Purchase this article with an account.
Lan Yue, Michal Pawlowski, Karol S. Bruzik, Haohua Qian, David R. Pepperberg; Photo-regulated Activity Of A Tethered Propofol Derivative At GABAA Receptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1166.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
GABAA receptors (GABAARs) shape visual signaling at several stages within the retina. In patients with photoreceptor degenerative diseases, establishing a light sensitivity of native postsynaptic receptors of inner retinal neurons might restore vision by bypassing the deteriorated photoreceptors. We recently described a light-regulated, freely diffusible, GABAAR modulator (MPC088) (Yue et al., 2010 Soc. for Neurosci. meeting). As a prototype approach to anchoring the compound to the receptor, we developed a MPC088 derivative containing thiol-reactive maleimide (MPC100), and tested MPC100 for light-regulated activity at cysteine-substituted GABAARs.
MPC100 was prepared by coupling a maleimide-terminated, 24-mer poly(ethylene glycol) (PEG) to an amino terminus of MPC088. Xenopus laevis oocytes expressing either wildtype α1β2γ2 or α1β2γ2A79C (γ-79C) GABAARs were studied by two-electrode voltage-clamp recording. Cis and trans azobenzene isomers were generated, respectively, by UV and visible (white) light. Typically, MPC100 treatment involved incubation with trans-dominant MPC100 (100 µM) for 7 min, followed by extensive perfusion with Ringer.
Treating γ-79C-expressing oocytes with MPC100 increased the baseline membrane current. This baseline current was sensitive to picrotoxin (a non-competitive GABAAR antagonist), reduced by UV illumination, and restored by visible light. In addition, MPC100 potentiated the GABA-elicited response. This potentiation was reduced by UV and largely restored by subsequent visible light in a manner similar to that displayed by MPC088 on wildtype GABAARs. On γ-79C-expressing oocytes, the effects of MPC100 persisted after prolonged Ringer perfusion, indicating covalent tethering. Furthermore, pre-treatment of γ-79C-expressing oocytes with a thiol-reactive agent blocked MPC100-induced modulation. On oocytes expressing wildtype GABAARs, co-application of MPC100 and GABA enhanced the GABA response, but these cells did not exhibit a persisting effect of MPC100.
Covalently tethering MPC100 to GABAARs directly activates and potentiates receptor activity, and both effects are light-regulated. The results encourage development of GABAAR-directed, MPC088-based structures as a retinal therapeutic device. In addition, studies involving MPC100 treatment of neurons engineered to express γ-79C GABAARs may afford photo-control of other neural circuits.
This PDF is available to Subscribers Only