Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

Abstract

The functional properties and cellular localization of the human neuronal ␣7 nicotinic acetylcholine (AcCho) receptor (␣7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mut␣7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wt␣7 receptors decay much faster than those elicited by the mut␣7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated ␣7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable ''run-down'' of the AcCho currents generated by mut␣7-GFP receptors, whereas those of the wt␣7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mut␣7-GFP oocytes was accompanied by a marked decrease of ␣-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wt␣7 and mut␣7 receptors provides powerful tools to study the distribution and function of ␣7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. F luorescent tagging of cellular proteins is a very useful, and aesthetically pleasant, method for studying the intracellular traffic and distribution of many proteins, including neurotransmitter receptors. It is commonly assumed that the tagged proteins function in exactly the same way as the native nontagged proteins. To determine whether that is the case for neurotransmitter receptors, we chose to study two human receptors that are homomeric: the GABA1 (1) and now the ␣7 nicotinic receptors.The neuronal ␣7 nicotinic acetylcholine receptor (␣7-AcChoR) is widely expressed in the central nervous system and, being located mainly at nerve terminals, it is believed to be involved in regulating the neurotransmitter release that mediates fast cholinergic neurotransmission (2, 3). In heterologous expression systems, including the Xenopus oocyte, the ␣7-subunit forms functional homomeric AcChoRs that show an unusually fast desensitization and high permeability to Ca 2ϩ (4-8). Even though it is not yet clear to what extent the ␣7 homomeric composition also applies in vivo, we and others have found it of interest to investigate the functional profile of homomeric ␣7 receptors, because they may be involved in the pathogenesis of many neurological disorders (9-16).For this study we constructed a chimera in which the enhanced form of the green fluorescent protein (GFP) was fused to the C...

Basic and modern concepts on cholinergic receptor: A review

“…Xenopus oocytes injected with wild-type, mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors [43] . Interplay of β2 nicotinic receptors and dopamine pathways in the control of spontaneous locomotion, and finds the acetylcholine (ACh) is a known modulator of the activity of dopaminergic (DAergic) neurons through the stimulation of nAChRs.…”

The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein

“…In a previous work we constructed human ␣7-GFP chimeras to study ␣7 receptors (28), and in the present work we have determined the single-channel properties of the chimeric homomeric ␣7 receptors formed in Xenopus oocytes. We confirm that chimeric receptors are functionally assembled in oocytes intranuclearly injected with ␣7-GFP-subunit cDNA and found that when these ␣7-GFP receptors are activated by the transmitter they exhibit unitary events with the same two classes of conductance but with different gating kinetics than those of the wt␣7 receptors.…”

“…Preparation of oocytes and nuclear injection procedures were as detailed elsewhere (8,25). Mutant ␣7-subunits and chimeras were obtained as described (28). wt␣7-subunit cDNA was a gift from the Janssen Research Foundation (Belgium).…”

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

“…Addition-ally, transgenes containing tagged subunits may be incorporated into viral vectors and used in subunit knockout animals to selectively reintroduce tagged subunits into a region of interest, thereby allowing a broad range of techniques to be applied to define subunit and receptor localization and function [48] . Potential drawbacks to fluorescent protein fusion do exist and have been enumerated by others [4,5,41] . Briefly, they include alterations in coassembly, trafficking and function.…”

“…Properties such as subunit assembly and chaperone/accessory protein interactions can be assessed using proteins labeled with different FPs and techniques such as fluorescence resonance energy transfer and colocalization [2,3] . However, whether it be an epitope tag or a FP, insertion of an extra sequence into a protein can affect target protein properties and function [2,4,5] . FPs are ~27-kDa proteins comprised of ~239 amino acids in a β-barrel conformation [2,6] .…”