Quantum Dot Conjugatesof GABA and Muscimol: Binding to α1β2γ2 and ρ1GABAA Receptors

摘要

GABAA receptors are ligand-gated ion channels that mediate inhibitory synaptic signaling in the CNS. Fluorescent probes with the ability to target these receptors can provide insights into receptor location, distribution and dynamics in live cells, while revealing abnormalities in their distribution and dynamics that could occur in a variety of diseases. We have developed fluorescent probes of GABAA receptors that are composed of a CdSe/ZnS core–shell nanocrystal (quantum dot; qdot) conjugated to pegylated derivatives of the GABA receptor agonists GABA and muscimol (GABA-qdots and muscimol-qdots, respectively). Quantitative fluorescence imaging was used to analyze the binding activity of these conjugates to α1β2γ2 GABAA and ρ1 GABAA receptors expressed in Xenopus oocytes. The selectivity of these conjugates for α1β2γ2 GABAA and ρ1 GABAA receptors was determined by their ability to compete with the antagonists bicuculline and methyl-(1,2,3,6-tetrahydropyridin-4-yl)phosphinic acid (TPMPA). Both GABA- and muscimol-qdots exhibited robust bindingto both α1β2γ2 and ρ1 GABAA receptors.At α1β2γ2 receptors, pretreatment with bicucullinereduced conjugate binding by ≥8-fold on average, an extentfar exceeding the reduction produced by TPMPA (∼30%). Conversely,at ρ1 receptors, pretreatment with TPMPA inhibited binding by∼10-fold, an extent greatly exceeding the change produced bybicuculline (∼50% or less). These results indicate specificbinding of muscimol-qdots and GABA-qdots to α1β2γ2GABAA and ρ1 GABAA receptors in a mannerthat preserves the respective pharmacological sensitivities of thesereceptors to TPMPA and bicuculline, and encourage the use of qdot-conjugatedneurotransmitter analogs as labeling agents at GABAA receptors.