Characterizing the Architecture of Nicotinic Receptors with Quantum Dot-Based Fluorescence Microscopy

Ion channel localization and trafficking is important for regulating excitability and synaptic transmission. Quantum dots (Qdots) coated with streptavidin were previously used to label membrane proteins that are recognized by biotinylated antibodies or by biotinylation of an acceptor peptide sequence. We report strategies, suitable for living cells, using strepatavidin-coated Qdots to count and locate extracellular domains of muscle and neuronal nicotinic acetylcholine receptors (nAChRs). Receptors were expressed in Xenopus oocytes. (1) The nonsense suppression methodology was used to incorporate the unnatural amino acid biocytin in the muscle nAChR α subunit, in the main immunogenic region, in place of the Asp70 residue. To accomplish this, the T. thermophila Gln amber suppressor (TQAS) was chemically aminoacylated with biocytin and co-injected with α70UAG:β:δ:γ mRNA. Functional expression was measured 24 - 48 hours post-injection. The muscle nAChR stoichiometry is (α)2(β)1(δ)1(γ/ɛ)1; in agreement, two colocalized Qdots were measured by blinking analysis with previously reported algorithms (Pantoja et al, Biophys J in press). (2) The muscle nicotinic receptor was labeled with α-bungarotoxin monoconjugated to biotin (α-Btx-Bio) and subsequently exposed to Qdots; some receptors exhibited the expected two Qdots. (3) The homopentameric α7 nAChR was labeled with α-Btx-Bio and subsequently labeled with Qdots. One to 3 Qdots per α7 receptor were detected, as expected from previous data. In all cases, the robust Qdot fluorescence enabled subunit localization with nanometer accuracy. Strategies (2) and (3) confirm that strategy (1), site-specific unnatural amino acid incorporation combined with Qdot labeling, provides a one-step, specific, efficient labeling approach to investigate composition and real-time trafficking of nicotinic receptors. Grants: NS11756, NS34407, HL79350. Fellowships: Ford and APA-DPN (RP), NSF (EAR).