-Aminobutyric Acid Type A (GABA A ) Receptor  Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting * □ S  Received for publication, March 9, 2012, and in revised form, May 31, 2012 Published, JBC Papers in Press, June 18, 2012, DOI 10.1074/jbc.M112.360461 Xia Wu ‡ , Zheng Wu ‡ , Gang Ning ‡ , Yao Guo ‡ , Rashid Ali § , Robert L. Macdonald ¶ , Angel L. De Blas § , Bernhard Luscher ‡ , and Gong Chen ‡1 From the Departments of ‡ Biology and  Biochemistry and Molecular Biology, Huck Institutes of Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, the § Department of Physiology and Neurobiology, University of Connecticut, Storrs, Connecticut 06269, and the ¶ Department of Neurology, Vanderbilt University Medical Center, Nashville, Tennessee 37212 Background: GABA A receptor 2 and  subunits are thought to be responsible for synaptic and extrasynaptic targeting. Results: We demonstrate here that 2 and 6 subunits can target /2 chimeras to synaptic and extrasynaptic sites. Conclusion: The  subunits play a direct role in GABA A receptor targeting. Significance: Different subunits of GABA A receptors encode intrinsic signals to control subcellular targeting. GABA A receptors (GABA A -Rs) are localized at both synaptic and extrasynaptic sites, mediating phasic and tonic inhibition, respectively. Previous studies suggest an important role of 2 and  subunits in synaptic versus extrasynaptic targeting of GABA A -Rs. Here, we demonstrate differential function of 2 and 6 subunits in guiding the localization of GABA A -Rs. To study the targeting of specific subtypes of GABA A -Rs, we used a molecularly engineered GABAergic synapse model to precisely control the GABA A -R subunit composition. We found that in neuron-HEK cell heterosynapses, GABAergic events mediated by 232 receptors were very fast (rise time 2 ms), whereas events mediated by 63 receptors were very slow (rise time 20 ms). Such an order of magnitude difference in rise time could not be attributed to the minute differences in receptor kinetics. Interestingly, synaptic events mediated by 63 or 632 receptors were significantly slower than those medi- ated by 23 or 232 receptors, suggesting a differential role of  subunit in receptor targeting. This was confirmed by differ- ential targeting of the same -2 chimeric subunits to synaptic or extrasynaptic sites, depending on whether it was co-assem- bled with the 2 or 6 subunit. In addition, insertion of a gephyrin-binding site into the intracellular domain of 6 and  subunits brought 63 receptors closer to synaptic sites. Therefore, the  subunits, together with the 2 and  sub- units, play a critical role in governing synaptic versus extra- synaptic targeting of GABA A -Rs, possibly through differen- tial interactions with gephyrin. Neural inhibition in the brain is mostly mediated by GABA A receptors (GABA A -Rs). 2 To date, 19 isoforms of GABA A -R subunits have been identified as follows: 1– 6, 1–3, 1–3, , , , , and 1–3 (1, 2). Most GABA A -Rs expressed in the brain are composed of two , two , and one  subunits, of which the  subunit can be substituted by , , , or  (3, 4). There are two forms of GABAergic inhibition, phasic and tonic (5, 6). Phasic inhibition is mediated by postsynaptically clustered GABA A -Rs composed of 1–3, 2–3, and 2 sub- units. Tonic inhibition is mediated by extrasynaptic GABA A -Rs typically composed of 4/6 (and possibly 1),  and  subunits (7–9), as well as 52 subunits (10 –12). Blocking tonic inhi- bition significantly enhanced neuronal excitability (5, 13–15). Malfunction of tonic inhibition is implicated in epilepsy, abnor- mal cognition and memory, sleep disorders, anxiety, depres- sion, schizophrenia, and alcohol addiction (16 –23). Although the mechanisms for synaptic receptor targeting have been extensively studied, little is known about the molec- ular mechanisms specifying extrasynaptic targeting of  sub- unit-containing GABA A -Rs. Neurons deficient in the 1 or 2 or 3 subunits showed diminished postsynaptic GABA A -R clusters in different subcellular localizations (24 –27). The 2 subunit, and particularly its intracellular loop (IL) and the fourth transmembrane domain (TM4), plays a critical role in synaptic clustering of GABA A -Rs (28 –31). In contrast, the  subunit-containing GABA A -Rs are mainly localized at extra- synaptic membranes (7, 8). Thus, the 2 and  subunits have been thought to be involved in the synaptic versus extrasynaptic targeting of GABA A -Rs. However, the mostly extrasynaptic 52 and punctated 1 GABA A -Rs suggest that 2 and  subunits cannot be solely responsible for guiding GABA A -R targeting (9, 10, 12). Here, we employed a molecularly engineered synapse model to investigate the mechanism of -GABA A -R targeting. We * This work was supported, in whole or in part, by National Institutes of Health Grants NS054858 from NINDS, MH083911 from NIMH (to G. C.), NS38752 (to A. L. D.) and NS33300 from NINDS (to R. L. M.), and MH062391 from NIMH (to B. L.).  This article was selected as a Paper of the Week. □ S This article contains supplemental Fig. 1 and Tables 1. 1 To whom correspondence should be addressed. Tel.: 814-865-2488; E-mail: gongchen@psu.edu. 2 The abbreviations used are: GABA A -R, GABA A receptor; IPSC, inhibitory post- synaptic current; THDOC, 3,21-dihydroxy-5-preganan-20-one; THIP, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol; GBS, gephyrin-binding site; IL, intracellular loop; TM4, fourth transmembrane domain; ANOVA, analysis of variance; GAD, glutamic acid decarboxylase; sIPSC, spontaneous IPSC. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 33, pp. 27417–27430, August 10, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. AUGUST 10, 2012 • VOLUME 287 • NUMBER 33 JOURNAL OF BIOLOGICAL CHEMISTRY 27417 by guest on June 8, 2020 http://www.jbc.org/ Downloaded from