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

Zhu, Yujie; Ripps, Harris; Qian, Haohua

doi:10.1016/j.neulet.2007.03.020

Cited by 9 publications

(11 citation statements)

References 36 publications

(44 reference statements)

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“…3). In addition, using noise analysis techniques we estimated the single channel properties of the GABA C receptors present on rod bipolar cells; the derived values were comparable to those published previously (Feigenspan et al, 1993;Qian and Dowling, 1995;Palmer, 2006;Zhu et al, 2007). Although noise analysis tends to underestimate the value of single channel conductance, the same bias should apply results obtained from normal and diabetic cells.…”

Section: Discussionsupporting

confidence: 72%

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Ramsey¹,

Ripps²,

Qian³

2007

Experimental Eye Research

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Neural deficits suggestive of involvement of the GABA signaling pathway can often be detected early in the course of diabetic retinopathy, a leading cause of blindness in the United States. To examine in greater detail the nature of the neuronal changes associated with hyperglycemia, we investigated GABA receptor activity on retinal bipolar cells in streptozotocin-induced diabetic rats; cells from age-matched normal rats served as controls. Patch-clamp recordings from isolated rod-bipolar cells revealed that diabetes enhanced the whole cell currents elicited by GABA. Responses of the GABA(C) receptor, the predominant GABA receptor on rat rod bipolar cells, exhibited a greater sensitivity to GABA, larger maximum current responses, slower response kinetics, and a smaller single channel conductance among diabetic cells relative to those recorded from normal controls. Compared with the properties of homomeric rho1 and heteromeric rho1rho2 receptors formed in a heterologous expression system, these results suggested that there was a greater contribution from the rho1 subunit in the GABA(C) receptor-mediated response of diabetic cells. The levels of mRNA, measured with real-time RT-PCR, were consistent with this finding. There was a significant enhancement in the ratio of rho1/rho2 subunit expression in the retina of diabetic animals, although the levels of GABA rho1 subunit expression were comparable in diabetic and normal retinas. Taken together, the results suggest that diabetes modifies the subunit composition of the GABA(C) receptor on retinal neurons, most likely through its effect on the efficacy of gene transcription.

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Section: Discussionsupporting

confidence: 72%

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Ramsey¹,

Ripps²,

Qian³

2007

Experimental Eye Research

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“…The activation rate is roughly half that of wild type, and the deactivation rate is significantly decreased to one-fourth of the value for wild-type receptors. The 1 GABA C receptor used in this study has a single-channel conductance of Ͻ2 pS (26,27). The resulting small currents vitiate systematic kinetic studies using single-channel analysis.…”

Section: Resultsmentioning

confidence: 93%

Establishing an Ion Pair Interaction in the Homomeric ρ1 γ-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

Wang

Lester

Dougherty

2007

Journal of Biological Chemistry

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␥-Aminobutyric acid type A (GABA A ) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric 1 GABA A receptor. We have identified an ion pair interaction between two conserved charged residues, Glu 92 in loop 2 of the extracellular domain and Arg 258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily.Fast inhibitory neurotransmission in the adult mammalian central nervous system is primarily mediated by the amino acid ␥-aminobutyric acid (GABA).2 So far, three types of GABA receptors have been identified, termed GABA A , GABA B , and the homopentameric 1 GABA A receptor, also known as GABA C (1, 2). Although GABA B is a G protein-coupled receptor, GABA A and GABA C receptors are homologous but distinct members of the Cys-loop superfamily of ligand-gated ion channels, which also includes the nicotinic acetylcholine (nAChR), serotonin, and glycine receptors. Members of this superfamily are composed of five subunits arranged around a central ionconducting pore, with each subunit consisting of a large extracellular domain, four transmembrane helices (M1-M4), and a large intracellular loop. The newest member of this family, the GABA C receptor (3), is expressed predominantly on retinal neurons, although recent studies indicate a wide distribution throughout the central nervous system (4 -6).The binding of agonist to a Cys-loop receptor triggers a complex structural transition that results in the opening of a "gate," allowing ions to flow through the channel (7). Identifying the linkage pathway has been limited by the lack of a complete atomic-resolution structure of any fast synaptic receptor. However, two breakthroughs have propelled the field into the structural age. The first is determination of the crystal structure of acetylcholine-binding protein (8), which is homologous to the extracellular domain of the nAChR and, by extension, all Cysloop receptors. This structural template provides critical insights into the nature of the binding site, but, of course, the ion channel and its gate are missing from such structures. Second, a refined electron microscopy structure of the Torpedo acetylcholine receptor by Unwin and co-workers (Protein Data Bank code 2BG9) has shed light onto the global structure and has suggested molecular determinants of functional mechanisms in Cys-loop receptors (9 -11).The available structural informati...

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“…Initially, we found that exchanging the N‐terminal extracellular domains of perch ρ1 and ρ2 subunits failed to alter the CTZ sensitivity profile (data not shown). However, it is important to recall that GABA ρ1 and ρ2 subunits exhibit a consistent difference at the 2′ position in the second transmembrane domain (Zhu et al 2007). The amino acid sequences of this region of the four perch ρ subunits (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cyclothiazide: a subunit‐specific inhibitor of GABA_C receptors

Xie¹,

Song²,

Ripps

et al. 2008

The Journal of Physiology

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We tested the effects of cyclothiazide (CTZ), an agent used to block desensitization of AMPA-type glutamate receptors, on heterologously expressed GABA C receptors formed by homomeric ρ subunits. CTZ inhibition of GABA C receptors was subunit specific; it produced a dose-dependent reduction of the GABA-elicited current on homomeric ρ2 receptors with an IC 50 of about 12 μM, but had no significant effect on homomeric ρ1 receptors. This differential sensitivity was attributable to a single amino acid located on the second transmembrane domain of the ρ subunits. Mutating the residue at this position from serine to proline on the ρ2 subunit eliminated CTZ sensitivity, whereas switching proline to serine on the ρ1 subunit made the receptor CTZ sensitive. The inhibitory properties of CTZ were consistent with its action as a channel blocker on the receptors formed by ρ2 subunits. The effect showed a small degree of voltage dependence, and was due mainly to a non-competitive mechanism that reduced the maximum response elicited by GABA. In addition, the prominent membrane current rebound when co-application of GABA and CTZ was terminated suggests that the binding site for CTZ on the GABA C receptor is distinct from that for GABA, and that CTZ acts as a non-competitive antagonist on the GABA C receptor. CTZ inhibited the open channel of the GABA C receptor with a time constant of about 0.4 s, but the kinetics were ∼10-fold slower when GABA is absent. The ability of CTZ to interact with various types of neurotransmitter receptors indicates that the drug has multiple actions in the CNS.

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

Cited by 9 publications

References 36 publications

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Establishing an Ion Pair Interaction in the Homomeric ρ1 γ-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

Cyclothiazide: a subunit‐specific inhibitor of GABA_C receptors

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

Cited by 9 publications

References 36 publications

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Streptozotocin-induced diabetes modulates GABA receptor activity of rat retinal neurons

Establishing an Ion Pair Interaction in the Homomeric ρ1 γ-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

Cyclothiazide: a subunit‐specific inhibitor of GABAC receptors

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Cyclothiazide: a subunit‐specific inhibitor of GABA_C receptors