentration gradient, are powered by cotransport of Na+ down its own transmembrane concentration gradient. For small neutral amino acids these systems include System N, System A, and System ASC. System N and System A belong to the same solute carrier (SLC) protein family (SLC38) and the known cloned members of this family have collectively been termed sodium-coupled neutral amino acid transporters (SNAT) (2). Members 1, 2 and 4 of this family are thought to belong to the System A class (3, 4), whereas members 3 and 5 belong to the System N class (5, 6). In contrast, System ASC transporters belong to the SLC1 family of transport proteins and the cloned members ASCT1 and ASCT2 have a high degree of sequence similarity with glutamate transporters (7–9). Pre-steady-state kinetic techniques have been used in the past to study details of the transport mechanism of ion pumps (10, 11) and secondary transporters (12–14). One means to obtain pre-steady-state kinetic data is to disturb an existing transporter steady state by application of step changes in the membrane potential and to monitor the relaxation to a new steady state (15). This technique has been widely applied to many types of secondary transporters, including SNAT1 (16). A second method is based on applying concentration jumps of the transported substrate to the carrier while keeping the membrane potential constant, which also leads to relaxation to a new steady state. This method has been used with glutamate (12, 17) and GABA (γ-aminobutyric acid) transporters (18) and has yielded a wealth of new information regarding the transport mechanism. However, rapid reactions of these secondary transporters were found to take place on the sub-millisecond to millisecond time scale (14), which makes substrate concentration jumps difficult to achieve with rapid mixing techniques. The technique of flash photolysis of a photolabile (“caged”) precursor can produce faster concentration jumps, with release of substrates in less than 100 μs being achievable in some cases (19). Obviously, this requires availability of a suitable reagent and hitherto there have been no reports of the synthesis and development of caged neutral amino acids as potential caged substrates for SNATs and ASCTs. Here, we report the synthesis and characterization of a new caged alanine derivative (MNI-alanine 4) that is based on protection with the 4-methoxy-7-nitroindolinyl caging group. The MNI and related nitroindoline caging groups have been used previously to generate caged derivatives of glutamate and glycine (20, 21) and the mechanism and kinetics of product release from these caging groups have been studied. The rate constant for amino acid release is 5 ×106 s−1 (22). We tested MNI-alanine with three neutral amino acid transporters, namely ASCT2, SNAT1 and SNAT2, and found that the compound is biologically inactive with respect to these transport systems. Photolysis of MNI-alanine results in the release of free alanine which activates specific currents in cells expressing ASCT2, SNAT1 or SNAT2. These currents have significant pre-steady-state components, which have not been observed in the past by using slower transport assays.