Synthesis and Photochemistry of Photolabile Derivatives of .gamma.-Aminobutyric Acid for Chemical Kinetic Investigations of the .gamma.-Aminobutyric Acid Receptor in the Millisecond Time Region

Abstract

The gamma-aminobutyric acid (GABA) receptor is an abundant neuronal receptor in the mammalian and invertebrate nervous systems and is associated with an inhibitory chloride ion channel. GABA is the endogenous neurotransmitter for the receptor and can trigger both fast activation and a reversible desensitization of the receptor. A series of photolabile amine-linked o-nitrobenzyl derivatives of GABA were synthesized that photolyze rapidly to release free GABA. The photochemical properties of the GABA precursors were determined; the compounds undergo rapid photolysis, initiated with UV irradiation at 308 nm, and release free GABA on a millisecond time scale. The pH of the photolysis medium affects both the quantum yield and the rate of photolysis. For example, the quantum yield observed for N-(alpha-carboxy-2-nitrobenzyl)-gamma-aminobutyric acid increases from 0.06 at pH 5.0 to 0.1 at pH 10.5, and the half-life for the photolytic reaction decreases from 1.0 to 2.5 ms in the same pH range. Photolysis of the compounds induces rapid onset of transmembrane ion currents in mouse cortical neurons. The potential of the new compounds for use in rapid chemical kinetic investigations of the neuronal GABA receptor is demonstrated.

Site-specific, photochemical proteolysis applied to ion channels in vivo

“…Given the established photochemistry of the 2-nitrobenzyl group (8)(9)(10)(11)(12)(13)(14)(15)(16)) and the well characterized Shaker-IR phenotype (26)(27)(28)(29)(30)(31)(32)(33)(34)(35), these results provide convincing evidence that irradiation of Npg leads to peptide backbone cleavage of functional ion channels in vivo. Photolysis of the Npg-containing channels resulted in the liberation of the inactivation ball from the remainder of the channel, thereby slowing inactivation.…”

“…The long irradiation times required here indicate that Npg photochemistry may be less efficient than in other related systems (8)(9)(10)(11)(12)(13)(14)(15)(16). Additional work from our labs establishes that more conventional nitrobenzyl caging groups can undergo very efficient photolysis when incorporated into ion channel proteins expressed in Xenopus oocytes (J. Miller, S. K. Silverman, and P.M.E., unpublished work).…”

“…Compounds of this type, including Npg itself, have been used as protecting groups in organic synthesis and to produce ''caged'' neurotransmitters, ions, and second messengers that can then be liberated photochemically, but, to our knowledge, Npg has not been incorporated into peptides or proteins (8)(9)(10)(11)(12)(13)(14)(15)(16)(17). As shown in Fig.…”

The free radical site in pea seedling copper amine oxidase probed by resonance Raman spectroscopy and generated by photolysis of caged substrate

“…Measurements over the range of 15-55°C gave linear Arrhenius plots and activation energies of 40 + 2 kJ mol -~ for both compounds. These uncaging rates are approximately an order of magnitude faster than those for N-(2-nitrobenzyl) cages of glycine [14], glutamine [15] and 7-aminobutyric acid [16], but slower than for the uncaging of N-(1-(2-nitrophenyl)ethyl)carbamoylcholine iodide [13]. The activation energy for decay of the aci-nitro intermediates of I and II is slightly smaller than the value of 55 kJ mol -] measured for caged ATP [11] under similar conditions.…”

“…'Caged' compounds have a functional group modified with a suitable photoremovable protecting or caging group, such as the 2-nitrophenyl substituent [13]. Rates of uncaging of 2-nitrobenzyl-substituted molecules, including amines, via aci-nitro intermediates vary widely [13][14][15][16] but in some cases occur on a sub-millisecond timescale, enabling kinetic measurements to be undertaken on timescales similar to or faster than those using the stopped flow technique. We describe steady-state resonance Raman spectroscopy of the free radical site in pea seedling copper amine oxidase (PSAO).…”