A Role for the 2′ Residue in the Second Transmembrane Helix of the GABAA Receptor γ2S Subunit in Channel Conductance and Gating

Luu, Tien; Cromer, Brett A.; Gage, Peter W.; Tierney, Mary L.

doi:10.1007/s00232-005-0759-2

Cited by 10 publications

(5 citation statements)

References 43 publications

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“…This is in keeping with earlier studies showing that the amino acid at position 2′ of GABA ρ subunits influences response kinetics [25], receptor pharmacology [4,29], and ion selectivity [5]. The importance of the amino acid at the 2′ position has also been reported in a recent study on the GABA A receptor, in which mutating the serine residue in the γ2 subunit significantly altered the single channel conductance of the receptor [16]. Thus, the results of the present study are consistent with the view that residues at position 2′ influence the conformational geometry of the channel and significantly affect receptor gating.…”

Section: Discussionsupporting

confidence: 88%

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

Zhu

Ripps

Qian

2007

Neuroscience Letters

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GABAC receptors, expressed predominately in vertebrate retina, are thought to be formed mainly by GABA rho subunits, each of which exhibits distinct physiological and pharmacological properties. In this study, the receptors formed by perch GABA rho subunits were expressed in HEK cells, and their single channel conductances were determined using noise analysis techniques. The receptors formed by the perch rho1A subunit gate a channel with a conductance of 0.2 pS, whereas the receptors formed by GABA rho2 subunits exhibit much higher channel conductances, i.e., 3.2 and 3.5 pS for perch rho2A and rho2B receptors, respectively. A comparison of the amino acid sequences of the channel-forming TMII regions of the various subunits suggested that a single amino acid at position 2' was a potential site for the large differential in conductance. We found that switching the serine residue at that site in the GABA rho2 subunit to the proline residue present in the rho1 subunit reduced the channel conductance to a level similar to that of the wild type rho1 receptor. Conversely, mutating proline to serine in the amino acid sequence of the rho1 receptor significantly increased its unitary conductance. These results indicate that a single amino acid in the TMII region plays an important role in determining the single channel conductance of the GABAC receptors.

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Section: Discussionsupporting

confidence: 88%

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

Zhu

Ripps

Qian

2007

Neuroscience Letters

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“…that they were often high (Ն40 pS) and they displayed a range of conductances (2-5 states), characteristics never seen in control cells expressing GABA A receptors alone (13). To illustrate the variation in the single channel conductances of the spontaneous currents and the frequency of the amplitudes within a single patch, a typical example is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Cells were transfected using a lipid-mediated reagent (Lipofectin; Sigma) as described in detail previously (13). For the identification of successfully transfected cells, the plasmid encoding enhanced green fluorescent protein (EGFP-NI; Clontech) was used in every transfection.…”

Section: Recombinant Expression Systemmentioning

confidence: 99%

GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

Luu

Gage

Tierney

2006

Journal of Biological Chemistry

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The single channel properties of recombinant ␥-aminobutyric acid type A (GABA A ) ␣␤␥ receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing ␣␤␥ receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA A receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (>40 pS), and GABA (>10 M) was able to increase their conductance, up to a maximum of 60 pS Inhibitory signals in human brains are mediated primarily by ␥-aminobutyric acid type A (GABA A ) 2 receptors. These ligandgated ion channels are composed of multimembrane-spanning subunits that assemble into pentamers and function by gating a pore selective for chloride ions. The targeting and organization of GABA A receptors at specific membrane locations are critical for their normal function. For example, GABA A receptors are clustered at inhibitory synapses but are also found both clustered and nonclustered at other sites on the neuronal cell surface (1, 2). These synaptic and nonsynaptic (extrasynaptic) sites reflect GABA A receptor involvement in both phasic and tonic signaling, respectively. The functional behavior of native GABA A receptors is complex. Much of the receptor's functional complexity has been attributed to its extensive structural heterogeneity as indicated by the 19 different genes identified to date (␣1-6, ␤1-3, ␥1-3, ␦, 1-3, ⑀, , and ).Recombinant GABA A receptors are different from native GABA A receptors in that they never display single channel conductances greater than 40 pS, nor do drugs modulate their conductance, properties we and others have described for native nonsynaptic (extrasynaptic) GABA A receptors (3-8). We have, however, been able to mimic the behavior exhibited by neuronal extrasynaptic GABA A receptors in a recombinant system and change the dispersion of receptors in the membrane simply by co-expressing the trafficking protein GABARAP with GABA A receptors (9). GABARAP (GABA A receptor-associated protein) was originally identified because of its physical association with GABA A receptors following their isolation by immunoprecipitation from solubilized rat brain (10). Subsequent immunolocalization data and the biochemical identification of the GABARAP interaction partners has led to the suggestion that it participates in trafficking and membrane fusion events underlying organizational processes at GABAergic synapses but does not remain associated with receptors once they are inserted at the synapse (11). In heterologous expression systems, recombinant GABARAP has been shown to promote clustering of ␥ subunit-containing GABA A receptors in the plasma membrane (9, 12). As a consequence of this ordered packing arrangement, the recombinant GABA ...

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“…Channels transitioning to high conductance states also exhibit significantly longer mean open times, and hence we refer to them as superchannels (5). By contrast, recombinant ␣␤␥ GABA A receptors never display high-conductance channels and drugs increase only open-channel probability (6,7). To mimic the conductance properties of native GABA A receptors, recombinant receptors must be coexpressed with the trafficking protein GABARAP (GABA A receptor associated protein) (8,9).…”

mentioning

confidence: 99%

Protein interactions involving the γ2 large cytoplasmic loop of GABA _A receptors modulate conductance

et al. 2009

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Native GABA(A) channels display a single-channel conductance ranging between approximately 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the gamma2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma2-GABARAP interaction did not affect the conductance of recombinant alphabetagamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.

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A Role for the 2′ Residue in the Second Transmembrane Helix of the GABAA Receptor γ2S Subunit in Channel Conductance and Gating

Cited by 10 publications

References 43 publications

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

Protein interactions involving the γ2 large cytoplasmic loop of GABA _A receptors modulate conductance

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A Role for the 2′ Residue in the Second Transmembrane Helix of the GABAA Receptor γ2S Subunit in Channel Conductance and Gating

Cited by 10 publications

References 43 publications

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

A single amino acid in the second transmembrane domain of GABA ρ receptors regulates channel conductance

GABA Increases both the Conductance and Mean Open Time of Recombinant GABAA Channels Co-expressed with GABARAP

Protein interactions involving the γ2 large cytoplasmic loop of GABA A receptors modulate conductance

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Protein interactions involving the γ2 large cytoplasmic loop of GABA _A receptors modulate conductance