Anna Sedelnikova

α1β2δ, a silent GABAA receptor: recruitment by tracazolate and neurosteroids

This study investigated the α1β2δ isoform of the GABAA receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown.

Stoichiometry of a pore mutation that abolishes picrotoxin‐mediated antagonism of the GABAA receptor

Picrotoxin, a potent antagonist of the inhibitory central nervous system GABAA and glycine receptors, is believed to interact with residues that line the central ion pore. These pore‐lining residues are in the second transmembrane domain (TM2) of each of the five constituent subunits. One of these amino acids, a threonine at the 6′ location, when mutated to phenylalanine, abolishes picrotoxin sensitivity. It has been suggested that this threonine, via hydrogen bonding, directly interacts with the picrotoxin molecule. We previously demonstrated that this mutation, in the α, β or γ subunit, can impart picrotoxin resistance to the GABA receptor. Since the functional pentameric GABA receptor contains two α subunits, two β subunits and one γ subunit, it is not clear how many α and β subunits must carry this mutation to impart the resistant phenotype. In this study, by coexpression of mutant α or β subunits with their wild‐type counterparts in various defined ratios, we demonstrate that any single subunit carrying the 6′ mutation imparts picrotoxin resistance. Implications of this finding in terms of the mechanism of antagonism are considered.

Structural rearrangements in loop F of the GABA receptor signal ligand binding, not channel activation.

Structure-function studies of the Cys loop family of ionotropic neurotransmitter receptors (GABA, nACh, 5-HT(3), and glycine receptors) have resulted in a six-loop (A-F) model of the agonist-binding site. Key amino acids have been identified in these loops that associate with, and stabilize, bound ligand. The next step is to identify the structural rearrangements that couple agonist binding to channel opening. Loop F has been proposed to move upon receptor activation, although it is not known whether this movement is along the conformational pathway for channel opening. We test this hypothesis in the GABA receptor using simultaneous electrophysiology and site-directed fluorescence spectroscopy. The latter method reveals structural rearrangements by reporting changes in hydrophobicity around an environmentally sensitive fluorophore attached to defined positions of loop F. Using a series of ligands that span the range from full activation to full antagonism, we show there is no correlation between the rearrangements in loop F and channel opening. Based on these data and agonist docking simulations into a structural model of the GABA binding site, we propose that loop F is not along the pathway for channel opening, but rather is a component of the structural machinery that locks ligand into the agonist-binding site.

Function and modulation of δ-containing GABAA receptors

alphabetadelta-Containing GABA(A) receptors are (1) localized to extra- and perisynaptic membranes, (2) exhibit a high sensitivity to GABA, (3) show little desensitization, and (4) are believed to be one of the primary mediators of tonic inhibition in the central nervous system. This type of signaling appears to play a key role in controlling cell excitability. This review article briefly summarizes recent knowledge on tonic GABA-mediated inhibition. We will also consider the mechanism of action of many clinically important drugs such as anxiolytics, anticonvulsants, and sedative/hypnotics and their effects on delta-containing GABA receptor activation. We will conclude that alphabetadelta-containing GABA(A) receptors exhibit a relatively low efficacy that can be potentiated by endogenous modulators and anxiolytic agents. This scenario enables these particular GABA receptor combinations, upon neurosteroid exposure for example, to impart a profound effect on excitability in the central nervous system.

Stoichiometric Pore Mutations of the GABAAR Reveal a Pattern of Hydrogen Bonding with Picrotoxin

Picrotoxin (PTX) is a noncompetitive antagonist of many ligand-gated ion channels, with a site of action believed to be within the ion-conducting pore. In the A-type gamma-aminobutyric acid receptor, a threonine residue in the second transmembrane domain is of particular importance for the binding of, and ultimate inhibition by, PTX. To better understand the relationship between this residue and the PTX molecule, we mutated this threonine residue to serine, valine, and tyrosine to change the structural and biochemical characteristics at this location. The known subunit stoichiometry of the A-type gamma-aminobutyric acid receptor allowed us to create receptors with anywhere from zero to five mutations. With an increasing number of mutated subunits, each amino acid substitution revealed a unique pattern of changes in PTX sensitivity, ultimately encompassing sensitivity shifts over several orders of magnitude. The electrophysiological data on PTX-mediated block, and supporting modeling and docking studies, provide evidence that an interaction between the PTX molecule and three adjacent uncharged polar amino acids at this position of the pore are crucial for PTX-mediated inhibition.

alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids.

This study investigated the α1β2δ isoform of the GABAA receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown.

Recombinant GABAC receptors expressed in rat hippocampal neurons after infection with an adenovirus containing the human ρ1 subunit

1 A recombinant adenovirus was generated with the human ρ1 GABAC receptor subunit (adeno‐ρ). Patch‐clamp and antibody staining were employed to confirm functional expression of recombinant ρ1 receptors after infection of human embryonic kidney cells (HEK293 cell line), human embryonic retinal cells (911 cell line), dissociated rat hippocampal neurons and cultured rat hippocampal slices. 2 Standard whole‐cell recording and Western blot analysis using ρ1 GABAC receptor antibodies revealed that recombinant ρ1 receptors were expressed in HEK293 and 911 cells after adeno‐ρ infection and exhibited properties similar to those of ρ1 receptors after standard transfection. 3 Cultured rat hippocampal neurons (postnatal day (P)3‐P5) did not show a native GABAC‐like current. After adeno‐ρ infection, however, a GABAC‐like current appeared in 70‐90 % of the neurons. 4 Five days after infection, expression of GABAC receptors in hippocampal neurons significantly decreased native GABAA receptor currents from 1200 ± 300 to 150 ± 70 pA (n= 10). The native glutamate‐activated current was unchanged. 5 Hippocampal slices (P8) did not show a native GABAC‐like current, although recombinant ρ1 receptors could be expressed in cultured hippocampal slices after adeno‐ρ infection. 6 These data indicate that an adenovirus can be used to express recombinant GABAC receptors in hippocampal neurons. This finding could represent an important step towards the gene therapy of CNS receptor‐related diseases.

Phosphorylation of the recombinant ρ1 GABA receptor

γ‐Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. While a growing body of literature indicates that postsynaptic GABA receptors are regulated by phosphorylation, there is discrepancy as to the specific effects of phosphorylation on GABA receptor function. Here, we have identified phosphorylation sites on the human ρ1 GABA receptor for six protein kinases widely expressed in the brain: protein kinase C (PKC); cAMP‐dependent protein kinase (PKA); calmodulin‐dependent kinase (CaMKII); casein kinase (CKII); mitogen‐activated protein kinase (MAPK); and cGMP‐dependent protein kinase (PKG). We demonstrate that in nearly all cases, the consensus sites and actual phosphorylation sites do not agree supporting the risk of relying on a sequence analysis to identify potential phosphorylation sites. In addition, of the six kinases examined, only CKII phosphorylated the human ρ2 subunit. Site‐directed mutagenesis of the phosphorylation sites, or activation/inhibition of select kinase pathways, did not alter the receptor sensitivity or maximal GABA‐activated current of the ρ1 GABA receptor expressed in Xenopus laevis oocytes suggesting phosphorylation of ρ1 does not directly alter receptor properties. This study is a first and necessary step towards elucidating the regulation of ρ1 GABA receptors by phosphorylation.

Fluorescence lifetime imaging of calcium flux in neurons in response to pulsed infrared light

Pulsed infrared light can excite action potentials in neurons; yet, the fundamental mechanism underlying this phenomenon is unknown. Previous work has observed a rise in intracellular calcium concentration following infrared exposure, but the source of the calcium and mechanism of release is unknown. Here, we used fluorescence lifetime imaging of Oregon Green BAPTA-1 to study intracellular calcium dynamics in primary rat hippocampal neurons in response to infrared light exposure. The fluorescence lifetime of Oregon Green BAPTA-1 is longer when bound to calcium, and allows robust measurement of intracellular free calcium concentrations. First, a fluorescence lifetime calcium calibration curve for Oregon Green BAPTA-1 was determined in solutions. The normalized amplitude of the short and long lifetimes was calibrated to calcium concentration. Then, neurons were incubated in Oregon Green BAPTA-1 and exposed to pulses of infrared light (0-1 J/cm2; 0-5 ms; 1869 nm). Fluorescence lifetime images were acquired prior to, during, and after the infrared exposure. Fluorescence lifetime images, 64x64 pixels, were acquired at 12 or 24 ms for frame rates of 83 and 42 Hz, respectively. Accurate α1 approximations were achieved in images with low photon counts by computing an α1 index value from the relative probability of the observed decay events. Results show infrared light exposure increases intracellular calcium in neurons. Altogether, this study demonstrates accurate fluorescence lifetime component analysis from low-photon count data for improved imaging speed.

All optical experimental design for neuron excitation, inhibition, and action potential detection

Recently, infrared light has been shown to both stimulate and inhibit excitatory cells. However, studies of infrared light for excitatory cell inhibition have been constrained by the use of invasive and cumbersome electrodes for cell excitation and action potential recording. Here, we present an all optical experimental design for neuronal excitation, inhibition, and action potential detection. Primary rat neurons were transfected with plasmids containing the light sensitive ion channel CheRiff. CheRiff has a peak excitation around 450 nm, allowing excitation of transfected neurons with pulsed blue light. Additionally, primary neurons were transfected with QuasAr2, a fast and sensitive fluorescent voltage indicator. QuasAr2 is excited with yellow or red light and therefore does not spectrally overlap CheRiff, enabling imaging and action potential activation, simultaneously. Using an optic fiber, neurons were exposed to blue light sequentially to generate controlled action potentials. A second optic fiber delivered a single pulse of 1869nm light to the neuron causing inhibition of the evoked action potentials (by the blue light). When used in concert, these optical techniques enable electrode free neuron excitation, inhibition, and action potential recording, allowing research into neuronal behaviors with high spatial fidelity.