Neuron,

Vol. 4, 919-928,

Functional Receptors

June, 1990, Copyright

0 1990 by Cell Press

Properties of Recombinant Rat GABA* Depend upon Subunit Composition

Todd A. Verdoorn: Andreas Draguhn: Sanie Ymer,+* Peter H. Seeburg,+ and Bert Sakmann* *Abteilung Zellphysiologie Max-Planck-lnstitut fur medizinische Forschung Heidelberg Federal Republic of Germany *Laboratory of Molecular Neuroendocrinology Center for Molecular Biology University of Heidelberg Heidelberg Federal Republic of Germany

Summary GABA-gated chloride channels were expressed in human embryonic kidney cells following transfection of cDNAs encoding the al, 82, and y2 subunits of the rat GABAA receptor (CABAR). Functional properties were determined using patch-clamp techniques in the whole-cell and outside-out configurations. large whole-cell currents were observed in cells expressing the al(32, aly2, and alB2y2 subunit combinations. The unique characteristics of GABAR channels consisting of these subunit combinations depended upon the presence or absence of (32 and y2 subunits. GABA-activated currents in cells expressing GABARs with the (32 subunit desensitized faster and showed greater outward rectification, and the channels had a shorter mean open time than GABARs composed of aly2 subunits. When the 72 subunit was present the resulting GABAR channels had a larger conductance. The slope of the concentration-response curve was significantly steeper for GABARs composed of alB2y2 subunits compared with GABARs consisting of al(32 or aly2 subunit combinations. Introduction The structurally related family of multimeric ligandgated ion channels, including receptors for y-aminobutyric acid (GABA), glycine, and acetylcholine, is becoming increasingly complex with the isolation of cDNAs encoding numerous subunits within each group (for reviews, see Schofield, 1989; Steinbach and Ifune, 1989). Heterogeneity of GABARs was previously postulated based largely upon receptor binding studies (Sieghart and Karobeth, 1980; Nielsen and Braestrup, 1980), but the much greater diversity implied by the existence of a wide range of subunits and subunit variants (Schofield et al., 1987; Levitan et al., 1988b; Lolait et al., 1989; Ymer et al., 1989; Khrestchatisky et al., 1989) was unexpected. In fact, physio-

*Present address: Division of Biochemistry and ogy, The John Curtin School of Medical Research, Australia.

Molecular Canberra

BiolCity,

logical studies on neurons have failed to establish functional GABAR heterogeneity clearly. The multiple conductance state channels opened by GABA (Hamill et al., 1983; Smith et al., 1989) may be due to activation of multiple GABAR subtypes, but this behavior could theoretically arise from a single protein. Therefore, it is important to determine whether structural diversity results in comparable functional diversity and to examine the role of different GABAR subunits in the expression of functionally distinct GABAR subtypes. The function of GABARs constructed from several subunit combinations and homomeric subunits has been studied using Xenopus laevis oocytes (Levitan et al., 1988a; Blair et al., 1988; Ymer et al., 1989) and transfected mammalian cells (Pritchett et al., 1988, 1989; Moss et al., 1990). In most cases GABA-activated currents mediated by these receptors are blocked by bicuculline potentiated by pentobarbital and carried mainly by Cl- ions. However, the functional information currently available is incomplete, and meaningful comparisons with neuronal GABARs are not yet possible. Indeed, most subunit combinations produce GABARs that lack functional features of the native receptor. To facilitate comparisons with GABARs in native membranes and to determine the functional role of particular subunits, we characterized the conductance and gating properties of GABARs expressed from three combinations of cloned subunit-specific cDNAs. Mammalian cells transiently transfected with various combinations of cDNA clones encoding the rat al, 82, and y2 subunits were used because these particular subunits are abundantly expressed in the rat central nervous system and are colocalized in a number of brain regions (l? H. S., unpublished data). The use of wholecell voltage clamp technique on small cells allows rapid application of known concentrations of GABA with a U-tube (Krishtal and Pidoplichko, 1980; Fenwick et al., 1982). Thus, differences in desensitization and concentration-response relationships can be measured. GABA-activated single-channel currents can be unambiguously identified in outside-out membrane patches with little interference from endogenous stretch-activated channels, a problem that is particularly acute in X. laevis oocytes (Methfessel et al., 1986). These techniques have revealed that both conductance and gating properties depend upon the subunit composition of the GABAR channel. Results Comparison of Subunit Assembly and Expression We initially examined receptors in cells expressing combinations of three cloned subunit cDNAs: al, (32, and ~2. Because single subunits (Pritchett et al., 1988; Blair et al., 1988) and various combinations of cloned subunits (Levitan et al., 1988b; Pritchett et al., 1988,

NC!UKMl 920

1989) produce GABA-gated channels, we compared the expression efficiency of each possible combination by measuring the amplitude of GABA-activated currents in electrically coupled clusters of transfected cells. Such measurements helped estimate the density of functional receptors produced upon transfection because a population of cells (100-500) could be tested for GABA responsiveness simultaneously. Ceils in clusters were not suitable for quantitative functional characterization, however, because voltage clamp of events occurring in distant cells was inadequate. Only ceils that did not contact other cells, which we termed lone cells, were used for quantitative experiments. Assuming that transfection of different subunit combinations does not affect the degree of electrical coupling, the amplitude of GABA-activated currents in cell clusters depends upon the percentage of cells expressing functional GABARs and the number of receptors expressed per cell. The highest level of expression was found for the aly2, alB2, and alB2y2 subunit combinations. The amplitudes of currents activated by 10 PM GABA (mean i SEM) in clusters of cells transfected with these combinations were 1100 & 250 pA (n = 5), 1500 + 360 pA (n = 6), and 1400 + 500 pA (n = IO), respectively. In cultures with this level of expression ~40% of the lone cells tested contained functional GABARs. Cells transfected with single subunit cDNAs or B2y2 combinations also expressed functional receptors, but the level of expression was much lower. In these instances currents activated by IO uM GABA in clusters of cells were approximately an order of magnitude smaller: 220 + 30 pA (n = 5) for al, 75 & 18 pA (n = 5) for B2,96 k 40 pA (n = 5) for ~2, and 85 2 22 pA (n = 5) for (32~2 cDNA-transfected cells. This large difference in expression level was not due to differences in sensitivity to GABA, because higher concentrations of GABA (100 PM) did not significantly increase the amplitude of the responses measured in these cells. More importantly, no lone cells containing receptors were found (n = 13 for al, n = 9 for ~2; the other combinations were not tested), suggesting that low cluster responses reflected low efficiency expression and assembly of these constructs. Untransfected or sham-transfected cells never showed responses to any concentration of GABA. The low density of receptors in cells transfected with single subunits or the B2y2 combination made proper functional characterization impractical, and these constructs were not further studied. In marked contrast, functional GABAR expression in cells transfected with the aly2, alB2, and alB2y2 combinations is robust, suggesting that transfection and assembly of these subunit combinations into functional GABARs are more efficient. Therefore, when these preferred combinations are transfected together, the population of GABAR channels formed from homooligomeric subunits or from the (32~2 subunit combination is probably low, because free subunits are more likely to be incorporated into the preferred com-

Bath Perfusion IO uMGABA

Figure 1. Rapid Application of GABA Improves Resolution Amplitude and Time Course of Whole-Cell Currents

of the

inward currents evoked by 10 pM CABA applied by bath perfusion (top trace) and a U-tube (bottom trace; Krishtal and Pidoplichko, 1980; Fenwick et al., 1982) were recorded from the same cell. The cell expressed GABARs composed of the alp2 subunit combination. The holding potential was -60 mV. The bars above the traces indicate duration of CABA application. Note the increased peak amplitude and more rapid onset and decay of currents seen when U-tube application was used.

binations. Of the highly expressing combinations, GABARs composed of the dual subunit combinations aly2 and alB2 showed the most pronounced functional differences. We thus focused on the properties of these combinations in the following descriptions. Desensitization Rapid application of GABA with a U-tube (Figure 1) allowed more accurate measurements of amplitude and time course of whole-cell currents as compared with previous studies in Xenopus oocytes (Schofield et al., 1987; Levitan et al., 1988a, 198813; Blair et al., 1988) and transfected cells (Pritchett et al., 1988,1989). Use of this application technique revealed a striking difference in desensitization properties between GABARs composed of aly2 subunits and those composed of alB2 (Figure 2). Currents mediated by alp2 GABARs desensitized more rapidly in response to lower concentrations of GABA (3-10 PM) than those mediated by the aly2 combination (Figure 2A). The time course of desensitization could be best fit by the sum of two exponentials in al82 GABARs, whereas the aly2 GABARs usually exhibited oniy one obvious component (Figure 2B). The desensitization time constant was dependent upon GABA concentration in both cases, but higher concentrations of GABA were required to desensitize aly2 GABARs compared with a?(32 GABARs (Figure 2C). The rate of desensitization was 10-fo!d

Recombinant 921

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2. Desensitization

of GABA-Activated

Tim:

Whole-Ceil

10

(s)

Currents

Mediated

by CABARs

Composed

of Two Combinations

of Subunits

(A) Superimposed current traces elicited at -60 mV by 10 s applications of various GABA concentrations (indicated in PM to the left of trace) in cells expressing alp2 (top) and aly2 (bottom) GABARs. (B) Plots of amplitude (log scale) as a function of time after the start of drug application (10 PM GABA for alp2 and 30 PM GABA for aly2) taken from the same cells as described in (A). Two time constants (1.1 and 20 s) can be clearly distinguished in the cell expressing a162 GABARs (top) whereas only one component is apparent with respect to aly2 CABARs (bottom, time constant = 12 s). (C) A double logarithmic plot of time constant as a function of CABA concentration. Each point represents the mean f SEM of 3-7 determinations for cells expressing aly2 (circles) or alp2 (squares) CABAR subtypes. The points describing alp2 GABAR behavior represent the faster of the two components of desensitization. The time constants are plotted on a logarithmic scale to facilitate comparison between the two groups, which are nearly IO-fold different.

faster in alp2 GABARs (fast time constant at 10 VM GABA = 1.2 k 0.5 s, n = 7) than in aly2 GABARs (time constant at 10 PM GABA = 10.8 k 1.2 s, n = 4). The differing desensitization behavior was reflected in the apparent forward rate constants and Kds of the desensitization reactions, which were estimated by linear regression of the relationship between the reciprocal of the time constant and the GABA concentration (Bernasconi, 1976). For a182 GABARs the apparent forward rate constant of fast desensitization was 0.44 s-l and the Kd was 12 PM, whereas aly2 GABARs exhibited an apparent forward rate constant of 0.10 s-l and a Kd of 66 PM. Concentration-Response Relationships GABA concentration-response relationships were determined for receptors composed of each subunit combination (Figure 3). The slope of the relationship between peak current response (Figure 3, insets) and GABA concentration in double logarithmic coordinates was slightly above the one in cells expressing alp2 or aly2 GABARs. This suggests that channel activation was cooperative in GABARs consisting of each

combination. Although the slope measured in cells expressing aly2 GABARs was slightly higher than that found for the alp2 GABARs, the difference was not statistically significant (Table 1). The sensitivity to GABA was also different in alp2 and aly2 receptors. Cells expressing al(32 GABARs usually responded well to 1 WM GABA, whereas 5-10 PM GABA was normally required to activate aly2 GABARs. Current-Voltage Relationships Current-voltage curves showed that GABA opened Cl--selective ion channels for each combination of subunits. In solutions containing symmetrical Clthe reversal potential was essentially 0 mV (aly2,1.4 f 1.5 mV; alfl2,1.8 f 2.0, n = 7 in both cases) and shifted according to the Nernst relationship for a Cl--selective channel when half of the extracellular Cl- was replaced by the impermeant anion isethionate (data not shown). Whole-cell current-voltage relationships showed only slight outward rectification in cells containing aly2 GABARs. The alp2 subunit combination produced receptors with markedly more outward rectification (Figure 4), which resulted in a ratio of cord

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Figure rents

3. Concentration

Dependence

3b (FM)

of GABA-Activated

Cur-

Shown are double logarithmic plots of peak whole-ceil currents as a function of GABA concentration for individual cells expressing either aID2 (A) or aly2 (B) subunit combinations. The insets show superimposed inward currents at -60 mV evoked by 2 s applications of GABA that were used to construct the plots (1, 2, 3, and 5 PM GABA in A; 3, 5, IO, and 30 PM GABA in B; 20 PM GABA point is not shown in inset). The calibration bars represent 200 pAand 1.7s in (A)and 100 pAand 1.7s in(B). The peakamplitudes were reproducible throughout the experiment, and thus the points represent the results from at least two GABA applications. Linear regression of the concentration-response relationship are shown as solid lines. The slopes of the lines are 1.07 (A) and 1.38 (6) in these particular cells.

(+I00 mV/-100 mV) -2-fold higher than of the aly2 GABARs (Table 1). Analogous to neuronal GABARs (Bormann et al., 1987), this indicates that channel gating may be voltage dependent. conductances

that

Channel Gating and Conductance We wished to determine whether the diverse characteristics of macroscopic currents mediated by al(32 and aly2 GABARs were similarly apparent with respect to single-channel behavior. Therefore, we investigated the conductance and gating properties of recombinant GABAR channels using fluctuation analysis of whole-cell currents and single-channel recording in outside-out membrane patches.

Fluctuation Analysis The marked increase in current noise upon application of low concentrations of GABA to cells expressing recombinant GABARs lent itself well to fluctuation analysis (Figure SA, top). The main time constants governing kinetic behavior were determined from power density spectra fitted with the sum of two Lorentzians, and the relationship between current variance and amplitude provided an estimate of the elementary conductance (Figure 5A, bottom). The derived time constant of the slower component (35 ms) did not vary between combinations and was similar to slow time constants observed in a number of neuronal preparations (Bormann et al., 1987; MacDonaid et al., 1989). The faster time constant was significantly different between GABAR channels composed of alp2 and al+! subunit combinations (Table 1). Receptors consisting of al82 subunits showed a time constant of 1.3 i 0.1 ms, nearly 3-fold faster than that observed in cells expressing the ally2 combination (3.6 + 0.5 ms). The relative contribution of each component w/as aiso different, with the fast component being significantly more prominent in a1[32 receptors (ratio fast/slow: 1.1 f 0.2, n = 5) than in GABARs consisting of aly2 subunits (ratio: 0.37 + 0.09, n = 5, p < 0.01, two-tailed t test). The elementary conductance estimated by the relationship between mean amplitude of whole-cell current and variance was likewise significantly different for aID2 receptors compared with aly2 receptors (Figure 5, Table I). A relatively low conductance of 8.4 pS was observed in cells expressing al!32 GABARs, whereas aly2 GABARs mediated currents with an elementary conductance of 22 pS. These results indicate that the gating properties and conductance of GABARs depend upon the subunit composition, with alp2 GABAR channels having a smaller conductance and shorter mean open time than aI?2 GABAR channels. Single-Channel Currents Direct measurement of single GABA-activated channels in outside-out membrane patches confirmed the results obtained with fluctuation analysis. Figure 6A (top) shows the currents activated by application of 1 t.rM GABA to an outside-out membrane patch at -50 mV with GABAR channels consisting of all32 subunits. The middle trace at higher time resolution illustrates that elementary events are brief and of relatively small amplitude. An all-points histogram (below,) taken from the same patch indicates that the most frequently OCcurring amplitude is 0.55 pA, corresponding to a conductance

of

11

pS.

Openings

to

other

amplitudes

were also apparent (0.7 and 0.9 PA), but they were not clearly resolved in the histograms. Histograms constructed from measurements at patch potentials of -90 mV were similarly unable to resolve additional closely spaced conductance states. We were uncertain

whether

they

are

due

to

unresolved

superim-

posed channel openings or resulted from openings to separate conductance states. In contrast, GABAR channels composed of aly2 subunits have higher conductances and longer open time (Figure 6B, top and mid-

Recombinant 923

Table

GABAR

Channels

1. Characteristics

of Currents

Mediated

Property

by Recombinant

CABA

ulB2

Slope Concentration-Response Rectification Ratio Channel Kinetics bw Cm) hiI (ms)

aly2

1.18 & 0.08 (n = 5) 2.87 f 0.46 (n = 7)

Elementary Conductance (pS) Main Single-Channel Conductance

(pS)

The rectification ratio is estimated from the conductance at -100 mV. Channel kinetics conductance is estimated from fluctuation B Value is significantly different from those

35.6 1.29 (n = 8.38 11.3

Receptors

k * 5) f +

4.4 0.12" 1.6d (n = 6) 0.23d (n = 6)

alB2y2

1.46 * 0.10 (n = 8) 1.47 + 0.2ga (n = 7) 35.9 3.64 (n = 22.2 30.8

k 6.6 f 0.51" 5) k 3.2 (n = 5) +l.O(n = 7)

current-voltage relationship by dividing the cord is estimated from fluctuation analysis of whole-cell analysis. The main single channel conductance is obtained with the other subunrt combinations (p

die). The better signal to noise ratio afforded by these channels allowed a clear distinction between two conductance states. In three of seven patches a single conductance state of 30.8 + 1.0 pS (n = 7) was observed. However, in four of the patches an additional subconductance state of 16.8 k 0.7 pS was seen (Figure 6B, bottom). CABAR Channels Formed in Cells Expressing Three Different Subunits Previous studies have indicated that GABARs comprised of al, (31, and ~2 subunits exhibit pharmacological differences compared with double subunit combinations (Pritchett et al., 1989). To determine whether functional differences exist, we studied the properties of alp2y2 GABARs. Currents mediated by al(32y2 GABARs were similar in time course to those mediated by aly2 GABARs (Figures 7A and 38, inset). However, a notable difference was seen with respect to the concentration-response relationship (Figure 7A), the

2.12 * 0.12d (n = 9) 2.84 k 0.36 (n = 5) 35.7 2.10 (n = 17.1 32.0

k * 4) * +

4.8 0.21" 3.1 (n = 4) 0.8 fn = 4)

conductance at 100 mV by the cord currents. r = li(2rrfJ. The elementary measured in outside-out patches. < 0.05, two-tailed t test).

slope of which (in double logarithmic coordinates) was 2.12 k 0.12 (n = 9) for alb2y2 GABARs and significantly different from those found for the dual combinations (Table 1). The current-voltage relationship of GABARs composed of al(32y2 subunits showed strong outward rectification similar to that exhibited by alp2 GABARs (Figure 7B, Table 1). The predominant singlechannel conductance was large (32.0 k 0.8 pS, n = 4) compared with that of the aly2 GABAR (Figure 7C). A subconductance state of 17.5 k 0.1 pS (n = 3) was also observed in al(32y2 GABARs. Thus, the alfi2y2 CABARS showed a distinct combination of properties. Rectification of the current-voltage relationship was similar to that produced by alp2 GABARs, and the single-channel conductancewas nearly indistinguishable from that of aly2 GABARs. Further, the alb2y2 GABAR showed faster desensitization (time constant = 6.3 f 0.5 s, 10 WM GABA, n = 4) and channel kinetics (fast time constant = 2.10 * 0.21 s, n = 4) than aly2 GABARs. These results are consistent with a previous report (Pritchett et al., 1989) which suggests that the three subunits form a novel GABAR subtype. Discussion

-120

mV I

Figure 4. Current-Voltage Cell Currents

Relations

of CABA-Activated

Whole-

Current-voltage curves for responses to GABA are shown from two cells, one expressing GABARs composed of alB2 (dots) subunits, and the other of aly2 (crosses) subunits. The current was normalized to the maxima found for each cell. The maxima were2950pAat +I20 mVforthecell expressing thealB2subunit combination (3 BM CABA) and 490 pA at +I20 mV for the cell expressing aly2 CABARs (30 BM GABA).

The results demonstrate that GABARs assembled from al(32, aly2, or alfi2y2 subunits exhibit clear functional differences, suggesting that specific functional properties may be conferred by particular subunits. The data presented here provide a basis for comparison between the functional properties of GABARs of known subunit composition with GABARs found in native membranes. Further, the distinct properties of recombinant receptors made from alp2 as opposed to aly2 subunits will allow more detailed studies of structural determinants for GABAR properties. The actual subunit stoichiometry of the functional entity formed upon cotransfection is unknown. Thus, the possibility that some subunits affect assembly or processing without actually becoming part of the structure cannot be ruled out. However, because the dual combinations show such marked functional differences, we believe that the most parsimonious explanation for our results is that subunit structure de-

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Figure 5. Fluctuation Analysis of GABA-Activated Whole-Cell Currents Currents activated by CABA were subjected to fluctuation analysis to estimate the conductance and gating properties of rhe elementary events. (A) The top trace shows the inward current evoked by application of 3 pM GABA (indicated by arrows) in a cell expressing the alp2y2 subunit combination at a holding potential of -60 mV. The lower trace is the same record filtered with an AC-coupled filter (0.2-500 Hz, -3 dB) that was used for noise analysis. The power density spectrum (lower panel) was composed of two components with corner frequencies of 3.3 and 80 Hz (arrows). The relative contribution of each component was determined by calculating the ratio of the areas under each single curve. Here the valve of A&Aslow was 0.22. The inset shows the relationship between mean current amplitude and variance. The linear regression over these data (solid line) represents the amplitude of the elementary current event, which in this case was 1.2 PA. (B) Results of similar analysis from currents activated by 3 PM GABA in a cell containing alp2 GABARs. The corner frequencies here were 6 and 140 Hz with A faSt/A Il0w equalling 1.4. The amplitude of the elementary current was 0.45 pA. (C) A spectrum Hz, Aia,Jhow

produced from current evoked by 10 VM GABA in a cell expressing was 0.52, and the elementary current amplitude was 1.6 PA.

termines the functional characteristics of the receptor. In fact, this has already been demonstrated in the case of the muscle nicotinic acetylcholine receptor by a series of experiments involving exchange of particular subunits expressed at different developmental stages and in different species (Mishina et al., 1986; lmoto et al., 1986). We observed GABA-activated currents in cells transfected with cDNAs encoding each possible combination of the al, (32, and y2 subunits, but the level of expression depended heavily upon the combination of subunits that was used. This is similar to previous studies carried out using Xenopus oocytes and transfected cells. Large currents were produced by activation of GABARs made from af3 (Schofield et al., 1987;

aly2

CABARs.

Corner

frequencies

were

4 and 60

Levitan et al., 1988a, 198813; Pritchett et al., 1988) and aby (Pritchett et al., 1989) combinations, whereas only small numbers of functional receptors were produced after injection or transfection of nucleic acids encoding single a or 0 subunits (Blair et al., 1988; Pritchett et al., 1988). These results together with our data indicate that GABARs formed from homooligomeric subunits and the (321/Z subunit combination assemble poorly, are not inserted into the membrane properly, or are largely nonfunctional when expressed in oocytes or transfected cells, particularly as compared with ap, ay, or aby combinations. Our observations indicate that particular subunit combinations produce channels with a limited range of conductance states. For example, the aI@2 combi-

Recombinant 925

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Figure 6. Single-Channel Currents Activated by GABA in Outside-Out Patches

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nation showed only states smaller than 20 pS. Other results from Xenopus oocyte experiments (Levitan et al., 1988a; Blair et al., 1988) have suggested that the albl subunit combination as well as homooligomeric a or fi subunits produce receptors with many conductance states ranging from 10 to 40 pS, and the properties of these channels were very similar regardless of the subunit or subunits that were expressed. Oocytes and transfected cells may process or assemble the CABAR subunits differently, which may account for the different properties we observed. However, channel opening was not shown to be dependent upon GABA application in the Xenopus oocyte studies, and thus it was not completely clear whether these channels were actually GABARs or some endogenous oocyte channel. Moss et al. (1990) have reported results from studies on hamster ovary cells stably transfected with al(31 subunits that are more similar to ours with the exception of the main conductance state, which was 18 pS. This difference may be due to the different species from which the clones were derived (rat vs. bovine) or the different b subunit variant studied (82 vs. PI). In fact, we previously reported that the human al(31 subunit combination forms a channel with a comparable higher main conductance state (Verdoorn et al., 1989, Sot. Neurosci., abstract). Functional Properties Are Determined by the b and y Subunits The functional properties of alp2 GABARs are very different from aly2 GABARs, indicating that the (32 and y2 subunits confer specific properties to the resulting channel subtype. The replacement of the b2 subunit with the ~2 subunit in GABARs containing at least the al subunit has significant effects on the rec-

2 (PA)

3

(A) Elementary currents recorded from outside-out patches containing alp2 GABARs. The top trace illustrates single-channel current activity elicited by application of 1 PM GABAduringthe time indicated bythe bar. The lower trace is a section of this record at higher time resolution. The membrane potential was -50 mV. All-points histograms compiled as dkscribed in Experimental Procedures are shown at the bottom. The histogram was fitted with the sum of the two Gaussians. One Gaussian represents the baseline current and the other the amplitude of the openings. The difference between the amplitude of openings and the baseline was 0.53 PA. (B) The results of a similar experiment performed on an outside-out patch taken from a cell expressing aly2 CABARs. Note the differences in amplitude and time course of GABA-activated channels. Three Caussians were fit to the histograms derived from currents mediated by aly2 GABAR channels. The derived amplitudes of the elementary events were 0.78 and 1.49 PA.

tification of the whole-cell current-voltage relationship, the desensitization and channel closing kinetics, as well as single-channel conductance. These properties may be characteristic of the fi and y subunit families and not only to the specific combinations tested here. When cDNA encoding the a3 subunit is used instead of cDNA encoding the al subunit, similar differences with respect to desensitization, rectification, and gating time constants are seen. Further, the function of alp1 and alyl combinations are analogous to those of the combinations containing (32 or ~2 (T. A. V., unpublished data). Previous results (Pritchett et al., 1989) and evidence presented here suggest that the alp2y2 combination also appears to form a novel receptor subtype. We were concerned that expression of dual combinations would hinder interpretation of alB2y2 characteristics, but we did not observe 11 pS channels in cells transfected with al(32y2 subunit-specific cDNA or very fast components of desensitization. Therefore, it seems likely that receptors composed only of alp2 subunits are rare in cells transfected with all three subunits. However, because the differences between alb2y2 and aly2 GABARs are more subtle, the presence of aly2 GABARs cannot be ruled out. Nevertheless, the unique combination of properties observed in cells expressing alp2y2 GABARs with respect to currentvoltage rectification, single-channel conductance and kinetics, and concentration-response relationship demonstrates that a separate GABAR type is formed in cells expressing all three subunits. Overall, the results help to determine the functional properties that are contributed to GABARs by the p2 and y2 subunits. The larger single-channel conductance caused by adding y2 or replacing 82 with ~2

Neuron 926

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1

10

[GABAl (F W Normalized

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10

T

mV

Figure

7. Novel

GABAR

Subtype

Formed

by alp2y2

Subunits

(A) Concentration-response characteristics of the alp2y2 subtype. On the left are superimposed inward currents evoked by 1, 3, 5, and 10 PM GABA at -60 mV holding potential in a cell transfected with cDNA encoding alp272 subunits. The right side is the double logarithmic plot of that data. The slope is 2.2. The dotted and broken lines are the average slopes, respectively, of the ale2 and aly2 CABARs for comparison. (B) Whole-cell current voltage relationship of GABARs composed of alP2y2 subunits (open circles). For comparison, results from a cell expressing aly2 GABARs are also shown (crosses). The current is normalized to maxima of 2450 pA for alp2y2 and 1100pAforaly2. GABA(lO,uM)wasapplied in both experiments. (C) Single-channel current activated by CABA in outside-out patches isolated from cells expressing aly2 (top) and alp2y2 (bottom) GABARs. The channels in the top trace were activated by IO PM GABA, and 3 PM GABA opened the channels shown on the bottom side. The patch potential was -50 mV in both cases.

suggests that the size of the open ion channel or charged amino acids affecting conductance may be determined by y2 subunit structure, whereas the increase in rectification and shortening of channel mean open time caused by addition of or replacement with the B2 subunit indicate that this subunit may control channel gating properties. The increase in concentration-response slope seen upon expression of the triple combination could possibly be due to an additional CABA binding site on the receptor. Further, the fact that the (32~2 cDNA combination expresses poorly, in marked contrast to the aly2 receptors, suggests that the a subunit is required to produce a GABA receptor with functional properties comparable to GABARs in native membrane.

Relation of Recombinant GABARs to GABARs in Native Membranes The physiological properties of native GABARs have been examined in a variety of tissues from different species. interestingly, the main conductance state observed in mammalian neurons varies between tissues with the most distinct difference being between hippocampal (Bormann et al., 1983; Gray and Johnston, 1985; Allen and Albuquerque, 1987) and spinal cord neurons (Bormann et al., 1987; MacDonald et al., 1989; Smith et al., 1989). The lower conductance state channels (20 pS or less) seen in hippocampus are similar to that found in recombinant GABARs constructed from the alp2 subunit combination, and the 30 pS channel most often observed in spinal cord compares favorably with that of the $I-containing combinations. The whole-cell current properties, such as desensitization, and the slope of the concentration-response relationship of the y2-containing combinations are similar to those of GABARs studied in bovine adrenal chromaffin cells (Bormann and Clapham, 1985) and pancreatic islet cells (Rorsman et al., 1989). It is, however, premature to assign putative subunit structures to the GABARs expressed in native tissue. Recombinant GABAR channels fail to exhibit the multiplicity of conductance states often seen in native membranes (Bormann and Clapham, 1985; Bormann et al., 1987; Smith et al., 1989), perhaps because the subunit combinations examined do not match those constituting GABARs in native membranes. Alternatively, each neuron may express numerous subunit combinations, each giving rise to GABAR subtypes with particular conductance states. Localization of the mRNA encoding a, b, and ~2 subunits by in situ hybridization (Wisden et al., 1988; Skquier et al., 1988; Shivers et al., 1989) suggests that they are colocalized in some brain regions and differentially distributed in others. However, the correlation between the sites of GABAR subunit expression and possible functional heterogeneity is incomplete. Ultimately, the structure of native GABARs must be determined directly with biochemical techniques or immunocytochemistry However, such structural probes currently lack the resolution necessary to identify the subunits composing functional GABARs in neuronai membranes. Moreover, studies of receptor structure alone cannot clarify the relationship between structural and functional diversity. Functional studies are necessary to provide information concerning the extent and possible significance of GABAR subunit diversity. Examination of the physiological characteristics of GABARs expressed from other combinations of cloned subunits can reveal the potential range of functional diversity. The properties conferred by various subunits or subunit families can similarly be examined. More importantly, sensitive physiological tests carried out in parallel on cells expressing recombinant GABARs and in brain slices (Edwards et al., 1989) will greatly facilitate classification of native GAB-

Recombinant 927

CABAR

ARs, particularly tic transmission. Experimental

Channels

those

that

mediate

inhibitory

synap-

Procedures

Cell Culture and Transfection Human embryonic kidney cells (293) were grown and transfected as described (Pritchett et al., 1988). Briefly, cells were grown on plastic cover slips in minimum essential medium (MEM, Gibco, Eggenstein, FRG) supplemented with 10% fetal calf serum. Approximately 48 hr after plating, the cells were transfected using the method of Chen and Okayama (1987). Subunit-specific cDNAs were each on separate CDM-8 plasmids. When combinations of subunits were transfected, equal weights of each plasmid were used. Forty-eight hours after beginning the transfection cells were used for electrophysiological recording. Electrophysiology Transfected cells on cover slips were transferred to the stage of an inverted microscope and bathed in normal rat Ringer’s solution (NRR) containing 135 mM NaCI, 5.4 mM KCI, 1.0 mM M&b, 1.8 mM CaCI,, 5 mM HEPES (pH 7.2). Patch-clamp techniques were used to measure GABA-activated currents in the whole-cell and outside-out configuration (Hamill et al., 1981) using a List EPC-7 amplifier (Darmstadt, FRG). The pipet solution contained 140 mM CsCI, 1.0 mM MgClz, 11 mM EGTA, 10 mM HEPES (pH 7.3). Confluent clusters of these kidney cells are electrically coupled (Pritchettet al., 1988). Therefore, signals recorded from one cell in such a cluster may arise from events occurring in one or more of its neighbors. We used the amplitude of GABA-activated currents in electrically coupled cell clusters to determine the overall effectiveness of the transfection (see Results). Because responses in distant cells were not adequately voltage clamped and because of uncertainties about the extent of coupling, quantitative measurements of whole-cell currents were performed onlyon cells that did not contact other cells upon visual inspection. CABA (Research Biochemicals Inc., Natick, MA) was dissolved in NRR and applied by U-tube to allow rapid application of known concentrations. The use of this fast application system greatly improved the measurement of time course and amplitude of the response. Data Collection and Analysis In general, currents were recorded on a video cassette recorder (Panasonic NVH65) via a pulse code modulator (Sony, PCM701ES) for later computer analysis. Alternatively, the currents were digitized on a VME-bus computer system during the experiment. For determination of dose-response relationships and desensitization properties whole-cell currents were sampled at 500 Hz after filtering (8 pole Bessel, Frequency Devices, Haverhill, MA) at 200 Hz (-3 dB). For dose-response experiments GABA was applied for 2 s and the log of maximum amplitude was plotted as a function of log of CABA concentration. The slope of this relationship was determined by linear regression. Only cells in which the response amplitudes were reproducible throughout the experiment were used. Desensitization time constants were measured using longer applications of GABA (IO-20 s) and were fit to an exponential function by eye or by linear regression of the relationship between log (current) and time. The results were similar in either case. The holding potential for these experiments was -60 mV. Power density spectra were produced from whole-cell current recordings (-60 mV) using a FFT routine (kindly provided by S. Heinemann, Gottingen, FRC). Currentswere sampled at 1000 Hz after filtering (AC-coupled filter, Krohn-Hite Corp., Avon, MA; 0.2-500 Hz band width). Data points (1024) were used to produce single spectra, and 50-100 of these were averaged. Averaged soectra derived durine recording in the absence of added GABA were subtracted from-those derived from currents activated by GABA to produce spectra such as those shown in Figure 5. The data were fitted to double Lorentzians to estimate the corner fre-

quencies. The slope of the relationship between current variante and amplitude was used to estimate the amplitude of the elementary event (Neher and Stevens, 1977). Over the range studied (2-5 PM GABA for alB2 and alB2y2, IO-30 J.~M CABA for aly2 GABARs) the parameters estimated by fluctuation analysis did not depend upon the GABA concentration. Current-voltage curves were produced by means of a 2 s voltage ramp from -120 to +I20 mV. Resting current values were subtracted from the values obtained during applications of CABA and averaged over 5 mV intervals. The resulting curves, representing GABA-activated current, were fit to fourth-order polynomial functions using a Simplex fitting routine to minimize the squared deviation. The reversal potentials and cord conductances were then calculated using the resulting polynomial equation. Single-channel currentswere measured at -50 or -90 mVand filtered at 1000 Hz before sampling at a frequency of 5000 Hz. Only patches that lacked activity in the absence of added GABA and showed a robust adding effect when GABA was applied from the U-tube were used. Amplitude histograms were constructed by binning all points in selected sections of records (0.06 pA bin width). Effort was made to avoid sections containing overlapping openings, but in some cases such events were difficult to identify (see Figure 6, middle left). The histograms were fit to the sum of two or three Caussians to determine the mean amplitude of single-channel currents. Acknowledgments We thank Markus Ewert and Dr. Dolan Pritchett for advice on the transfection procedure and help with plasmid preparation, U. Warncke for cell culture, and Drs. J. P Ruppersberg and R. Kass for helpful comments on the manuscript. T. A. V. is an Alexander von Humboldt Foundation Fellow. Received

January

29, 1990;

revised

March

16, 1990.

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