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

Abstract

Neuronal nicotinic acetylcholine (AcCho) receptors composed of ␣7-subunits (␣7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their singlechannel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) ␣7-AcChoRs, we identified two classes of channel conductance: a low conductance (␥L) of 72 pS and a high one (␥H) of 87 pS, with mean open-times (op) of 0.6 ms. The same classes of conductances, but longer op (3 ms), were seen in experiments with chimeric ␣7 receptors in which the wt␣7 extracellular C terminus was fused to the green fluorescent protein (wt␣7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing ␣7 receptors carrying a Leu-to-Thr 248 mutation (mut␣7) or oocytes expressing chimeric mut␣7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wt␣7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter op, than those activated by AcCho. Accordingly, human homomeric wt␣7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mut␣7 receptors open channels of lower conductance and longer lifetime than those gated by wt␣7-AcChoRs, and these parameters are not greatly altered by fusing the mut␣7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function. N euronal nicotinic acetylcholine receptors (AcChoRs) are pentameric proteins that constitute a family of ligand-gated ion channels, which are widely distributed in the mammalian brain and are built by combinations of ␣-and ␤-subunits (heteromeric AcChoRs) or by an assembly of ␣-subunits (homomeric AcChoRs). In particular, AcChoRs composed of ␣7-subunits (␣7 AcChoRs) are believed to be involved in various physiological activities: acting presynaptically to modulate neurotransmitter release (1); acting postsynaptically to generate postsynaptic currents (2, 3); and in cognitive functions, development, pain, and aging (4-7). Moreover, ␣7-AcChoRs exhibit unusual properties, such as (i) sensitivity to ␣-bungarotoxin (␣-BuTx) (8) and ␤-amyloid peptide (9); (ii) having a highly Ca 2ϩ permeability (10, 11); (iii) exerting striking antiapoptotic effects on central nervous system nerve cells (12); and (iv) transducing signals to phosphatidylinositol 3-kinase (13). Despite this pleiotropic action, relatively little is known on the behavior of the ␣7-containing AcChoR channels expressed in native nerve cells (14-17) and nothing is known about the channel kinetics of human homomeric ␣7-AcChoRs. To begin to fill this deficiency, we made single-channel recordings in outside-out patches from human homomeric ␣7-AcChoRs expressed in Xenopus oocytes.To facilitate their study, ligand-ga...

Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

“…The observed kinetic parameters are compatible with those reported for α7 AChRs transiently expressed in oocytes [19] and in BOSC 23 cells [20] . A recent report [21] describing the functional properties of α7 AChRs heterologously expressed in CHO cells failed to observe receptor-mediated Ca 2+ fluxes when nicotinic agonists were used to activate the AChR in the absence of the positive allosteric modulator PNU-120596 or after pretreatment of cells with the tyrosine kinase inhibitor genistein.…”

“…The low levels of cell-surface expression have limited our understanding of the kinetic activation of α7 AChRs. Few studies have reported single-channel currents in oocytes from wild-type and mutant α7 AChRs [18,19] . Recently, the human α7 AChR and the Ric-3 protein were transiently transfected in BOSC 23 cells [20] .…”

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] .…”

“…These subunit-FP fusions have revealed much about nicotinic receptors, such as their subunit stoichiometry, changes in expression levels and location of subtypes in the brain [9,10] . Whereas tagging the extracellular region with an FP provides an accessible tag for FP-targeted antibody staining and for trafficking studies [11,12] , it has been reported to interfere with endocytosis [8] and to modify receptor function [4] . Alternatively, placing the FP in an intracellular domain might obviate these concerns; however, inserting it in this location presents other challenges including interruption of trafficking domains and phosphorylation sites, which could disrupt function [7] .…”