Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Groot-Kormelink, Paul J.; Broadbent, Steven; Boorman, James P.; Sivilotti, Lucia G.

doi:10.1085/jgp.200409042

Cited by 36 publications

(42 citation statements)

References 33 publications

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“…[37][38][39][40][41][42][43][44][45][46]. The subunit composition of each individual multimeric channel, however, could be different from that of the tandem constructs (examples in refs.…”

Section: Discussionmentioning

confidence: 99%

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Jin

Sui²,

Rosenhouse‐Dantsker³

et al. 2008

Channels

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Phosphatidylinositol bisphosphate (PIP 2 ) is the most abundant phosphoinositide in the plasma membrane of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP 2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP 2 . We first showed that PIP 2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP 2 levels. Moreover, by controlling the number of PIP 2 -sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP 2 , where each of the four open states corresponds to the channel activated by one to four PIP 2 molecules.

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“…[37][38][39][40][41][42][43][44][45][46]. The subunit composition of each individual multimeric channel, however, could be different from that of the tandem constructs (examples in refs.…”

Section: Discussionmentioning

confidence: 99%

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Jin

Sui²,

Rosenhouse‐Dantsker³

et al. 2008

Channels

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“…The lack of current produced by expressing tandem subunits alone or with the ␤ subunit suggests that they do not produce monomers (Fig. 1) that can incorporate into receptors, nor do they "loop out" with only one of the two subunits incorporated into the pore-forming pentamer (Zhou et al, 2003;Groot-Kormelink et al, 2004). Furthermore, the similarity of ␣␤tanϩ␥2 to ␣1␤2␥2 1:1:10 transfections suggests that the formation of both a functional ␣1 and ␤2 subunit from a tandem subunit does not occur, because a mixture with ␣1␤2 receptors should be detectable in that case.…”

Section: Discussionmentioning

confidence: 99%

Tandem Subunits Effectively Constrain GABA_AReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABA_AReceptor-Associated Protein

Boileau¹,

Pearce²,

Czajkowski³

2005

J. Neurosci.

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GABAergic synapses likely contain multiple GABA A receptor subtypes, making postsynaptic currents difficult to dissect. However, even in heterologous expression systems, analysis of receptors composed of ␣, ␤, and ␥ subunits can be confounded by receptors expressed from ␣ and ␤ subunits alone. To produce recombinant GABA A receptors containing fixed subunit stoichiometry, we coexpressed individual subunits with a "tandem" ␣1 subunit linked to a ␤2 subunit. Cotransfection of the ␥2 subunit with ␣␤-tandem subunits in human embryonic kidney 293 cells produced currents that were similar in their macroscopic kinetics, single-channel amplitudes, and pharmacology to overexpression of the ␥ subunit with nonlinked ␣1 and ␤2 subunits. Similarly, expression of ␣ subunits together with ␣␤-tandem subunits produced receptors having physiological and pharmacological characteristics that closely matched cotransfection of ␣ with ␤ subunits. In this first description of tandem GABA A subunits measured with patch-clamp and rapid agonist application techniques, we conclude that incorporation of ␣␤-tandem subunits can be used to fix stoichiometry and to establish the intrinsic kinetic properties of ␣1␤2 and ␣1␤2␥2 receptors. We used this method to test whether the accessory protein GABA A receptor-associated protein (GABARAP) alters GABA A receptor properties directly or influences subunit composition. In recombinant receptors with fixed stoichiometry, coexpression of GABARAP-enhanced green fluorescent protein (EGFP) fusion protein had no effect on desensitization, deactivation, or diazepam potentiation of GABA-mediated currents. However, in ␣1␤2␥2S transfections in which stoichiometry was not fixed, GABARAP-EGFP altered desensitization, deactivation, and diazepam potentiation of GABA-mediated currents. The data suggest that GABARAP does not alter receptor kinetics directly but by facilitating surface expression of ␣␤␥ receptors.

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“…However, potential problems with using concatemers include dimer proteolysis 13 , dipentamer formation 14 and incomplete incorporation of fusion protein into individual pentamers 15 . These problems are more likely to occur when dimers are co-transfected with monomers than when fully concatenated pentamers are used 16,17 .…”

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confidence: 99%

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

et al. 2012

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ABSTRACT:The glycine receptor is an anion-permeable member of the Cys-loop ion channel receptor family. Synaptic glycine receptors predominantly comprise pentameric α1β subunit heteromers. To date, attempts to define the subunit stoichiometry and arrangement of these receptors have not yielded consistent results. Here we introduced FLAG and 6-His epitopes into α1 and β subunits, respectively, and imaged single antibody-bound α1β receptors using atomic force microscopy. This permitted us to infer the number and relative locations of the respective subunits in functional pentamers. Our results indicate an invariant stoichiometry of 2α1:3β with a subunit arrangement of β−α−β−α−β.Glycine receptor (GlyR) chloride channels mediate inhibitory transmission in the spinal cord and brain stem 1 . As they are considered potential therapeutic targets for indications including inflammatory pain, hyperekplexia, spasticity, tinnitus and breathing disorders [2][3][4][5] , there is widespread interest in understanding the molecular structure of their ligand binding sites for therapeutic development. GlyRs are members of the pentameric Cys-loop ion channel receptor family. Because ligandbinding sites are located at subunit interfaces, the pharmacological properties of each binding site are defined by the contributing subunits 6 . It is thus of interest to establish the subunits stoichiometry and arrangement in functional receptors to determine the number and type of subunit interfaces. Synaptic GlyRs are αβ heteromers, and receptors resulting from the cotransfection of varying amounts of α and β subunit RNA have uniform functional properties, implying a fixed stoichiometry 7,8 . Depending on their stoichiometry, heteromeric GlyRs may contain a mixture of α−α, α−β, β−α and β−β interfaces.The initial characterization of affinity-purified native GlyRs inferred that heteromeric GlyRs comprised 3α and 2β subunits 9,10 . This stoichiometry was supported by a subsequent functional analysis that showed the effect of a pore mutation was more dramatic when inserted into the α relative to the β subunit, implying an excess of α subunits per pentamer 11 . However, a study that compared the effects of mutations to corresponding glycine binding residues in α1 and β subunits concluded that β subunits predominated in α1β GlyRs 12 . They then showed that α1-β concatemers produced functional heteromers when co-expressed with β monomers but not when expressed alone or with α1 monomers. This was consistent with either a 2α:3β or a 1α:4β stoichiometry. Quantitation of radio-labeled methionine levels in recombinant α1 and α1β GlyRs allowed the authors to deduce a 2α1:3β stoichiometry, with an arrangement of β−α−β−α−β 12 .However, potential problems with using concatemers include dimer proteolysis 13 , dipentamer formation 14 and incomplete incorporation of fusion protein into individual pentamers 15 . These problems are more likely to occur when dimers are co-transfected with monomers than when fully concatenated pentamers are used 16,17 . Thus, as the ...

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Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Cited by 36 publications

References 33 publications

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Tandem Subunits Effectively Constrain GABA_AReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABA_AReceptor-Associated Protein

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

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Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Cited by 36 publications

References 33 publications

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Tandem Subunits Effectively Constrain GABAAReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABAAReceptor-Associated Protein

Stoichiometry and Subunit Arrangement of α1β Glycine Receptors As Determined by Atomic Force Microscopy

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Tandem Subunits Effectively Constrain GABA_AReceptor Stoichiometry and Recapitulate Receptor Kinetics But Are Insensitive to GABA_AReceptor-Associated Protein