Lateral Mobility and Anchoring ofRecombinant GABAA Receptors Depend on

Subunit Composition

Macarena Peran,1 Barry W. Hicks,2 Nancy L. Peterson,3 Helen Hooper,4 and Ramiro Salas5

1Departamento de Bioquimica, Facultad de Medicina, Universidad de Malaga,Malaga, Spain

2Department of Chemistry, US Air Force Academy, USAFA, Colorado3Department of Biochemistry, North Central College, Naperville, Illinois

4School of Applied and Molecular Sciences, University of Northumbria atNewcastle, Newcastle, Great Britain

5 Division of Neuroscience, Baylor College of Medicine, Houston, Texas

The clustering of type Ag-aminobutyric acid receptors (GABAAR) at discrete andfunctionally significant domains on the nerve cell surface is an important determinantin the integration of synaptic inputs. To discern the role that the subunits of theGABAAR play in determining the receptor’s cell surface topography and mobility, thea1, b1, b3, andg2s subunits were transfected into COS7, HEK293, and PC12 cellsand the distribution and cell surface mobility of these recombinant receptors wereexamined. Our results show thata1 subunits are retained in the endoplasmic reticulumwhile b1 and b3 subunits are sorted to the plasma membrane where they formclusters. Co-expression and co-assembly ofa1 andb3 subunits result in the rescue ofintracellulara1 subunits, which are transported asab subunit complexes to the cellsurface where they formed clusters. Fluorescence photobleach recovery and singleparticle tracking of recombinant receptors show that, despite clustering,b3 subunithomooligomers are mobile within a cell surface domain. Inclusion ofa1 in b3 orb3g2s complexes, however, dramatically reduces the receptor’s lateral mobility inCOS 7 and PC12 cells and anchors GABAARs on the cell surface, suggesting theformation of a direct link to a component of the cytoskeleton. The mobility ofrecombinant receptors that include thea1 subunit mirrors the mobility of GABAARson cell bodies and dendrites of cortical and spinal cord neurons. The results suggestthat incorporation ofa1 subunits give rise to a population of GABAARs that areimmobilized on the cell surface. Cell Motil. Cytoskeleton 50:89–100, 2001.© 2001 Wiley-Liss, Inc.

Key words: single particle tracking; fluorescence photobleach recovery; receptor mobility; cytoskeleton;anchoring

INTRODUCTION

In the mammalian central nervous system (CNS),fast inhibitory neurotransmission is largely mediated bytype A g-aminobutyric acid receptors (GABAAR). Allneurons in the CNS appear to be responsive to GABAand approximately 20% of CNS synapses utilize thisneurotransmitter. GABAARs are clustered at dendrites,cell bodies, and axonal hillocks. The maintenance ofGABAARs in these discrete and functionally significantdomains is an important feature of their integrative func-tion in neurons.

Abbreviations used: CNS: central nervous system; DC: lateral diffu-sion coefficient; ER: endoplasmic reticulum; FITC: fluorescein iso-thiocyanate; FPR: fluorescence photobleach recovery; FS: fluorescentmicrospheres; GABAAR: Type Ag aminobutyric acid receptor; MSD:mean square displacement; NGF: nerve growth factor; SPT: singleparticle tracking; TRITC: tetramethylrhodamine isothiocyanate.

Grant sponsor: Durham University, Durham, UK; Grant sponsor: NIH.

*Correspondence to: Ramiro Salas, Division of Neuroscience, BaylorCollege of Medicine, Houston, TX 77030.E-mail: rsalas@bcm.tmc.edu

Received 3 April 2001; Accepted 31 July 2001

Cell Motility and the Cytoskeleton 50:89–100 (2001)

© 2001 Wiley-Liss, Inc.

The GABAAR is a pentameric membrane protein[Nayeem et al., 1994] that, upon activation, opens achloride channel. Molecular cloning has revealed a di-verse, but inter-related, set of subunits that are divided onthe basis of sequence homology into five classes, withmultiple members:a(1-6); b(1-4); g(1-4); d; and e[McKernan and Whiting, 1996; Whiting et al., 1997].Recently, a novel GABAAR subunit, namedQ, has beenfound in rat brain [Bonnert et al., 1999]. Thea subunitsexhibit the greatest diversity in cellular distribution [Wis-den et al., 1992] and function, often conferring uniquekinetic and pharmacological properties. For example, thetype of alpha subunit present in the GABAAR determinesthe type of benzodiazepine pharmacology [Pritchett etal., 1989a; Wafford et al., 1993] and the affinity andefficacy of activation by barbiturates [Thompson et al.,1996b]. Theb subunit is essential for channel functionand some differences in receptor physiology and phar-macology have been noted for recombinant receptorscontaining differentb subunits [Sigel et al., 1990; Had-ingham et al., 1993]. Inclusion of theg subunit into thecomplex confers benzodiazepine sensitivity to the recep-tor, with some differences noted between alternativelyspliced forms of theg2 subunit [Wafford et al., 1991].

The relatively limited number of different GABA-mediated currents measured in neurons suggests that theobserved structural diversity of GABAAR subunits mayhave evolved for alternative purposes [McKernan andWhiting, 1996]. Differential receptor sorting could rep-resent one such role. The subunit composition of a givenreceptor has been reported to specify their sorting andcell surface distribution [Perez-Velazquez and An-gelides, 1993; Connolly et al., 1996b] with receptors ofdifferent subunit composition selectively clustered andsequestered in different domains [Nusser et al., 1998].Recent work in cerebellar granule cells has shown thata6 anda1 subunit containing receptors are co-localizedat many GABAergic Golgi synapses. However,a6, butnot a1 subunit containing receptors, are concentrated atglutamatergic mossy fiber synapses [Somogyi et al.,1989; Baude et al., 1992; Nusser et al., 1996]. Thesestudies suggest differential targeting and maintenance ofGABAAR subunits on the surface of the same type ofneuron.

Even in the absence of discernible synaptic contact,GABA/benzodiazepine receptors are clustered and im-mobilized at discrete regions of cortical and hippocampalneurons [Velazquez et al., 1989]. However, other thanthe inclusion of the benzodiazepine sensitivity-confer-ring g subunit, the molecular composition of the recep-tors measured in these mobility studies was not known.In this work, we have expressed recombinant subunits inCOS7, HEK293, and PC12 cells and used immunocyto-chemistry, fluorescence photobleach recovery (FPR) [El-

son et al., 1976; Watson et al., 1999], and single particletracking (SPT) [Cherry et al., 1998; Brown et al., 2000]to gain insight into the role that individual subunits playand mechanisms by which receptors are clustered andimmobilised.

RESULTS

Expression of GABAAR a, b, and g Subunits inHEK293 and COS7 Cells: Intracellular Retentionof a1 and Its Rescue by b Subunits

Immunocytochemistry of fixed and permeabiliseda1 GABAAR subunit cDNA transfected COS7 andHEK293 cells shows that thea1 subunit peptide is re-tained in an intracellular compartment that has the mor-phological characteristics of the endoplasmic reticulum(ER) (Figs. 1A, 2B). The absence of labelling on livecells confirms that homomerica1 complexes do notreach the cell surface (Figs. 1B, 2A).

In contrast,b1 and b3 homomeric subunit com-plexes were observed at the cell surface of live cellsdistributed in a punctate rather than homogeneous pattern(Fig. 1C and D). This clustered distribution was con-firmed in cells that had been fixed and permeabilized (notshown). Thus, information contained within theb1 andb3 subunits, but not thea1 subunit, codes for cell surfaceexpression.

When thea1 subunit was co-transfected withb1 orb3 subunits, it was re-routed from its intracellular loca-tion and transported to the cell surface where it assumeda clustered distribution pattern similar tob3. Both sub-units co-expressed on the surface of live labelled cells asshown in Figures 1E,F and 2C,D. Co-expression of theg2s subunit with thea1 andb3 subunits did not alter cellsurface expression or clustering ofa1b3 complexes(Figs. 1G,H, 2E,F). Furthermore, labelling of either liveor fixed cells co-transfected withg2s anda1 subunitsshowed that thea1 subunit was not expressed on the cellsurface, suggesting that unlike theb1/3 subunits, theg2ssubunit was not capable of rescuing thea1 subunit fromits intracellular location (not shown).

Expression of GABAAR Subunits in PC12 Cells:Association of the a1 Subunit With the b3Subunit in PC12 Cells Does NotCompartmentalise GABAAR to Cell Bodies or toNeurites

The a1 subunit was not observed on the cell sur-face of live transfected PC12 cells (not shown). Whenfixed and permeabilised, immunocytochemistry showsthat thea1 subunit protein is localized within the cellbody of PC12 cells (Fig. 3A), as in COS7 and HEK293cells.

90 Peran et al.

Fig. 1. Assembly and cell surface expression of GABAAR subunitsin COS7 cells.A,B: Expression of thea1 subunit.a1 cDNA subunittransfected COS7 cells stained with bd24 and visualised using FITCconjugated anti-mouse IgG. A: Fixed and permeabilized cells. B: Livecells.C,D: Cell surface expression ofb subunits. C: Cells expressingb1, live labelled with 102 and visualized with TRITC conjugatedanti-rabbit IgG. D: Cells expressingb3, live labelled with bd-17 andvisualized with TRITC conjugated anti-mouse IgG.E,F: Cell surfaceexpression ofa1b3 subunit complexes. Cells expressinga1b3 sub-

units live labelled with bd24 and 102 subunit-specific antibodies. E:a1subunit containing complexes visualized with cascade blue conjugatedanti-mouse IgG. F: Same cell visualized for theb3 subunit withTRITC-conjugated anti-rabbit IgG.G,H: Cell surface expression ofa1b3g2s complexes. Cells expressinga1b3g2s subunits live labelledwith bd24 and BODIPY FL Ro-1986. G:a1 subunit containing com-plexes visualized with TRITC conjugated anti-mouse IgG;. H:gsubunit containing complexes visualised using BODIPY FL Ro-1986.Scale bars5 20 mm.

Fig. 3. Assembly and cell surface expression of GABAAR subunitsin PC12 cells.A: Expression of thea1 subunit on fixed and perme-abilized cells.a1 cDNA subunit transfected differentiated cells stainedwith bd24 and visualised using FITC conjugated anti-mouse IgG.B,C:Cell surface expression of theb3 subunit. B:b3 subunit expressingcells labelled fixed with bd17 and visualised with TRITC conjugatedanti-mouse IgG. C: Same cell as shown in B, double stained forspectrin and visualised with FITC conjugated anti-rabbit.D,E: Cell

surface expression ofa1b3 complexes. Same differentiated cells ex-pressinga1andb3 subunits live labelled with bd24 and 102. D:a1subunit containing complexes visualized with TRICT-conjugated anti-mouse IgG. E: same cell visualized for theb3 subunit with FITC-conjugated anti-rabbit IgG. F: Cell surface expression ofa1b3g2scomplexes. Differentiated cells expressinga1b3g2s subunits live la-belled with BODIPY FL Ro-1986. Scale bars5 10 mm.

Fig. 2. Assembly and cell surface expression of GABAAR subunitsin HEK293 cells. A,B: Expression of thea1 subunit. a1 cDNAsubunit transfected COS7 cells stained with bd24 and visualised usingTRITC-conjugated anti-mouse IgG. A: Live cells. B: Fixed and per-meabilized cells.C,D: Cell surface expression ofa1b3 subunit com-plexes. Cells expressinga1b3 subunits live labelled with bd24 and102 subunit-specific antibodies. C:a1 subunit containing complexesvisualized with cascade blue conjugated anti-mouse IgG. D: Same cellvisualized for theb3 subunit with TRITC-conjugated anti-rabbit IgG.E,F: Cell surface expression ofa1b3g2s complexes. Cells expressinga1b3g2s subunits live labelled with bd24 and BODIPY FL Ro-1986.E: a1 subunit containing complexes visualized with TRITC conju-gated anti-mouse IgG;. F:g subunit containing complexes visualisedusing BODIPY FL Ro-1986. Scale bars5 10 mm.

92 Peran et al.

In contrast,b3 subunit homomeric complexes weretransported to the plasma membrane (Fig. 3B) and dis-tributed in clusters on the cell body and along the entirelength of the neurite. The highly differentiated morphol-ogy of the transfected PC12 cells is illustrated by doublelabelling with anti spectrin antibodies (Fig. 3C). Thesame clustered pattern was observed in live cells (notshown) confirming that the distribution is not a result ofeither fixation or permeabilization. Control experimentsperformed at 4°C or with Fab9 fragments show that theclusters persist and are not the result of receptor inter-nalisation or capping by the primary antibody (data notshown). Differentiation and process extension are notnecessary for sorting theb3 subunit to the plasma mem-brane since undifferentiated cells also show cell surfaceexpression (not shown).

When co-expressed and co-assembled with theb3subunit, thea1 subunit was sorted to the cell surface oflive labelled cells, where it also assumed a clustereddistribution on both cell somas and neurites (Fig. 3D,E;note double-labelled clusters). Co-expression ofg2s witha1 andb3 alters neither the relative cell surface distri-bution of the receptor complex (e.g., cell bodies vs.neurites) nor the pattern of cell surface clustering(Fig. 3F).

Rescue of the a1 Subunit From the EndoplasmicReticulum by b Subunits Anchors Receptors onthe Cell Surface.

The lateral mobility of GABAAR complexes ex-pressed on the cell surface of COS7, HEK293, and PC12cells was measured using Fluorescence Photobleach Re-covery (FPR). FPR measurements ofb3 cDNA subunittransfected COS7 and PC12 cells showed that despiteclustering, a significant proportion ofb3 homooligomershad rapid and unrestricted mobility (Table I). A repre-sentative FPR recovery curve for theb3 subunit ex-pressed in PC12 cells is shown in Figure 4A. The mobilefraction was determined to be 396 4% in PC12 cells and

43 6 13% in COS7 cells (Table I) with diffusion coef-ficients probably limited only by the viscosity of the cellmembrane (DC 5 196 3 3 10

-10cm2sec-1 in COS7 cellsand 186 3 3 10-10 cm2sec-1 in PC12 cells). The re-mainder of complexes appeared immobile during theFPR timescale and within the 2mm2 domain defined bythe laser (Table I).

FPR of co-expresseda1b3 subunits, revealed adistinct change in the lateral mobility of recombinantcomplexes in COS7 and PC12 cells. Association of thea1 subunit with theb3 subunit resulted in a significantdecrease in the mobility of heteromeric complexes in themembrane (Fig. 4B). In PC12 cells, the mobile fractiondecreased to 206 7% and in COS7 cells to 86 4%(Table I). Inclusion of theg subunit ina1b3g2s subunitcomplexes did not significantly modify the lateral mo-bility of a1b3 heteromeric complexes in either COS7 orPC12 cells (Fig. 4C, Table I).

However, when the lateral mobility ofa1b3g2ssubunit complexes expressed in HEK293 cells was ex-amined, receptors had free and unrestricted mobility(Fig. 4D). The rapidly mobile fraction was 886 13%with Dc 5 4.46 2 3 10

-10 cm2sec-1 (Table I). These datacontrast with the mobility of the same subunit combina-tions expressed in PC12 and COS7 cells.

The mobility of recombinant GABAAR was furtherexamined using single particle tracking (SPT). Fab9 frag-ment anti-a1 andb2/3 antibodies were covalently cou-pled to 50–100 nm fluorescent spheres (a1-FS andb2/3-FS) and used to track the movement of singlerecombinant complexes over the cell surface in real time.Attempts to measure regional differences in mobilitybetween cell bodies and neurites in PC12 cells were notsuccessful due to the size and geometry of the highlydifferentiated morphology. SPT of recombinantb3 sub-unit complexes on the surface of transfected COS7 cellsis shown in Figure 5A–C. The SPT paths are character-istic of unrestricted Brownian movement (Fig. 5A) withsome complexes experiencing long-range movements

TABLE I. Summary of the Lateral Mobility of Recombinant GABA AR Expressed in PC12, COS7, and HEK293 Cells*

GABAAR-subunitstransfected Cell type Label

Mobile fraction(%)

Diffusion coef.(310210 cm2/sec)

b3 COS7 Fab9-b3(bd17) 436 13 196 3b3 PC12 Fab9-b3(bd17) 396 4 186 3a1b3 COS7 Fab9-a1(bd24) 86 4 1.36 0.4a1b3 PC12 Fab9-a1(bd24) 206 7 1.26 0.5a1b3g2s COS7 Bodipy-Ro-1986 176 4 2.76 1.7a1b3g2s PC12 Bodipy-Ro-1986 276 11 1.36 0.4a1b3g2s HEK293 Bodipy-Ro-1986 886 13 4.46 2None Cortical neurona Bodipy-Ro-1986 27 1.2None Spinal corda Bodipy-Ro-1986 276 0.08 1.96 0.7

The number of FPR measurements taken from each preparation of transfected cells was 10–30, to permit statistical sampling.aVelazquez et al. [1989].

Anchoring of GABA AReceptors and Subunit Composition 93

over the cell surface (exceeding 2 mm within the 16-secSPT timescale). The mean square displacement (MSD)of 10 FS bound tob3 subunit complexes (colored linescorrespond to different paths) are shown in Figure 5B.The straight thick line represents the theoretical value forunconstrained diffusion and the black plotted points arethe average for the rapidly mobile fraction ofb3 subunitbound FS. It is initially coincident with the theoreticalvalue and thereafter remains within statistical deviationeven at large time intervals (not shown) suggesting thatmostb3 subunit homomeric complexes are freely diffus-ing. From the initial one-half second of movement, dif-fusion coefficients were estimated for each FS labeledcomplex and are shown as a histogram in Figure 5C. Themajority (66%) were rapidly mobile with a mean Dc 54.396 0.663 10-10 cm2sec-1and a mean displacement of1.4 mm. The remaining third had a slower diffusion

coefficient, and would appear by FPR as the “immobile”fraction (Table I). However, SPT measurements showthat there is movement within this region and taken incombination with FPR data suggest thatb3 subunit ho-momericb3 complexes are not “tethered” but restrictedin their mobility by a cell domain or barrier.

When thea1 subunit was included in the complexthere was a distinct change in receptor mobility measuredby SPT (Fig. 5D–F). Heteromerica1b3 subunit com-plexes display a stationary behavior (Fig. 5D; note thatboth axes scales in Fig. 5D are an order of magnitudesmaller than in Fig. 5A for homomericb3 subunit com-plexes), moving about a fixed origin. The MSD ofa1b3subunit bound FS are shown with increasing time inFigure 5E. The plotted points are the ensemble average(note the difference in magnitude in the y axis scalebetween Fig. 5E and B; the gradient for unconstrained

Fig. 4. FPR of recombinant GABAA receptors expressed in PC12 and HEK293 cells.A–C: The mobilityof GABAAR complexes expressed in PC12 cells. Representative FPR recovery curves are shown. A: FPRof the b3 subunit probed with TRITC conjugated anti-b2/3 Fab9. B: FPR ofa1b3 subunit complexesprobed with FITC conjugated anti-a1 Fab9. C: FPR ofa1b3g2s subunit complexes probed with BODIPYFL Ro-1986. D: FPR ofa1b3g2s subunit complexes expressed in HEK293 cells, probed with BODIPYFL Ro-1986. The e-2 beam radius5 0.85mm.

94 Peran et al.

diffusion tends towards the vertical). Diffusion coeffi-cients for eacha1-FS were estimated from the initialone-half second of movement (Fig. 5F) and the contrastin lateral mobility betweenb3 anda1b3 subunit com-plexes is shown by the difference in rapidly mobile and

immobile histogram populations (on the same scale).About 90% ofa1b3 subunit complexes are immobile,with a mean Dc 5 2.68 6 0.28 3 10

-12 cm2sec-1 andmean displacement of 50 nm or less (Fig. 5E). This isconsistent with the large immobile fraction ofa1b3

Fig. 5. Single particle tracking /fluorescence nanovid microscopy ofsingle recombinant GABAARs expressed on the surface of transfectedCOS7 cells.A–C: SPT ofb3 subunit complexes. A: The paths of fourdifferent fluorospheres (b3-FS) are shown by the red, blue, green, andorange lines that start from an arbitrary origin. B: Initial portion of themean square displacement (MSD) of 10b3-FS with increasing time;C: Histogram of the range of diffusion coefficients (Dc) for eachb3-FS

estimated from the initial one-half second of movement.D–F: SPT ofa1b3 subunit complexes. D: The paths of four different fluorospheres(a1-FS) are shown by the red, blue, green, and orange lines that startfrom an arbitrary origin. E: Initial portion of the MSD of 10a1-FSwith increasing time; F: Histogram of the range of Dc for eacha1-FSestimated from the initial one-half second of movement.

Anchoring of GABA AReceptors and Subunit Composition 95

subunit complexes measured by FPR (Table I), and sug-gests thata1b3 heteromeric complexes may be directlytethered to underlying elements of the cytoskeleton.

DISCUSSION

Expression of the a1 Subunit in HEK293, COS7,and PC12 Cells

This study demonstrates that expression of theGABAAR a1 subunit alone in HEK293, COS7, andPC12 cells results in retention in an intracellular com-partment. Although homomeric receptors have yet to bedescribed in neurons, previous studies have reported thatGABAAR subunits have the potential to assemble as bothhomo- and heteromeric receptors [Blair et al., 1988;Pritchett et al., 1988, Pritchett et al.,1989b; Verdoorn etal., 1990]. However, our results are consistent with theintracellular abundance ofa1 seen in CA1 hippocampalneurons and cerebellar granule neurons [Somogyi et al.,1989]. Intracellular retention of thea1 subunit inHEK293, COS7, and PC12 cells and in BHK cells [Con-nolly et al., 1996a; Gorrie et al., 1997], as it is in neurons,likely reflects a mechanism that ensures thata1 subunithomomers are not expressed on the cell surface.

We found that the distribution of intracellulara1subunit in all the cells analysed was typical of retentionwithin the ER. The flat morphology of COS7 cells clearlyhighlights the pattern, which is identical to cells stainedwith the ER markers DiOC6 and BiP [Terasaki andReese, 1992], suggesting that thea1 subunit does notacquire the necessary structural conformation requiredfor cell surface localization. Thea1 subunit lacks aclassical C-terminal KDEL ER retention signal [Pelham,1990]. However, althougha1 homopentamers have beenshown to create channels in oocytes [Blair et al., 1988],it is possible thata1 can not form pentamers in mam-malian cells and the neuron’s editing machinery will notallow membrane sorting of a non-pentameric GABAAR.

Rescue and Cell Surface Expression of the a1Subunit by b Subunits

Immunocytochemistry in COS7, HEK293, andPC12 cells show that ab subunit is required to transportthe a1 subunit from its intracellular localization to thecell surface. The role ofb subunits in targetingGABAARs has been shown previously [Perez-Velazquezand Angelides, 1993; Connolly et al., 1996a,b].

It is not known exactly how theb subunits aid incell surface expression of thea1 subunit. It is possiblethat the assembly ofb subunits with thea1 subunitmasks an ER retention signal and thus permits thea1subunit to be transported to the cell surface. Alterna-tively, a conformational change induced by co-assembly

might signal exit of thea1 subunits from the ER andconsistent with this is the report of chaperone-like effectsof K1 channels [Shi et al., 1996]. If the interactionbetweena1 and b subunits does produce a transportsignal upon assembly, then it must be common to allbsubunits because all appear equally capable of sortinga1to the cell surface (unpublished observations). Further,despite the potential of all subunits to assemble, it ispossible that neurons have an additional level of controlthat architecturally edit certain GABAAR complexesprior to their exit from the ER and/or Golgi [Klausner,1989]. This interpretation is consistent with our resultsand with experiments showing that despite consistentlyhigh a1 mRNA levels, a1 is only expressed on thecerebellar granule cell surface late in development, co-incident with expression of bothb2/b3 andg2 mRNAsand proteins [Nadler et al., 1996].

It has been shown thata1/g2s co-transfected HEKcells do not express functional channels, whilea1/b1/g2s do [Angelotti et al., 1993]. This is in agreement withour data, which suggest thata1/g2s transfected cells arenot able to sort the complexes to the plasma membraneand, therefore, no functional channels can be found onthe surface.

Role of Receptor Subunits in Clustering andAnchoring

In COS7, HEK293, and PC12 cells,b3, a1b3 anda1b3g2s complexes are clustered on the cell surface.The clustering of recombinant GABAARs in non-neuro-nal cells suggests that clustering might in part be encodedby the subunits contained within the receptor complex.Furthermore, clustered homomericb3 complexes sug-gest that this information is encoded by theb subunit. Infact, homo-oligomerization and cell surface expressionof b3 subunit are mediated by four amino acids locatedin the N-terminal domain of the subunit [Taylor et al.,1999]. However,b3 subunits do not appear to specifycell surface distribution. No differences were observedbetween the distribution of theb3 subunit expressed oncell bodies vs. neurites in PC12 cells despite their highlybranched morphology.

FPR and SPT were used to gain insight into themechanisms underlying receptor mobility. FPR measuresthe mobility of cell surface receptors in a region definedby the laser beam, in this case an area of 2mm2, inresponse to an imposed concentration gradient. SPT hasthe advantage of being able to track single receptors overdifferent parts of the cell, to determine regional differ-ences, while at the same time exploring how large thecell surface domain might be. The forces that restrictGABAAR mobility and regulate distribution may arisefrom several possible kinds of interactions. First, segre-gation to and confinement within an impermeable barrier

96 Peran et al.

or corral could define cell surface domains such as thecell body and the dendrite/axon. Examined using FPR,receptors within such a domain would appear clusteredand immobile, but by SPT would appear mobile. Second,the receptor may be linked to specialized cytoskeletalelements in the cell body, dendrite, or axon. Such a linkwould have to be highly selective for GABAARs sinceother proteins and lipids diffuse freely between theseregions [Winckler and Poo, 1996].

FPR and SPT experiments show that despite clus-tering, homomericb3 complexes are quite mobile. Thefact that ina1b3g2s transfected HEK cells a similar levelclustering is observed, while the mobile fraction is closeto 90%, suggests that clustering per se is not a majorplayer in receptor mobility determination. When thea1subunit is included,a1b3 subunit complexes are com-pletely immobilised in COS and PC12 cells, indicatingthe formation of a direct link to a component of thecytoskeleton. In fact, the interaction of the GABAAR a1subunit with two main cytoskeletal components, tubulinand actin, has been shown [Kannenberg et al., 1997].Furthermore, the data demonstrate that HEK293 cellslikely lack some critical factor that anchorsa1b3 anda1b3g2s GABA complexes in COS7, PC12, or neurons.

The results suggest thatb3 serves to cluster oraggregate receptors while thea1 subunit provides adirect link that reduces complex mobility. The “move-ment” of these anchored receptors, which is less than 50nm, is consistent with tensile cytoskeletal element(s)providing direct, but flexible attachment. The suggestionof direct anchoring is consistent with findings thatGABAARs are clustered and immobile on hippocampaland cortical neurons [Velazquez et al., 1989]. Further-more the immobility of thea1b3g2 complex is alsoconsistent with “Type I” receptors, to which botha1b1g2 anda1b3g2 belong [Lo et al., 1982]. By “op-erational” criteria, these complexes have been found inTriton X-100 insoluble fractions associated with the cy-toskeleton.

The exact molecular elements that interact with thea1 subunit and mediate clustering and restrict receptorlateral mobility are not known. The 94-kDa glycine re-ceptor associated cytoskeletal protein, gephyrin, has beenreported to co-localize with some GABAAR a subunits[Koulen et al., 1996], and to indirectly interact withg2subunit-containg GABAAR complexes [Essrich et al.,1998]. Gephyrin has been found at GABAergic but notglutamatergic terminals [Craig et al., 1996] and has beensuggested to participate in the clustering of GABAARssince expression of gephyrin in HEK293 induces clustersof b3-containing GABAARs [Kirsch et al., 1995].Rapsyn, the 43-kDa protein at neuromuscular junctionsthat clusters ACh receptors, has been reported to clusterrecombinant GABAARs composed ofa1, b1, and g2

subunits [Yang et al., 1997]. Recently the intracellulardomains ofb1, b2, b3 org2 subunits, but not those ofa1subunits have been proved to be able to form sitesmediating clustering by rapsyn [Ebert et al., 1999]. Al-though clustering of GABAARs by gephyrin and rapsynare attractive suggestions, we find thatb3 homomericreceptors form clusters in gephyrin’s and rapsyn’s ab-sence and theseb3 clusters, when examined by SPT, arenot directly attached to the cytoskeleton. The use of theyeast two-hybrid system has isolated a protein,GABARAP, that has been suggested as a candidate fortargeting and clustering of GABAAR via its interactionswith the cytoplasmic domain of theg2 subunit [Wang etal., 1999]. However, we have seen clustering ofb sub-units without cell surface immobilisation and have dem-onstrated the clustering and mobility ofa1b3g2s by FPRin HEK293 cells. The precise intermediate subplasmall-emal element(s) that interact witha1/ GABAAR com-plexes are likely to be unique because, while other ionchannels and receptors interact with the ankyrin-basedcytoskeleton, GABAARs do not [Srinivasan et al., 1988].

Significance of the Intracellular Retention andRegulated Cell Surface Expression of a1

This study shows that constraints to cell surfacelateral mobility of the receptor are specified by thea1subunit that is sequestered in an internal compartmentprior to assembly withb subunits. This is a novel func-tion for a GABAAR subunit and shows that in addition totheir role in specifying receptor function, assembly of aparticular subunit codes for receptor anchoring. There isemerging evidence that despite high levels of specificsubunit mRNAs expressed by individual neurons duringdevelopment, the corresponding GABAAR subunit is notalways expressed on the cell surface [Killisch et al.,1991].

The intracellular rescuing and sorting of thea1subunit byb subunits suggest that the ability of neuronsto control assembly and incorporate specific subunitsfrom an internally sequestered pool might give rise to apopulation of GABAARs that are spatially segregatedand immobilised on the cell surface. Thus, the composi-tion and location of GABAARs expressed on thecell surface during development or plasticity-inducedchanges might depend on the temporal availability of aspecific subunit required for assembly and may be con-trolled at the level of translation and/or subunit assembly,rather than at the transcriptional level. Indeed, recentstudies have shown that cerebellar granule cells are ableto modulate the expression ofa1 and a6-containingreceptors in response to cAMP-mediated signalling[Thompson et al., 1996a]. The recruitment of specificsubunits into complexes and their immobilization at spe-cific domains provides a mechanism by which receptors

Anchoring of GABA AReceptors and Subunit Composition 97

can be anchored at discrete sites on the neuron’s cellsurface.

MATERIALS AND METHODS

Cell Culture

PC12 cells are a well-characterized line derivedfrom the neural crest that have been used to modelmechanisms of vesicular sorting and neuronal differen-tiation [Jap Tjoen San et al., 1995; Smith et al., 1995].PC12 cells were used to explore what effects the acqui-sition of an arborized morphology and the formation ofdifferent membrane domains had on expression and lat-eral mobility of GABAAR subunits. Expression of en-dogenous GABAAR subunits were analyzed by PCR,electrophysiology, and immunocytochemistry. PCR re-vealed PC12 cells transcribe theb3 subunit. However,endogenousa1 and b3 subunits are not translated asneither protein is detected by immunocytochemistry norare whole cell GABA-mediated currents elicited fromPC12s (data not shown). HEK293 cells were cultured inEagle’s MEM supplemented with 10% fetal bovine se-rum and plated on poly-D-lysine-coated glass coverslips.COS7 cells were grown in Dulbecco’s MEM with 10%fetal bovine serum.

Differentiated PC12 Cells.PC12 cells were platedon collagen/poly-D-lysine-coated glass coverslips inRPMI medium with 10% horse serum and 5% fetalbovine serum. Differentiation of PC12 cells was inducedby the addition of 50 ng/ml nerve growth factor (NGF,7S form) and 1 mM dibutyryl cyclic AMP for 7 daysafter which cells extend processes.

Subunit Expression Vectors

Bovine GABAAR a1, rat b1-3 and murineg2ssubunit cDNAs were subcloned into mammalian expres-sion vectors pSVK3 (Pharmacia, Herts, UK) and pCDM8(Invitrogen, San Diego, CA).

Transfection of Cells

Lipofectamine: (GIBCO BRL, Paisley, Scotland)was used according to the manufacturer’s instructions.Electroporation: 400ml of cells (1–53 107 cells/ml) inserum-free medium were electroporated (Electro CellManipulator ECM 395) and plated at a density of 13105/cm2. Mock transfections were performed with vectorDNA alone. Analysis of cells was performed 48 h posttransfection.

Immunocytochemistry.

Antibodies. Monoclonal anti-a1 (bd24) and anti-b2/3 (bd17) (Boehringer, Germany) were used at a 1:50dilution; polyclonal anti-spectrin (gift from E.-H. Joe)was used at a 1:10 dilution and a polyclonalb-antibody

(102) raised against an N-terminal peptide from the ratb3 sequence: QSVNDPGNMSFVKET was used at a1:200 dilution. Antibody 102 was characterized by im-munoblot analysis and immunocytochemistry of trans-fected cells and recognizes all threeb subunits (data notshown). Secondary antibodies used were TRITC-conju-gated goat anti-mouse, FITC-conjugated goat anti-rabbit(Calbiochem, Nottingham, UK) or Cascade blue-conju-gated goat anti-mouse antibody (Molecular Probes, Lei-den, The Netherlands) at 1:200 dilution.

Labeling of fixed and permeabilized cells.Cellswere fixed with 4% paraformaldehyde in phosphate buff-ered saline (PBS) for 15 minutes, washed twice withPBS, blocked in 10% heat inactivated serum (HIS) for 15min, and then incubated for 1 h in primary antibodydiluted in buffer A (1 mg/ml BSA, 10% HIS, 0.5% TritonX-100, PBS) at room temperature. Cells were washedthree times in PBS and then incubated for 30 min withsecondary antibodies in buffer A. Controls were per-formed with mock transfected cells and using secondaryantibody only.

Labeling of live cells.The bottom of culture disheswere replaced by coverslips that permitted direct viewingof live cells using a high numerical aperture objective ofan inverted microscope. Transfected cells plated in thesedishes were washed twice in PBS, then incubated at 4°Cin primary antibody diluted in growth media for 15 minand secondary antibody for 10 min.g-subunit containingreceptors were labeled with 40 nM of BODIPY FLRo-1986 in PBS for 20 min at room temperature.

Image capture and analysis.Images were ob-tained using a Bio-Rad microRadiance confocal micro-scope or a Nikon FX-35A camera adapted to a Nikonfluorescence microscope. Images of BODIPY FL Ro-1986 labeled receptors were obtained through the Zeissphotomicroscope III used in the FPR system where theargon laser, tuned to 496 nm (100 mW), served as theillumination source. The laser beam was dispersed by adiffusion lens placed in the exciting light path. Photo-graphs were recorded through a Zeiss 633 water immer-sion 1.2NA objective on Ektachrome film pushed to ASA3200. Images were transferred to Adobe Photoshop 3.0and printed on a Kodak high resolution color printer.

Lateral Mobility of Recombinant GABAARs

Fluorescence photobleach recovery.Fab9 frag-ments of monoclonal antibodies bd17 (antib2/3) or bd24(anti a1) were prepared by papain digestion as describedpreviously [Harlow, 1988] and were used to label trans-fected cells. FITC or TRITC-conjugated goat anti mouseFab9 fragments were used as secondary antibodies (Cap-pel Research Products, Durham, NC). BODIPY FL Ro-1986 was used to probe the cell surface mobility ofa1b3g2s complexes. Controls to determine the level of

98 Peran et al.

non-specific binding were performed with 1 mM chloraz-epate to displace benzodiazepine binding. Initially, usinga 633 water immersion integrated into the FPR micro-scope (a Zeiss Photomicroscope III), bright field opticswere used to localise cells. Those transfected were iden-tified and selected for photobleaching by their high flu-orescence signal intensity. A 90/10 mirror was then usedto bleach a 2mm2 region of the cell surface with an argonlaser. FPR measurements were carried out using the 488nm (FITC), 496 nm (BODIPY FL Ro-1986), or 514 nm(TRITC) lines. Multiple measurements (N5 10–30)were routinely taken from a single preparation of trans-fected cells, thus permitting statistical sampling whencalculating the percentage mobile fraction (F) and lateraldiffusion coefficient (Dc).

Single Particle Tracking Fluorescence NanovidMicroscopy Of Single GABAARs

Paucivalent fluorescent microspheres (100 nm; FS)(Fluorospheres, Molecular Probes, Leiden, The Nether-lands) were prepared with antibodies bd24 and bd17(Boehringer, Germany) against thea1 (FS-a1) andb3(FS-b3) subunits as described previously [Hicks andAngelides, 1995]. Transfected COS7 cells were directlylabeled with FS-b3 or FS-a1 antibodies and abundantlylabeled cells were identified using a 100X 1.3 NA ob-jective. The movements of individual FS bound to recep-tors were monitored at high magnification by low lightvideo microscopy using a silicon intensified target cam-era. The resolution of the movement of the receptorsunder the conditions used for these experiments was7.5 6 0.4 nm and was limited primarily to cameradistortions.

ACKNOWLEDGMENTS

Macarena Peran was supported by a Marie CurieFellowship from the European Community. GABAARcDNAs were kind gifts from D. Burt, H. Luddens, and P.Seeburg. PC12 cells were a gift from L. Greene. The antispectrin antibody was a kind gift from E.H.Joe. Manythanks to Dr. C. Thompson and Prof. K. Bowler forscrutiny of this manuscript. This work was supported byDurham University (Durham, U.K.) and the N.I.H.

REFERENCES

Angelotti TP, Uhler MD, Macdonald RL. 1993. Assembly of GABAAreceptor subunits: analysis of transient single-cell expressionutilizing a fluorescent substrate/marker gene technique. J Neu-rosci 13:1418–1428.

Baude A, Sequier JM, McKernan RM, Olivier KR, Somogyu P. 1992.Differential subcellular distribution of the alpha 6 subunit ver-sus the alpha 1 and beta 2/3 subunits of the GABAA/benzodi-

azepine receptor complex in granule cells of the cerebellarcortex. Neuroscience 51:739–748.

Blair LA, Levitan ES, Marshall J, Dionne VE, Barnard EA. 1988.Single subunits of the GABAA receptor form ion channels withproperties of the native receptor. Science 242:577–579.

Bonnert TP, McKernan RM, Farrar S, le Bourdelles B, Heavens RP,Smith DW, Hewson L, Rigby MR, Sirinathsinghji DJ, BrownN, Wafford KA, Whiting PJ. 1999. theta, a novel gamma-aminobutyric acid type A receptor subnit. Proc Natl Acad SciUSA 96:9891–9896.

Brown FL, Leitner DM, McCammon JA, Wilson KR. 2000. Lateraldiffusion of membrane proteins in the presence of static anddynamic corrals: suggestions for appropriate observables. Bio-phys J 78:2257–2269.

Cherry RJ, Smith RP, Morrison IEG, Fernandez N. 1998. Mobility ofcell surface receptors: a re-evaluation. FEBS Lett 430:88–91.

Connolly CN, Krishek BJ, McDonald BJ, Smart TG, Moss SJ. 1996a.Assembly and cell surface expression of heteromeric and ho-momeric gamma- aminobutyric acid type A receptors. J BiolChem 271:89–96.

Connolly CN, Wooltorton JR, Smart TG, Moss SJ. 1996b. Subcellularlocalization of gamma-aminobutyric acid type A receptors isdetermined by receptor beta subunits. Proc Natl Acad Sci USA93:9899–9904.

Craig AM, Banker G, Chang W, McGrath ME, Serpinskaya AS. 1996.Clustering of gephyrin at GABAergic but not glutamatergicsynapses in cultured rat hippocampal neurons. J Neurosci 16:3166–3177.

Ebert V, Scholze P, Fuchs K, Sieghart W. 1999. Identification ofsubunits mediating clustering of GABA(A) receptors byrapsyn. Neurochem Int 34:453–463.

Elson EL, Schlessinger J, Koppel DE, Axelrod D, WW W. 1976.Measurement of lateral transport on cell surfaces. Prog ClinBiol Res 9:137–147.

Essrich C, Lorez M, Benson JA, Fritschy JM, Luscher B. 1998.Postsynaptic clustering of major GABAA receptor subtypesrequires the gamma 2 subunit and gephyrin. Nat Neurosci1:563–571.

Gorrie GH, Vallis Y, Stephenson A, Whitfield J, Browning B, SmartTG, Moss SJ. 1997. Assembly of GABAA receptors composedof alpha1 and beta2 subunits in both cultured neurons andfibroblasts. J Neurosci 17:6587–6596.

Hadingham L, Wingrove PB, Wafford KA, Bain C, Kemp JA, PalmerKJ, Wilson AW, Wilcox AS, Sikela JM, Ragan CI, et al. 1993.Role of the beta subunit in determining the pharmacology ofhuman gamma- aminobutyric acid type A receptors. Mol Phar-macol 44:1211–1218.

Harlow E LD. 1988. Antibodies, a laboratory manual. Cold SpringHarbor, NY: Cold Spring Harbor Laboratory.

Hicks BW, Angelides KJ. 1995. Tracking movements of lipids andThy1 molecules in the plasmalemma of living fibroblasts byfluorescence video microscopy with nanometer scale precision.J Membr Biol 144:231–244.

Jap Tjoen San ER, van Rozen AJ, Nielander HB, Oestreicher AB,Gispen WH, Schotman P. 1995. Expression levels of B-50/GAP-43 in PC12 cells are decisive for the complexity of theirneurites and growth cones. J Mole Neurosci 6:185–200.

Kannenberg K, Baur R, Sigel E. 1997. Proteins associated with alpha1-subunit-containing GABAA receptors from bovine brain. JNeurochem 68:1352–1360.

Killisch I, Dotti CG, Laurie DJ, Luddens H, Seeburg PH. 1991.Expression patterns of GABAA receptor subtypes in developinghippocampal neurons. Neuron 7:927–936.

Anchoring of GABA AReceptors and Subunit Composition 99

Kirsch J, Kuhse J, Betz H. 1995. Targeting of glycine receptor sub-units to gephyrin-rich domains in transfected human embryonickidney cells. Mol Cell Neurosci 6:450–461.

Klausner RD. 1989. Architectural editing: determining the fate ofnewly synthesized membrane proteins. New Biol 1:3–8.

Koulen P, Sassoe-Pognetto M, Grunert U, Wassle H. 1996. Selectiveclustering of GABA(A) and glycine receptors in the mamma-lian retina. J Neurosci 16:2127–2140.

Lo MM, Strittmatter SM, Snyder SH. 1982. Physical separation andcharacterization of two types of benzodiazepine receptors. ProcNatl Acad Sci USA 79:680–684.

McKernan RM, Whiting PJ. 1996. Which GABAA-receptor subtypesreally occur in the brain? Trends Neurosci 19:139–143.

Nadler LS, Raetzman LT, Dunkle KL, Mueller N, Siegel RE. 1996.GABAA receptor subunit expression and assembly in culturedrat cerebellar granule neurons. Brain Res Dev Brain Res 97:216–225.

Nayeem N, Green TP, Martin IL, Barnard EA. 1994. Quaternarystructure of the native GABAA receptor determined by electronmicroscopic image analysis. J Neurochem 62:815–818.

Nusser Z, Sieghart W, Somogyi P. 1998. Segregation of differentGABAA receptors to synaptic and extrasynaptic membranes ofcerebellar granule cells. J Neurosci 18:1693–1703.

Nusser Z, Sieghart W, Stephenson FA, Somogyi P. 1996. The alpha 6subunit of the GABAA receptor is concentrated in both inhib-itory and excitatory synapses on cerebellar granule cells. JNeurosci 16:103–114.

Pelham HR. 1990. The retention signal for soluble proteins of theendoplasmic reticulum. Trends Biochem Sci 15:483–486.

Perez-Velazquez JL, Angelides KJ. 1993. Assembly of GABAA re-ceptor subunits determines sorting and localization in polarizedcells. Nature 361:457–460.

Pritchett DB, Sontheimer H, Gorman CM, Kettenmann H, SeeburgPH, Schofield PR. 1988. Transient expression shows ligandgating and allosteric potentiation of GABAA receptor subunits.Science 242:1306–1308.

Pritchett DB, Luddens H, Seeburg PH. 1989a. Type I and type IIGABAA-benzodiazepine receptors produced in transfectedcells. Science 245:1389–1392.

Pritchett DB, Sontheimer H, Shivers BD, Ymer S, Kettenmann H,Schofield PR, Seeburg PH. 1989b. Importance of a novelGABAA receptor subunit for benzodiazepine pharmacology.Nature 338:582–585.

Shi G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, TrimmerJS. 1996. Beta subunits promote K1 channel surface expres-sion through effects early in biosynthesis. Neuron 16:843–852.

Sigel E, Baur R, Trube G, Mohler H, Malherbe P. 1990. The effect ofsubunit composition of rat brain GABAA receptors on channelfunction. Neuron 5:703–711.

Smith CJ, Anderton BH, Davis DR, Gallo JM. 1995. Tau isoformexpression and phosphorylation state during differentiation ofcultured neuronal cells. FEBS Lett 375:243–248.

Somogyi P, Takagi H, Richards JG, Mohler H. 1989. Subcellularlocalization of benzodiazepine/GABAA receptors in the cere-bellum of rat, cat, and monkey using monoclonal antibodies. JNeurosci 9:2197–2209.

Srinivasan Y, Elmer L, Davis J, Bennett V, Angelides K. 1988.Ankyrin and spectrin associate with voltage-dependent sodiumchannels in brain. Nature 333:177–180.

Taylor PM, Thomas P, Gorrie GH, Connolly CN, Smart TG, Moss SJ.1999. Identification of amino acid residues within GABA(A)receptor beta subunits that mediate both homomeric and het-eromeric receptor expression. J Neurosci 19:6360–6371.

Terasaki M, Reese TS. 1992. Characterization of endoplasmic reticu-lum by co-localization of BiP and dicarbocyanine dyes. J CellSci 101:315–322.

Thompson CL, Pollard S, Stephenson FA. 1996a. Bidirectional regu-lation of GABAA receptor alpha1 and alpha6 subunit expres-sion by a cyclic AMP-mediated signalling mechanism in cere-bellar granule cells in primary culture. J Neurochem 67:434–437.

Thompson SA, Whiting PJ, Wafford KA. 1996b. Barbiturate interac-tions at the human GABAA receptor: dependence on receptorsubunit combination. Br J Pharmacol 117:521–527.

Velazquez JL, Thompson CL, Barnes EM, Angelides KJ. 1989. Dis-tribution and lateral mobility of GABA/benzodiazepine recep-tors on nerve cells. J Neurosci 9:2163–2169.

Verdoorn TA, Draguhn A, Ymer S, Seeburg PH, Sakmann B. 1990.Functional properties of recombinant rat GABAA receptorsdepend upon subunit composition. Neuron 4:919–928.

Wafford KA, Burnett DM, Leidenheimer NJ, Burt DR, Wang JB,Kofuji P, Dunwiddie TV, Harris RA, Sikela JM. 1991. Ethanolsensitivity of the GABAA receptor expressed in Xenopus oo-cytes requires 8 amino acids contained in the gamma 2L sub-unit. Neuron 7:27–33.

Wafford KA, Whiting PJ, Kemp JA. 1993. Differences in affinity andefficacy of benzodiazepine receptor ligands at recombinantgamma-aminobutyric acidA receptor subtypes. Mol Pharmacol43:240–244.

Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW. 1999.GABA(A)-receptor-associated protein links GABA(A) recep-tors and the cytoskeleton. Nature 397:69–72.

Watson FL, Heerssen HM, Moheban DB, Lin MZ, Sauvageot CM,Bhattacharyya A, Pomeroy SL, RA S. 1999. Rapid nuclearresponses to target-derived neurotrophins require retrogradetransport of ligand-receptor complex. J Neurosci 19:7889–7900.

Whiting PJ, McAllister G, Vassilatis D, Bonnert TP, Heavens RP,Smith DW, Hewson L, O’Donnell R, Rigby MR, Sirinathsing-hji DJ, Marshall G, Thompson SA, Wafford KA, Vasilatis D.1997. Neuronally restricted RNA splicing regulates the expres-sion of a novel GABAA receptor subunit conferring atypicalfunctional properties [corrected; erratum to be published]. JNeurosci 17:5027–5037.

Winckler B, Poo M. 1996. No diffusion barrier at axon hillock. Nature379:213.

Wisden W, Laurie DJ, Monye H, Seeburg PH. 1992. The distributionof 13 GABAA receptor subunit mRNAs in the rat brain. I.Telencephalon, diencephalon, mesencephalon. J Neurosci 12:1040–1062.

Yang SH, Armson PF, Cha J, Phillips WD. 1997. Clustering ofGABAA receptors by rapsyn/43kD protein in vitro. Mol CellNeurosci 8:430–438.

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INTRODUCTIONRESULTSFig. 1.Fig. 2.Fig. 3.TABLE I.Fig. 4.Fig. 5.

DISCUSSIONMATERIALS AND METHODSACKNOWLEDGMENTSREFERENCES