Multiple functional neurosteroid binding sites on GABAA receptors

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(-) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action

Site-specific effects of neurosteroids on GABA(A) receptor activation and desensitization

This study examines how site-specific binding to three identified neurosteroid-binding sites in the

Steroid Interaction with a Single Potentiating Site Is Sufficient to Modulate GABA-A Receptor FunctionS⃞

Neuroactive steroids are efficacious potentiators of GABA-A receptors. Recent work has identified a site in the α1 subunit of the GABA-A receptor, that is essential for potentiation by steroids. However, each receptor contains two copies of the α1 subunit. We generated concatemers of subunits so that the α1 subunits could be mutated separately and examined the consequences of mutations that remove potentiation by most neurosteroids (α1 Q241L, α1 Q241W). Concatemers were expressed in Xenopus laevis oocytes, and activation by GABA, potentiation by neurosteroids, and the agonist activity of piperidine-4-sulfonic acid (P4S) were determined. When the α1 Q241L mutation is present in α1 subunits the EC50 for activation by GABA is shifted to higher concentration and potentiation by neurosteroids is diminished. When the α1 Q241W mutation is expressed, the EC50 for GABA is shifted to lower concentration, the relative efficacy of P4S is increased, and potentiation by neurosteroids is diminished. Mutation of only one α1 subunit does not produce the full effect of mutating both sites. Overall, the data demonstrate that at a macroscopic level, the presence of a single wild-type steroid-binding site is sufficient to mediate responses to steroid, but both must be mutated to completely remove the effects of steroids. Differences between the two sites seem to be relatively subtle

Introduced Amino Terminal Epitopes Can Reduce Surface Expression of Neuronal Nicotinic Receptors

<div><p>Epitopes accessible on the surface of intact cells are extremely valuable in studies of membrane proteins, allowing quantification and determination of the distribution of proteins as well as identification of cells expressing large numbers of proteins. However for many membrane proteins there are no suitable antibodies to native sequences, due to lack of availability, low affinity or lack of specificity. In these cases the use of an introduced epitope at specific sites in the protein of interest can often provide a suitable tool for studies. However, the introduction of the epitope sequence has the potential to affect protein expression, the assembly of multisubunit proteins or transport to the surface membrane. We find that surface expression of heteromeric neuronal nicotinic receptors containing the α4 and β4 subunits can be affected by introduced epitopes when inserted near the amino terminus of a subunit. The FLAG epitope greatly reduces surface expression when introduced into either α4 or β4 subunits, the V5 epitope has little effect when placed in either, while the Myc epitope reduces expression more when inserted into β4 than α4. These results indicate that the extreme amino terminal region is important for assembly of these receptors, and demonstrate that some widely used introduced epitopes may severely reduce surface expression.</p></div

Sequences of the amino-terminal regions of the subunits studied.

<p>The sequences of the mature subunit from the predicted amino-termini are shown for the human nicotinic α4 and β4 subunits are shown. The locations of insertions are shown by the boxed residues (e.g. insertions were made between α4 T4 and R5). The region indicated by gray shading is likely to be α-helical based on alignments to related subunits of known structure (see Text for details). The two leucine residues shown in <i>italics</i> are residues identified as important for expression in the nicotinic α7 subunit (see Text).</p

Sequences and predicted structures of the α4 constructs studied.

<p>The first column identifies the construct. Then the N-terminal amino acid sequence is shown aligned at the conserved RP motif, with the insert shown in bold letters. Immediately below the sequence is the secondary structure predicted by Sympred (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151071#sec002" target="_blank">Methods</a>). The last column shows the mean relative ELISA signal. The boxed region is likely to be helical based on alignments to related subunits of known structure.</p