Pre-steady-State Currents in Neutral Amino Acid Transporters Induced by Photolysis of a New Caged Alanine Derivative

Abstract

Na + -dependent transmembrane transport of the 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, ASCT1 and 2) and SLC38 families (SNAT1, 2 and 4). 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 and SNAT1 and 2. 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, τ, 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 (τ = 1.6 ms) from previously described pre-steady-state currents generated by applying steps in the membrane potential (τ ~ 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.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). † This work was supported by a grant by the Florida Department of Health (04NIR-07) awarded to CG. 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)(13)(14). One means to obtain pre-steady-state kinetic data is to disturb an existing transporter steady state by a...

Voltage-dependent processes in the electroneutral amino acid exchanger ASCT2

“…5 B in the presence of intracellular K + , a cation that is not thought to interact with ASCT2. Rapid alanine application through photolysis of caged alanine (Zhang et al, 2007) within less than a microsecond (Fig. 5 A; released [alanine], ∼600 µM) resulted in a small transient inward current that rapidly relaxed back to a steady state within ∼10 ms (Fig.…”

“…Control experiments with nontransfected cells demonstrate the absence of current signals upon application of the laser flash. Further control experiments are shown in Zhang et al (2007).…”

“…Laser-pulse photolysis experiments were performed as described previously (Zhang et al, 2007). In brief, the cells were equilibrated with neutral amino acids or methoxynitroindoline (MNI)-caged alanine using a fast solution exchange device.…”

Electrogenic Steps Associated with Substrate Binding to the Neuronal Glutamate Transporter EAAC1

“…Electrogenic Na ϩ binding appears to be a hallmark of many Na ϩ -driven, secondary active transporters and has been also demonstrated for glucose transporters (9), GABA transporters of the SLC6 family (10), amino acid transporters (11), and phosphate transporters (12).…”

Synthesis and photolytic evaluation of a nitroindoline-caged glycine with a side chain of high negative charge for use in neuroscience

“…and glycine 4, 4 L-aspartate 5, 5 and L-alanine 6, 6 which have been applied in various biological systems. Detailed pharmacological evaluation has found that caged glutamates 1 and 2 do not bind to glutamergic receptors prior to photolysis and are therefore suitable reagents for investigations of synaptic transmission.…”