Förster resonance energy transfer (FRET) correlates of altered subunit stoichiometry in cys-loop receptors, exemplified by nicotinic α4ß2.

We provide a theory for employing Förster resonance energy transfer (FRET) measurements to determine altered heteropentameric ion channel stoichiometries in intracellular compartments of living cells. We simulate FRET within nicotinic receptors (nAChRs) whose α4 and ß2 subunits contain acceptor and donor fluorescent protein moieties, respectively, within the cytoplasmic loops. We predict FRET and normalized FRET (NFRET) for the two predominant stoichiometries, (α4)(3)(ß2)(2)vs. (α4)(2)(ß2)(3). Studying the ratio between FRET or NFRET for the two stoichiometries, minimizes distortions due to various photophysical uncertainties. Within a range of assumptions concerning the distance between fluorophores, deviations from plane pentameric geometry, and other asymmetries, the predicted FRET and NFRET for (α4)(3)(ß2)(2) exceeds that of (α4)(2)(ß2)(3). The simulations account for published data on transfected Neuro2a cells in which α4ß2 stoichiometries were manipulated by varying fluorescent subunit cDNA ratios: NFRET decreased monotonically from (α4)(3)(ß2)(2) stoichiometry to mostly (α4)(2)(ß2)(3). The simulations also account for previous macroscopic and single-channel observations that pharmacological chaperoning by nicotine and cytisine increase the (α4)(2)(ß2)(3) and (α4)(3)(ß2)(2) populations, respectively. We also analyze sources of variability. NFRET-based monitoring of changes in subunit stoichiometry can contribute usefully to studies on Cys-loop receptors.

Probing the non-canonical interface for agonist interaction with an α5 containing nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (nAChRs) containing the α5 subunit are of interest because genome-wide association studies and candidate gene studies have identified polymorphisms in the α5 gene that are linked to an increased risk for nicotine dependence, lung cancer, and/or alcohol addiction. To probe the functional impact of an α5 subunit on nAChRs, a method to prepare a homogeneous population of α5-containing receptors must be developed. Here we use a gain of function (9') mutation to isolate populations of α5-containing nAChRs for characterization by electrophysiology. We find that the α5 subunit modulates nAChR rectification when co-assembled with α4 and ß2 subunits. We also probe the α5-α4 interface for possible ligand-binding interactions. We find that mutations expected to ablate an agonist-binding site involving the α5 subunit have no impact on receptor function. The most straightforward interpretation of this observation is that agonists do not bind at the α5-α4 interface, in contrast to what has recently been demonstrated for the α4-α4 interface in related receptors. In addition, our mutational results suggest that the α5 subunit does not replace the α4 or ß2 subunits and is relegated to occupying only the auxiliary position of the pentameric receptor.

Nicotine exploits a COPI-mediated process for chaperone-mediated up-regulation of its receptors.

Chronic exposure to nicotine up-regulates high sensitivity nicotinic acetylcholine receptors (nAChRs) in the brain. This up-regulation partially underlies addiction and may also contribute to protection against Parkinson's disease. nAChRs containing the α6 subunit (α6* nAChRs) are expressed in neurons in several brain regions, but comparatively little is known about the effect of chronic nicotine on these nAChRs. We report here that nicotine up-regulates α6* nAChRs in several mouse brain regions (substantia nigra pars compacta, ventral tegmental area, medial habenula, and superior colliculus) and in neuroblastoma 2a cells. We present evidence that a coat protein complex I (COPI)-mediated process mediates this up-regulation of α6* or α4* nAChRs but does not participate in basal trafficking. We show that α6ß2ß3 nAChR up-regulation is prevented by mutating a putative COPI-binding motif in the ß3 subunit or by inhibiting COPI. Similarly, a COPI-dependent process is required for up-regulation of α4ß2 nAChRs by chronic nicotine but not for basal trafficking. Mutation of the putative COPI-binding motif or inhibition of COPI also results in reduced normalized Förster resonance energy transfer between α6ß2ß3 nAChRs and εCOP subunits. The discovery that nicotine exploits a COPI-dependent process to chaperone high sensitivity nAChRs is novel and suggests that this may be a common mechanism in the up-regulation of nAChRs in response to chronic nicotine.