Gustav Akk, John Bracamontes and Joe Henry Steinbach- Pregnenolone sulfate block of GABA-A receptors: mechanism and involvement of a residue in the M2 region of the alpha subunit

8/3/2019 Gustav Akk, John Bracamontes and Joe Henry Steinbach- Pregnenolone sulfate block of GABA-A receptors: mechani

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Steroids can have rapid effects on mood and behaviour ofanimals, and on the function of neurons. A number ofsteroids have been identified that are produced in thebrain; these steroids have been termed neurosteroids(Mensah-Nyagan et al. 1999). Pregnenolone sulfate (PS) isa sulfated neurosteroid that is present in brain at arelatively high concentration compared with many otherneurosteroids (e.g. Wang et al. 1997). It has been foundthat the level of PS is reduced in the hippocampus of someaged rats and that there is a correlation between the

reduction in PS levels and reduced performance in abehavioural test for memory of a novel place (Vallee et al.1997), which could be restored by injection of PS (Vallee etal. 1997). Even in young adult rats, injection of PS intothe brain can improve performance on multiple tests ofmemory (Mayo et al. 1993; Flood et al. 1995). Thefunctional role of PS in the brain is not known, but it hasbeen reported to have direct effects on two receptors forneurotransmitters: it blocks activation ofy-aminobutyricacid A (GABAA) receptors (Majewska et al. 1988) and itpotentiates activation ofN-methyl-D-aspartate (NMDA)

receptors (Wu et al. 1991). We are particularly interestein the blocking of GABAA receptors, since many otheneurosteroids are known to potentiate the activation othese receptors (Lambert et al. 1995).

Most previous studies of the action of PS and othesulfated steroids (in particular dehydro-epiandrosteronsulfate, DHEAS) on GABAA receptors have utilisestudies of macroscopic currents elicited from whole cell(Majewska et al. 1988; Woodward et al. 1992; Zaman eal. 1992; Park-Chung et al. 1999; Shen et al. 1999). Thes

studies have shown that PS can block receptors whenapplied before GABA (Zaman et al. 1992), indicating thareceptors without bound transmitter and with closedchannels can be blocked. However, PS or DHEAS block ina non-competitive fashion (Majewska et al. 1988Majewska et al. 1990; Woodward et al. 1992), indicatinthat receptors with bound GABA can also be blockedExperiments using steroids that potentiate GABAreceptor responses have shown that potentiating steroidare equally effective in the presence of PS (Zaman et a1992) and that PS or DHEAS is equally effective in th

Pregnenolone sulfate block of GABAA receptors:

mechanism and involvement of a residue in the M2 regionof the a subunit

Gustav Akk, John Bracamontes and Joe Henry Steinbach

Department of Anesthesiology, Washington University School of Medicine,660 South Euclid Avenue, Saint Louis, MO 63110, USA

(Received 20 September 2000; accepted after revision 9 January 2001)

1. Neurosteroids are produced in the brain, and can have rapid actions on membrane channels of

neurons. Pregnenolone sulfate (PS) is a sulfated neurosteroid which reduces the responses of the

y-aminobutyric acid A (GABAA) receptor. We analysed the actions of PS on single-channel

currents from recombinant GABAA receptors formed from a1, b2 and y2L subunits.

2. Currents were elicited by a concentration of GABA eliciting a half-maximal response (50 M)

and a saturating concentration (1 mM). PS reduced the duration of clusters of single-channel

activity at either concentration of GABA.

3. PS had no discernable effect on rapid processes: no effects were apparent on channel openingand closing, nor on GABA affinity, and a rapidly recovering desensitised state was not

affected. Instead, PS produced a slowly developing block which occurred at a similar rate for

receptors with open or closed channels and with one or two bound GABA molecules.

4. The rate of block was independent of membrane potential, implying that the charged sulfate

moiety does not move through the membrane field.

5. Change in a specific residue near the intracellular end of the channel lining portion of the a1

subunit had a major effect on the rate of block. Mutation of the residue a1 V256S reduced the

rate of block by 30-fold. A mutation at the homologous position of the b2 subunit (b2A252S)

had no effect, nor did a complementary mutation in the y2L subunit (y2L S266A). It seems

likely that this residue is involved in a conformational change underlying block by PS, instead

of forming part of the binding site for PS.

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presence of potentiating steroids (Zaman et al. 1992;Park-Chung et al. 1999), indicating that the potentiatingand blocking steroids do not have a common binding siteor mechanism of action. A single report has been made ofthe effects of PS on single-channel currents elicited fromGABAA receptors (Mienville & Vicini, 1989). In thatstudy, a high concentration of PS (50 M) had no effect onthe duration or amplitude of openings elicited by a lowconcentration of GABA (1 M), but did reduce the

frequency of openings (by about 25%). Although thesestudies have provided insights into the actions of PS, nodetailed study has been made of the possible mechanismby which PS has its effects, and several possiblemechanisms are consistent with these observations. Forexample, the channel opening rate could be reduced, aslowly developing block of both open and closed channelscould be produced, or desensitisation could be altered(Shen et al. 2000).

We examined the mechanism of action of PS usingrecordings of single-channel activity from recombinantGABAA receptors. Our first goal was to determine

whether PS affected rapid steps in receptor activation,including agonist binding or channel opening and closing.The second was to explore with more precision thepossibility that block by PS occurred from some specificstate of the receptor (e.g. unliganded, liganded closed,liganded open). The third was to examine, at the single-channel level, whether PS block was affected bypotentiation produced by other steroids or bybarbiturates. The final goal was to determine residues inthe GABAA receptor that are required for the action ofPS. The overall objective was to provide additionalunderstanding of the mechanism of action for thisinhibitory steroid on the GABA

Areceptor.

METHODSRat GABA receptor cDNA was generously provided by Drs A. Tobin(University of California Los Angeles; a1, b2) and D. Weiss(University of Alabama, Birmingham; y2L) and subcloned into theexpression vector pcDNAIII (Invitrogen Corp., San Diego, CA, USA).Point mutations were produced using QuikChange (Stratagene, SanDiego, CA, USA). The mutated subunits were sequenced to confirmthat only the desired mutation had been produced.

The methods used are described in Akk & Steinbach (2000). In brief,GABA receptors were expressed in HEK 293 cells using transienttransfection based on calcium phosphate precipitation (Ausubel et al.

1992). Electrophysiological experiments were usually performedusing the cell-attached patch-clamp configuration (Hamill et al. 1981)although in some cases inside-out patches were used. The interior ofthe pipette was held at +60 mV unless indicated otherwise. Weassume that the cell membrane potential was about _40 mV , thusthe total potential difference across the patch was usually about_100 mV. Experiments were performed at 22 C. Single-channelcurrents were recorded with an Axopatch 200B amplifier (AxonInstruments, Foster City, CA, USA), low-pass filtered at 10 kHz,acquired with a Digidata 1200 Series Interface at 50 kHz usingpCLAMP 7 software (Axon Instruments) and stored on a PC harddrive for further analysis.

All of the analysis was performed on events in clusters of activityelicited by 50 M or 1 mM GABA. In the present experiments, mosclusters were separated from each other by prolonged periods oinactivity lasting several seconds. Files that contained persistenoverlapping activity were not analysed. We assume that during thintercluster silent periods all the receptors in the patch ardesensitised (cf. Sakmann et al. 1980). We could not estimate the ratof recovery from desensitisation from these data, since the longclosed times will depend on both the intrinsic rate of recovery and thnumber of receptors in the patch (which is not known). A cluster wa

defined as a series of openings separated by closed intervals shortethan a critical duration (tcrit). We chose tcrit to be 200500 ms, whicis ~10 times longer than the mean duration of the longest closed-timconstant within clusters, and ~10 times shorter than the meainterval between clusters.

The cluster duration was defined as the time between the firsopening and the last closing transition. We imposed a minimumduration for a cluster to be accepted (tmin). This was done to eliminatthe contribution of isolated events (clusters with a single opening oartifacts). The value for tmin ranged from 25 ms (in the presence o50 M PS) to 200 ms (in the absence of PS). As a result of thapplication of a minimal duration, the apparent mean clusteduration will be longer that the true duration. Assuming that thcluster durations are well described by a single exponentia

distribution (data not shown), the mean cluster duration (T([PS])) ithe apparent cluster duration minus tmin. For all data sets excepthose obtained in the presence of 50 M PS, tmin < 0.1T([PS]), and sthe correction was negligible. Accordingly, the correction was onlyapplied to the data obtained in the presence of 50 M PS.

Open and closed intervals were measured for events within clustersTo do this, the data in accepted clusters were digitally low-pasfiltered at 24 kHz and idealised using the segmented-k-meanalgorithm (program SKM; www.qub.buffalo.edu). Data from eachpatch was fitted with kinetic schemes incorporating three open (foopen duration histograms) or three closed states, respectively, tobtain the mean durations and relative occurrences presente(programme MIL; Qin et al. 1996, 1997). Cluster durations as function of [PS] were fitted using Nfit (UTMB, Galveston, TX, USA

Parameters are presented as best fitting value S.D. of the fit.

All chemicals used were obtained from Sigma (St Louis, MO, USA).

RESULTS

Action of pregnenolone sulfate on clusters ofopenings

Our studies were made by analysing the properties oclusters of openings elicited by relatively highconcentrations of agonists. A cluster results from thactivity of a single receptor. At the start of a cluster areceptor recovers from the long-lived desensitised state(sand begins a rapid series of sojourns in open and closedchannel states. At the end of a cluster, the receptor reenters a long-lived desensitised state. During the clusterit is possible to measure the probability that the channeis open (Popen). The distribution of the durations oopenings gives insight into the number of kineticallydistinct open states and their intrinsic rates of closingThe durations of the closed periods within a clusteprovides information about the closed states in rapidequilibrium with the open states. At the agonisconcentrations which produce clusters, these closed state

G. Akk, J. Bracamontes and J. H. Steinbach674 J. Physiol. 532.

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involve states with various numbers of ligands bound,and short-lived desensitised states. Finally, the overallduration of the cluster provides information on the rateat which the long-lived desensitised state(s) develop.Accordingly, we reasoned that examination of the effectsof PS on the properties of clusters would provide thelargest amount of information on the kinetic mechanismfor its action.

We used a high concentration of GABA (1 mM) and alower concentration (50 M). Clusters of openings are clearat both concentrations (Fig. 1). Activation of the GABAAreceptor by GABA is known to have a relativelycomplicated mechanism (Macdonald et al. 1989; Weiss &Magleby, 1989; Twyman et al. 1990), which we haveconfirmed (G. Akk & J.H. Steinbach, in preparation). AGABA concentration of 1 mM appears to saturateactivation, producing the maximal Popen of about 0.8, sothat the channel is about 4 times more likely to be openthan closed. A GABA concentration of 50 M producesabout a half-maximal Popen (0.4) and, accordingly, thereceptor has a closed channel for more than half the time

during the cluster. The clusters at 1 mM and 50 M arevery similar in duration (Figs 1 and 2). Since terminationof the cluster under control conditions is the result ofentry into a long-lived desensitised state, this observationindicates that long-lived desensitisation can arise fromboth partially and fully liganded states.

We then made recordings in the presence of differentconcentrations of PS. As shown in Fig. 1, PS reduced thedurations of the clusters elicited by either 50 M or 1 mMGABA. The combined data show no differences in theconcentration effect curve for PS-induced shortening forclusters elicited at the two GABA concentrations (Fig. 2).

The data shown in Fig. 2 were fitted with two simpleequations. Equation 1 was the Hill equation,

T([PS]) = Tmax/(1 + ([PS]/IC50)nH), (1)

where T([PS]) is the mean cluster duration in the presenceof a given concentration of PS, Tmax is the maximal(control) mean cluster duration, IC50 is the concentration

of PS producing half-maximal reduction in T([PS]), annH is the Hill coefficient. Equation (1) does not imply anyparticular mechanism, but can be used to describe thshape of the curves. The parameter estimates for the datat the two GABA concentrations agree well (Fig. 2).

The second equation was derived from a very simplpicture of block by PS. In the control condition, the meanduration of a cluster is given by the inverse of the overal

rate of entering long-lived desensitised statesT([0]) = 1/(k+D). In the presence of PS, it is assumed thathere is an additional mechanism for terminating cluster, that in the simplest case has a rate which idirectly proportional to the concentration of PS. Hencein the presence of PS the total rate for leaving a clustewill be the sum (k+D + k+PS[PS]), where k+PS is thassociation rate constant for the hypothesised blockingstep. Following this idea, the second equation used was

T([PS]) = 1/(k+D + k+PS[PS]). (2

As the lines in Fig. 2 show, eqn (2) can describe the datwell, and k+PS is very similar for clusters elicited by thtwo concentrations of GABA (Fig. 2). We will use thparameter k+PS to characterise the action of PS in the resof this paper, keeping in mind that we do not know thablock is actually described by a first-order reactionEquation (2) is appropriate to describe the concentrationdependence for the rate of development of blockproduced by an occupancy mechanism (e.g. direcocclusion of the channel). In addition, it describes the ratof development for other mechanisms of block so long athe concentration of PS is low compared with its affinityat the binding site mediating block. For example, ibinding and dissociation were rapid and block wer

produced by a slower conformational change, the rate odevelopment of block would increase linearly with [PS] alow concentrations. Thus, k+PS is a suitable parameter tdescribe block by PS by a variety of mechanismsalthough it should not be interpreted as a true first-ordeassociation rate constant. It is straightforward testimate k+PS, since it can be obtained simply bymeasuring the mean cluster duration at a concentration

Pregnenolone sulfate block of GABAA receptorsJ. Physiol. 532.3 67

Figure 1. PS has similar effects on clusters at twoGABA concentrations

Single clusters elicited by 50 M or 1 mM GABA areshown in the top row (the current through an openchannel is downwards). The middle row shows typicalclusters in the presence of 10 M PS, while the bottomrow shows two clusters in the presence of 50 M PS. Notethat the clusters are of similar duration at the twoGABA concentrations, and are reduced similarly byincreasing concentrations of PS. The probability of beingopen is about twice as high in the presence of 1 mMGABA compared with 50 M, as can be seen in the topand middle traces. Data recorded cell attached with amembrane potential of about _100 mV (see Methods).



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of PS that greatly reduces the cluster duration. In thiscase, k+PS[PS] >> k+D, and k+PS can be calculated directlyfrom the mean cluster duration and the concentration ofPS (k+PS = 1/{(T([PS]))[PS]}).

The results using 50 M and 1 mM GABA show that PSblocks equally rapidly at half-maximal and maximalconcentrations of GABA, confirming macroscopicmeasurements showing non-competitive block by PS. In

addition, the single-channel data suggest that PS canblock essentially equally well when the receptor has oneor two GABA molecules bound to it. At a highconcentration of PS, the cluster duration reflects theoverall rate of block for receptors in all states within acluster. The overall rate can be approximately separatedinto block of receptors with two bound GABA moleculescompared with other states of ligation, in the followingway. IfF2 is the fraction of time spent with two boundGABA molecules,s is the rate of blocking receptors withtwo bound GABA molecules and Qis the relative rate forblocking receptors with less than two bound GABA, thenthe aggregate rate for blocking a cluster would be

(F2s + ( 1 _ F2)Qs). In applying this approach, multiplestates of the receptor (open channel, closed channel,desensitised) with the same degree of ligation will belumped together. When 1 mM GABA is used to activate,the receptor is doubly-liganded essentially throughoutthe cluster (see below), so F2 = 1. On the other hand, when

50 M GABA is used the channel is open about 40% of thtime and closed 60 %. If we assume that half of the timwhen the channel is closed at 50 M GABA it is doublyliganded (and closed or desensitised), then F2 = 0.7. Thmean cluster duration at 50 M PS did not differ fo50 M and 1 mM GABA (Fig. 2), so there is less than 2-fold difference in cluster durations. With thesassumptions, the rate for PS block must differ by lesthan 4-fold for receptors with two GABA molecule

bound compared with those with fewer.

An analogous calculation can be made to examine the ratof block of receptors with open or closed channels. IfFc ithe fraction of time spent closed,s is the rate of blockingreceptors with closed channels and Q is the relative ratfor blocking open channels, then the aggregate rate foblocking a cluster would be (Fcs + ( 1 _ Fc)Qs). As anexample, if the rate of block were 10-fold higher for openchannels (Q= 10), then the mean cluster duration shouldhave been only half as long with 1 mM GABA (where Fc i0.2) than at 50 M GABA (where Fc is 0.6). This analysiindicates, therefore, that the difference in rates is les

than 10-fold between receptors with open and closedchannels.

We used an additional approach to examine the questionof selectivity between open and closed channel statesPiperidine-4-sulfonic acid (P4S) has been described as anagonist at the GABAA receptor that binds with high


Figure 2. Dependence on [PS] of the reduction in clusterduration and the cluster Popen

A, mean cluster duration (T([PS])) for data from individualpatches plotted against [PS] (note logarithmic abscissal co-ordinates; the points on the left are for GABA with no PS). Thedata for activity elicited by 50 M and 1 mM GABA overlap at all[PS]. The lines show the fits of eqn (2). When eqn (1) is fitted tothe data, the best-fitting parameter estimates are: 50 M GABA,Tmax 2652 1158 ms, nH _0.8 0.2, IC50 0.9 1.1 M; 1 mMGABA, Tmax 2149 362 ms, nH _1.0 0.1, IC50 2.1 1.0 M. Foreqn (2), the values are k+D 0.52 0.11 s

_1, k+PS 0.21 0.04 M_1 s_1

(50 M) and k+D 0.48 0.06 s_1, k+PS 0.20 0.03 M

_1 s_1 (1 mM).Bshows there is no change in Popen over the same range of [PS].Each point shows data from a single patch.



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affinity but has a low maximal response (Woodward et al.1993; Ebert et al. 1994). This suggested to us that P4S haslow efficacy for opening the channel, so that we couldobtain clusters of activity reflecting fully ligandedreceptors which spend most of the time with closedchannels (a low maximal Popen). This appears to be thecase, as clusters produced by 1 mM P4S are very sparse(Fig. 3), with an estimated Popen of about 0.1. In thepresence of 50 M PS the cluster duration was clearly

reduced, with a mean duration indistinguishable fromthose produced by GABA in the presence of 50 M PS(Table 1). Given the Popen values for 1 mM GABA and1 mM P4S, the qualitative analysis described aboveindicates that the difference in blocking rates forreceptors with open or closed channels is less than 3-fold.In sum, the use of a low efficacy agonist also gives noindication that the di-liganded closed state is blocked anydifferently than the di-liganded open state.

Lack of voltage dependence of block by PS

PS is an anion at physiological pH. We determinedwhether the charge on PS interacts with the membranefield during the blocking action by measuring the meancluster duration at different membrane potentials in thepresence of 1 mM GABA and 50 M PS. There was nodiscernable consequence of changing the membranepotential by 65 mV (data not shown; the regression slopegave a voltage dependence of e-fold per 4125 mV). Theimplication of this low slope is that the single negativecharge on PS must traverse a very small fraction of themembrane field (Woodhull, 1973), and the sign of thedependence is the opposite expected for movement of ananion from the external medium into the membrane field.Accordingly, it is unlikely that the action of PS involves

interaction of the sulfate group with a residue that is veryfar into the membrane field.

Actions of PS on rapid processes within the cluster

PS did not affect the Popen within a cluster (Fig. 2). Thisobservation suggests that PS has relatively little effect onthe rapid processes involved in receptor activation,although it could be that equal changes were made in boththe open and closed durations (for example, both could beprolonged).

However, PS did not alter the open times within a cluste(Fig. 4). The histograms of open-time durations contaithree components in control conditions (Fig. 4), whicreflect three distinct open states. Such complexity habeen reported before (Macdonald et al. 1989; Weiss &Magleby, 1989; Twyman et al. 1990), and we will nopropose a specific kinetic scheme to accommodate thobservations. However, PS did not affect the number oopen-time components, the fitted time constant of any o

the three components, or the relative prevalence of thcomponents (Fig. 5). The values of the three open-timconstants and the three closed-time constants in thabsence and presence of 50 M PS are given in Fig. 4Accordingly, there is no indication that PS actpreferentially on a particular open state. FurthermorePS did not appear to increase the rate of channel closingnor to produce a rapid open-channel block. The absence oany systematic changes in the mean open duration in thabsence and presence of various concentrations of PS ishown in Fig. 5. For a single patch recorded with 50 MGABA and no PS, the mean open duration was 3.7 ms an

the mean closed duration was 6.9 ms. For two patcherecorded with 50 M GABA + 50 M PS, the mean opendurations were 1.5 and 1.9 ms, while the mean closetimes were 2.4 and 2.9 ms. For a single patch recorded ithe presence of 1 mM GABA, the mean open duration wa2.3 ms and the mean closed duration was 0.7 ms. For onpatch obtained in the presence of 1 mM GABA + 50 MPS, the mean open time was 3.5 ms and the mean closetime was 1.1 ms.

The closed periods are informative because they canprovide some information on the agonist binding rate, thchannel opening rate and the rates for rapidly recoverindesensitisation. Again, three components were presenfor closed periods within a cluster at either GABAconcentration, and PS did not affect the mean durationor relative amplitudes for the closed periods at eitheGABA concentration. The lack of effect on thhistograms demonstrates that PS has little effect on thchannel opening rate, GABA association or dissociationrates, or a rapidly equilibrating desensitised state. Aquantitative demonstration of this lack of effect wilrequire a full model for GABAA receptor activation


Figure 3. PS reduces the durations of clusterselicited by a low efficacy agonist

A single cluster elicited by 1 mM piperidine-4-sulfonic acid (P4S) is shown in the upper row,while the lower row shows typical clusters elicitedby 1 mM P4S in the presence of 50 M PS. Theprobability of being open in clusters elicited byP4S is very low (compare with Fig. 1), but PSreduces the cluster duration. Data recorded cellattached with a membrane potential of about_100 mV (see Methods).



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which is not presently available. However, theseconclusions are supported by inspection of thehistograms. In particular, we used a high concentration ofGABA (1 mM) to examine the channel opening rate and arapidly equilibrating desensitised state. In contrast, theclosed-time histograms for data obtained using aconcentration of GABA producing a half-maximal valuefor Popen will contain information about GABAassociation and dissociation rates.

In qualitative terms, one of the two closed-timecomponents (C1 and C2 in Fig. 5) in the presence of 1 mMGABA reflects the channel opening process (durations of0.25 or 1 ms imply a channel opening rate of about10004000 s_1). There is no effect of PS on the duration orprevalence of either component. Accordingly, we find noevidence that PS acts by reducing the channel opening

rate. There is also a much longer component in the closedtime histograms at 1 mM GABA (identified as C3 inFig. 5), which is related to a short-lived desensitised stat(see Jones & Westbrook, 1995). This component has amean duration of 2050 ms and constitutes about 2 % othe closed periods within a cluster. If PS produced itblock by increasing the rate of occurrence of this type odesensitisation, we would have expected to see anincrease in its prevalence. Alternatively, if PS increased

the duration of this desensitised state we would expect tsee an increase in the duration of this component withincreased [PS]. Since there was no change in the presencof PS, we find no evidence that PS acts by affecting thicomponent of desensitisation.

The closed-time histograms obtained with 50 M GABAare harder to interpret. However, PS has no effect on th


Figure 4. PS does not change the histograms of open or closed times within clusters

Histograms of the open durations (first and second columns) and closed durations (third and fourth

columns) are shown for clusters elicited by 50 M (A)or 1 mM GABA (B)in the absence (first and third

columns) or presence (second and fourth columns) of 50 M PS. Each histogram shows data from a single

patch. In each case the data have been fitted with the sum of 3 exponential components (time constants

given in the panels), shown by the continuous lines. The collected data on the mean durations and

fractional areas of the 3 components are shown in Fig. 5. Note that the histograms are displayed with the

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distributions (Figs 4 and 5). This concentration of GABAproduces a Popen which is about half-maximal. At thisconcentration the closed times reflect both channelopening and GABA association and dissociation, asindicated most clearly by the observation that theapparent opening rate for channels is far from maximaland continues to increase with GABA concentration(e.g. Maconochie et al. 1994). The fact that no changes areobserved in the closed-time histograms at a half-maximally effective concentration of GABA suggeststhat PS does not exert its major effect by reducing theassociation rate for GABA to the second site on thereceptor, or by increasing the dissociation rate for GABAfrom this site.

These observations, taken together, indicate that PS doesnot have major effects on the channel opening rate,closing rate, nor on GABA binding or dissociation.Accordingly, the reduction of response by PS is the resultof a slow process which shortens clusters of activity. Weare unable to determine, from the present results,whether PS acts by increasing the rate of a slowdesensitisation process or by an independent mechanism.

Interaction of PS with potentiators of GABAAreceptors

Barbiturates and many steroids act at low concentrationsto potentiate activation of GABAA receptors. Themechanism of potentiation is not fully understood,although barbiturates and steroids appear to act afterbinding to sites which differ between the two classes of

drugs, as well as from the sites which bind GABA,benzodiazepines or convulsants (e.g. Amin & Weiss, 1993;Lambert et al. 1995; Ueno et al. 1997). We usedpentobarbital and 3a,5a,17b-3-hydroxyandrostane-17-carbonitrile (ACN) as representatives of these two classesof potentiating drugs.

There was no change in k+PS for PS when 50 M GABAand 40 M pentobarbital, or 50 M GABA and 1 M ACNwere co-applied (Table 1). These results, therefore,suggest that the mechanism by which PS reduces cluster

duration is independent of potentiation by barbiturateor potentiating steroids.

The reduction of cluster duration by PS is dependenton a particular residue in the a1 subunit

The ability of some non-competitive blocking drugs tproduce their effects is critically dependent on particularesidues in the second membrane-spanning region oreceptor subunits. This was first shown with noncompetitive blocking agents for the muscle nicotinireceptor (Charnet et al. 1990). The actions of sompositively charged local anaesthetics depended on thnature of the residues at the 6th (6) and 10th (10positions in the aligned M2 regions of all four subunitsFor convenience, the position in the M2 region will bindicated by a primed number, in which the 1st (1position is the closest to the N-terminus of the proteinand also closest to the cytoplasmic end of the M2 helix

Studies of GABAA receptors have identified the 6 residuas a critical residue for the blocking action of picrotoxin(Gurley et al. 1995), but additional studies have founthat a mutation of the 6 residue of the GABAA ysubunit which removes block by picrotoxin had no effecon the ability of PS to block responses (Shen et al. 1999).

Work with the GABA-activated rdl (ffrench-Constant eal. 1993) ands (Wang et al. 1995) receptors had indicatethat the 2 position can also have significant effects onpicrotoxin block. A mutation of the rdl subunit whicconverted an alanine at the 2 position to serine (A2Sreduced block by picrotoxin (ffrench-Constant et a

1993). Accordingly, we set out to determine whether thnature of the residue at the 2 position was critical for Paction. The GABAA a1 subunit has valine (V256) at the 2position, theb2 subunit has alanine (A252), while the ysubunit has serine (S266). We produced the point mutanta1(V2S),b2(A2S) and y2(S2A).

In all cases, the receptors containing mutated subunithad essentially normal single-channel amplitudes, meanopen times and Popen values (data not shown), indicatinthat the mutations did not have major effects on thesaspects of receptor function.


Table 1. Mean cluster duration and forward rate constant with 50 M PS

Condition T([0]) Nclusters T([50 M]) k+PS Nclusters(ms) (Npatches) (ms) (M_1 s_1) (Npatches)

1 mM GABA 2881 1978 33 (2) 101 80 0.20 135 (2)50 M GABA 3586 3150 25 (2) 81 78 0.25 46 (2)1 mM P4S n.d. 128 96 0.16 115 (3)50 M GABA + 40 M PB 6701 5716 9 (1) 100 81 0.20 131 (4)50 M GABA + 1 M ACN 10141 10639 * 5 (1) 84 60 0.24 89 (2)

The mean cluster durations ( S.D.) in the absence of PS (T([0])) and in the presence of 50 M PS T([50 M])are shown, with the calculated value for k+PS. P4S, piperidine-4-sulfonic acid; PB, pentobarbitone. Thenumber of clusters analysed in each condition is shown (from N patches). Note that for a singleexponential distribution the mean and standard deviation of the durations should be equal. * Data forT([0]) in the presence of ACN were obtained with 0.5 M ACN. n.d., not determined.



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Table 2. Mean cluster duration and forward rate constant for wild-type and mutatedsubunits

Receptor T([0]) Nclusters T([50 M]) k+PS Nclusters(ms) (Npatches) (ms) (M_1 s_1) (Npatches)

a1b2y2 2881 1978 33 (2) 81 78 0.25 46 (2)a1(V2S)b2y2 n.d. 2913 2502 0.007 38 (7)a1b2(A2S)y2 5396 2997 12 (2) 68 50 0.29 133 (5)a1b2y2(S2A) 3417 242 17 (2) 97 76 0.21 80 (3)

a1(V2S)b2y2(S2A) 3916 2977 23 (3) 7240 1678 0.003 5 (2)a1(V2S)b2(A2S)y2 n.d. 5140 7600 0.004 10 (3)

Data displayed as in Table 1.

Figure 5. PS does not change the distributions of open or closed times within clusters

The mean durations and relative areas of the components fitted to the duration histograms are shown for

clusters elicited by 50 M GABA (A) and 1 mM GABA (B)at a range of PS concentrations. The data from

a patch (100020 000 sojourns) were well fitted with the sum of 3 exponential components (identified as

O1, O2, O3 and C1, C2, C3, respectively) with time constants separated 3- to 10-fold. The plots show

results from individual patches plotted against [PS]; note that the points on the left in each panel are for

patches with no PS. The lines in panels showing mean durations denote the weighted average durationsof all 3 components of the respective histograms. The plots in the first column show the mean open times

(in ms) for each component while the plots in the second column show the fraction of the total open times

in each component. Note that PS has no effect on the durations or prevalence of open-time components

nor the weighted average duration. There is also no apparent difference between open-time distributions

in the presence of 50 M and 1 mM GABA. The third and fourth columns show the data for closed times in

the same format: the mean closed time for each component (third column), then the fraction of the total

closed times in each component (fourth column). In contrast to the data on open times, there is a change

in the distributions of closed times between the two GABA concentrations, most clearly seen in the

fractional representation in each component (fourth column; compare A with B). However, again PS has

no effect on the duration or prevalence of the closed-time components nor on the weighted average

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Cotransfection of a1(V2S)b2y2 subunits resulted inexpression of receptors with altered block by PS. Thecluster duration was prolonged. In many cases the extentof overlap of activity was such that it was not possible toestimate the cluster duration accurately. In one patchwith very low activity the mean cluster duration wasabout 11 s. When recordings were made in the presence of50 M PS the cluster duration was reduced enough toallow clear resolution of clusters, which had a mean

duration significantly longer than control (Fig. 6,Table 2). The apparent association rate (k+PS) for PS wasreduced by about 30-fold by the mutation. This indicatesthat the presence of serine at the 2fi position of the a1 andy2 subunits affects the ability of PS to block. It isimportant to note that clusters which showed overlappingactivity (the occurrence of periods when two or morechannels were open at the same time) were eliminated incalculating the mean duration. Since longer clusters have agreater likelihood of participating in an overlap, themeasured mean durations for clusters from these receptorsare actually a lower estimate of the mean duration (and so

the estimate for k+PS also is a lower limit).This action on PS block was quite specific to the mutationin the a1 subunit. Cotransfection of a1b2(A2S)y2subunits resulted in expression of receptors that hadsomewhat prolonged mean cluster durations (about 6 s,compared with control values of about 3 s). When clusterswere examined in the presence of 50 M PS, however, thecluster duration was reduced to the same extent as forwild-type receptors and k+PS was unchanged (Fig. 6,Table 2). Clearly, the presence of serine at the 2 positionof theb2 and y2 subunits does not affect the ability of PSto block.

Cotransfection of a1b2y2(S2A) subunits resulted inexpression of receptors with normal cluster durations(about 3 s), and the action of PS was not affected (Fig. 6,Table 2). Hence, removal of all serines at the 2 positiondid not affect PS block.

Cotransfection of a1(V2S)b2(A2S)y2 subunits ora1(V2S)b2y2(S2A) subunits produced receptors withproperties very similar to those seen followingtransfection with a1(V2S)b2y2 subunits (Fig. 6, Table 2).These observations demonstrate that the effect of theserine residue in the a1 subunit is independent of thepresence (or absence) of homologous serines in theb2 ory2

subunits.

DISCUSSION

Mechanism of block by PS

Our results indicate that PS produces block of GABAAreceptors by a slow process, with no discernable effects onrapid processes of GABA binding and unbinding orchannel opening and closing. The block also does notappear to involve change in a rapid form ofdesensitisation.

The apparent forward rate constant, k+PS, is abou2 w 105 M_1 s_1 , a slow value for an open channel blocke(cf. Charnet et al. 1990). We could not estimate thdissociation rate, but recovery times in whole-celexperiments suggest that the dissociation rate i~3 w 10_2 s_1 (Woodward et al. 1992). These valuegenerate a value for the apparent IC50 for steady-statblock of ~100 nM. This value is lower than estimates inthe literature obtained with recombinant a1b2yreceptors (e.g. 1.2 M; Shen et al. 1999), but it is nocertain that the steady-state level of block had beenreached at the lower concentrations of PS tested in thwhole-cell recordings. The total content of PS in rat brainis estimated to be 10100 nmol (kg wet weight of tissue)_

(Robel et al. 1987; Corpechot et al. 1997; Wang et a1997), which would suggest that some steady-state bloc


Figure 6. Mutation in the a subunit reduces the

ability of PS to shorten cluster duration

Representative clusters elicited by 1 mM GABA in the

presence of 50 M PS are shown. Note that the

clusters in the presence of PS are much longer when

the receptor contains the mutated a1(V2S) subunit,

while the mutated b2(A2S) or y2(S2A) subunits have

no effect by themselves, nor do they alter the

consequences of incorporating the a1(V2S) subunit.

The bottom trace has a different time scale from the

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might be present. However, it is not known what theeffective concentration of PS at the GABAA receptor inthe brain would be, so the significance of the comparisonis not clear.

Block appears to develop at the same rate independent ofwhether the channel is open or closed, and of whether thereceptor has one or two bound GABA molecules. Theseobservations confirm previous studies of macroscopic

currents, which had found that PS can block GABAAreceptors in the absence of agonist (Zaman et al. 1992) andthat block is not competitive with GABA (Majewska et al.1988, 1990; Woodward et al. 1992). However, theseprevious results were all obtained using steady-statemeasurements of block. We have extended the previousobservations using studies of clusters of single-channelevents which allowed us to identify the distinct states ofthe receptor with greater precision, and using conditions(GABA concentration or P4S as agonist) that altered theprobability a channel is open in a cluster.

At a microscopic level clusters are shortened to the same

mean duration irrespective of whether pentobarbital orACN is present as well as PS. These observations at thesingle-channel level agree with previous studies of evokedwhole-cell currents, which had found that blockingsteroids act independently of potentiating steroids(Zaman et al. 1992; Park-Chung et al. 1999). All of theseresults imply that there is no significant overlap betweenthe binding sites occupied by potentiating and blockingsteroids, in agreement with results from studies ofbinding interactions (e.g. Gee et al. 1989).

Structural basis for block by PS

In general, it is felt that the sulfate moiety (or another

negatively charged group) is critical in producing a steroidthat blocks rather than potentiates GABAA receptors(e.g. Park-Chung et al. 1999). This conclusion, however,must be tempered by the observations that dehydro-epiandrosterone has been reported to be essentially aspotent at blocking responses as its sulfated derivative(Demirgoren et al. 1991; LeFoll et al. 1997), and that somesulfated steroids are weak blocking agents (El-Etr et al.1998). In any case, the idea that an anionic group wascritical had suggested to us that the sulfate mightactually interact with residues forming the binding sitewhich mediated block. We found that the rate ofdevelopment of block is independent of the membrane

potential, which suggests that the charged sulfate moietydoes not interact significantly with the membrane field asPS approaches the transition state between unbound andunblocked to bound and blocked (Woodhull, 1973). It hasalready been reported that equilibrium block by PS isindependent of membrane potential (Majewska et al.1988), so the reverse step is also voltage independent.Accordingly, the lack of voltage dependence renders ithighly unlikely that the sulfate moiety interacts with asite deep within the channel.

It was a surprise, therefore, to find that a residue deep inthe channel at the 2 position in the M2 region of the asubunit is critical for block by PS. In thinking about throle of this residue, we considered whether it was likelythat it formed part of a binding site for PS. It seems likelythat a large, rigid molecule such as a steroid, if it bound inthe channel, would bind end-on rather than with its longaxis across the channel. In this case, either the A ring(where the sulfate moiety is attached) or the D ring (the

other end of the molecule) would penetrate most deeplyThe lack of voltage dependence indicates that the A ringis unlikely to penetrate to the 2 position. However, thstructure of the D ring of PS is identical to that for manypotentiating pregnane steroids. Accordingly, we think iis less likely that other (uncharged) portions of the PSmolecule interact with the 2 residue. A morcircumstantial argument is the finding that the presenceof a serine residue is effective at altering PS block onlywhen in the a1 subunit, not when in theb2 or y2 subunitStudies of the 2 residue in nicotinic receptors have led tothe suggestion that the side-chains from all five subunit

form a ring in the channel (Villarroel et al. 1992), and imight be expected from symmetry arguments that therwould be closer equivalence in the subunits focontributions to a binding site. Studies of the effects omutations of the 6 and 10 residues in muscle nicotinireceptor subunits have also indicated that the residuefrom the different subunits participate relatively equallyin forming a binding site for local anaesthetics (Charnet eal. 1990).

Accordingly, we think that it is likely that the specifimutation we have found to affect block by PS, GABA Aa1(V256S), exerts its actions by altering an allosterimechanism rather than by directly altering a binding siteThe studies of local anaesthetic block of nicotinireceptors (Charnet et al. 1990) have clearly shown tharesidues in M2 can participate in binding sites. Howeverit is important to note that several reports have foundthat mutations in M2 also affect block by allosterimechanisms ranging from converting block by dihydro-berythroidine in nicotinic a7 receptors to activation(Devillers-Thiery et al. 1992), to altering block bypseudocompetitive blocking agents in GABA- andglycine-activated receptors (Wang et al. 1995; Steinbachet al. 2000).

Conclusions

The question remains, however, as to what the actuamechanism is for block by PS. We have demonstratedthat it is not the result of an effect on GABA binding ochannel opening or closing rates. Two alternatives arethat PS acts to produce a novel change in channestructure, or acts to enhance a normal mode of behaviourparticularly to increase the rate of development of slowdesensitisation (as has been proposed previously; Shen eal. 2000). An observation that might support thi




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conclusion is that receptors containing the mutated a1subunit have prolonged cluster durations as well asreduced k+PS. However, it seems unlikely that a simplemultiplicative increase in the rate of desensitisation byPS can explain all the observations. For example, theobservation that PS blocks receptors in the absence ofGABA as well as in its presence is difficult toaccommodate in a simple version of this idea. In addition,some of our observations are problematic. The a1(V2S)mutations effect of reducing k+PS (30-fold) is greater thanits effect of reducing k+D (~5-fold), and other conditionsthat prolonged clusters (presence of pentobarbitone orACN) did not affect k+PS. Thus, although our studiesindicate that PS blocks by an allosteric mechanism,further work will be required to identify the molecularmechanism by which PS acts.

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Acknowledgements

This research was supported by grant P01 GM47969 to J.H.S. J.H.Sis the Russell and Mary Shelden Professor of Anesthesiology, anG.A. is a fellow of the McDonnell Center for Cellular and MoleculaNeurobiology.

Corresponding author

G. Akk: Department of Anesthesiology, Washington UniversitSchool of Medicine, 660 South Euclid Avenue, Saint Louis, MO 63110

USA.

Email: [email protected]


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Gustav Akk, John Bracamontes and Joe Henry Steinbach- Pregnenolone sulfate block of GABA-A receptors: mechanism and involvement of a residue in the M2 region of the alpha subunit