identification of channel-lining residues in the m2 membrane-spanning segment of the gabaa

Identification of Channel-Lining Residues in the M2 Membrane-spanning Segment of the

GABAA Receptor 0L1 Subunit

MING X u a n d MYLES H. AKABAS

From the Center for Molecular Recognition and the Departments of Physiology and Cellular Biophysics and Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032

ABSTRACT The ~/-aminobutyric acid type A (GABAA) receptors are the major inhibitory, postsynaptic, neurotransmitter receptors in the central nervous system. The binding of~/-aminobutyric acid (GABA) to the GABAA receptors induces the opening of an anion-selective channel that remains open for tens of milliseconds before it closes. To understand how the structure of the GABA A receptor determines the functional properties such as ion conduction, ion selectivity and gating we sought to identify the amino acid residues that line the ion conducting channel. To accomplish this we mutated 26 consecutive residues (250-275), one at a time, in and flanking the M2 membrane-spanning segment of the rat c~ t subunit to cysteine. We expressed the mutant ~1 subunit with wild-type [31 and ~/2 subunits in Xenopus oocytes. We probed the accessibility of the engineered cysteine to covalent modification by charged, sulfhydryl-specific reagents added extracellularly. We assume that among residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction through the channel. We infer that nine of the residues, oqVa1257, ~lThr261, eqThr262, utLeu264, c~aThr265, alThr268, ~1Ile271, u1Ser272 and oqAsn275 are exposed in the channel. On a helical wheel plot, the exposed residues, except cqThr262, lie on one side of the helix in an arc of 120 ~ We infer that the M2 segment forms an a helix that is interrupted in the region of eqThr262. The modification of residues as cytoplasmic as eqVa1257 in the closed state of the channel suggests that the gate is at least as cytoplasmic as alVa1257. The ability of the positively charged reagent methanethiosulfonate ethylammonium to reach the level of ~Thr261 suggests that the charge-selectivity filter is at least as cytoplasmic as this residue.

I N T R O D U C T I O N

The ~/-aminobutyric acid type A (GABAA) receptors form anion-selective channels at inhibitory synapses in the mammalian central nervous system. The GABAA receptors are the targets for several classes of clinically useful drugs which potentiate GABA-induced currents including benzodiazepines, barbiturates, and several general anesthetics (Franks and Lieb, 1994; Macdonald and Olsen, 1994). In addition, epileptogenic drugs such as picrotoxin, penicillin and TBPS bind to the GABA A receptors and inhibit GABA-induced currents. In insects, the GABA a receptors are inhibited by the commonly used cyclodiene insecticides (ffrench-Con- stant et al., 1993). Multiple GABA a receptor subunits have been cloned (Burt and Kamatchi, 1991; Wisden and Seeburg, 1992; Macdonald and Olsen, 1994). The

Address correspondence and reprint requests to Dr. Myles H. Aka- bas, Center for Molecular Recognition, Columbia University, 630 West 168th Street, NewYork, NY 10032.

subunits are members of a ligand-gated ion channel gene superfamily which includes the subunits of the nicotinic acetylcholine (Numa, 1989), serotonin (Mar- icq et al., 1991), and glycine (Betz, 1992) receptors and the avermectin-sensitive, glutamate-gated, chloride channel (Cully et al., 1994). Studies of heterologously expressed GABAA receptors of defined subunit composi- tion have provided insights into the pharmacological diversity of GABAA receptors in situ (Burt and Kamat- chi, 1991; Wisden and Seeburg, 1992; Macdonald and Olsen, 1994), however, there have been few studies of the structure of the ion conduct ion pathway.

Based on studies of the GABAA receptor and the homologous nicotinic acetylcholine receptor, the GABAA receptors are presumably composed of five subunits ar- ranged pseudo-symmetrically around a central channel (Unwin, 1993; Nayeem et al., 1994); the subunit stoichiometry, however, is uncertain (Backus et al., 1993; Macdonald and Olsen, 1994). The subunits have a ~200 amino acid NH2-terminal extracellular domain, three closely spaced membrane-spanning segments

195 J. GEN. PHYSIOL. �9 The Rockefeller University Press �9 0022-1295/96/02/195/11 $2.00 Volume 107 February 1996 195-205

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(M1-M3), a cytoplasmic domain of variable length, a fourth membrane-spann ing segment (M4) and a short, extracellular C O O H terminus. The results of numer- ous exper iments on the acetylcholine receptor indicate that residues in the M2 membrane-spann ing segment line the ion conducting channel (Sakmann, 1992; Lester, 1992; Karlin, 1993), however, there is little information identifying the channel-lining residues in the GABAA receptors. We previously applied the scanning-cysteine- accessibility me thod to four residues, ~]Thr268 to 0qIle271, in the M2 membrane-spann ing segment of the rat ~1 subunit (Xu and Akabas, 1993). We showed that two residues near the extracellular end of the M2 segment, oqIle271 and oqThr268, are exposed in the channel lumen (Xu and Akabas, 1993). In addition, using a variation of this me thod on the residues cxlVa1257 to cxlThr261 we demons t ra ted that picrotoxin, a non- competit ive inhibitor, appears to bind in the channel at the level of~x~Val257 (Xu et al., 1995). We now present the results of a scanning-cysteine-accessibility analysis of all of the residues in and flanking the M2 membrane - spanning segment of the rat eq subunit.

The scanning-cysteine-accessibility me thod provides a systematic approach to the identification of the residues lining an ion channel. We mutate individual residues in largely hydrophobic, membrane-spanning segments to cysteine. We express the cysteine-substitution mutants in Xenopus oocytes and examine their functional propert ies in situ. If mutan t channels are near- normal in their responses, we proceed to test whether the new cysteine is on the water-accessible surface of the protein. We test the ability of small, charged, hydro- philic, sulfhydryl-specific reagents to react covalently with the new cysteine. We assume that of the residues in membrane-spann ing segments only those exposed in the channel lumen will be accessible to react with these sulfhydryl reagents. I f the reagents react with a cysteine in the channel lining we assume that they will irreversibly alter ion conduction. We infer that for a mutan t channel whose conduct ion is irreversibly al tered by the sulfhydiyl-specific reagents, the side chain of the corre- sponding wild-type residue lines the ion channel. The reagents we have used in this study include the organic mercurial derivatives p-chloromercuribenzenesulfonate (pCMBS) and p-chloromercur ibenzoate (pCMB), the methanethiosulfonate (MTS) derivatives MTS-ethylsul- fonate (MTSES) and MTS-ethylammonium (MTSEA), and iodoacetate. The structures of these reagents and their reactions with free sulfhydryls are shown in Fig. 1.

The scanning-cysteine-accessibility me thod has been used to study the acetylcholine receptor (Akabas et al., 1992, 1994a) the GABA a receptor (Xu and Akabas, 1993; Xu et al., 1995), the cystic fibrosis t ransmem- brane conductance regulator (Akabas et al., 1994b), potassium channels (Kurz et al., 1995; Lu and Miller,

A

m ~

S"

I +

X"

> i~ HgOH I

- - N C - - H

B

- - N H

S"

i

X

I

$

I O~---S=O

I CH3 - - N

H

X

I

S

I $

i

C

q N H

S- [ O = C - - O -

F

I

O = C - - O - I

>

i FIGURE 1. Chemical structures of the sulfhydryl reagents and their reactions with cysteine. The structure of the reactive ionized thiolate form of cysteine is shown on the left of each panel; the structure of the reagents follows the + sign and the structure of the products are to the left of the arrow. Note that all of these reagents transfer the charged portion of the reagent onto the sulfhydryl of cysteine resulting in a charged product. (A) Organic mercurial derivatives. For pCMBS, X = SO3 ; for pCMB, X- = CO0-. (B) Methanethiosulfonate derivatives. For MTSES, X = SO3-; for MTSEA, X = NH3 +. (C) Iodoacetate.

1995; Pascual et al., 1995), bacterial toxin channels (Mindell et al., 1994; Slatin et al., 1994), the lactose permease (Jung et al., 1993), and the dopamine D2 receptor (Javitch et al., 1995).

We now repor t on the analysis of 26 residues in and flanking the M2 segment f rom oqGlu250 to cx1Asn275, using GABA A receptors fo rmed by the expression ofeq, [3] and ~/2 subunits. We show that nine of these residues are exposed in the channel lumen. We found that the positively charged reagent, MTSEA, can penetra te f rom the extracellular end of the channel to the level of eqThr261 suggesting that the charge selectivity filter is at a more cytoplasmic position. Fur thermore , we show that both negatively and positively charged reagents can enter the channel in the closed state implying that the gate is near the cytoplasmic end of the M2 segment.

196 Residues Lining the Channel of the GABAA Receptor

The results for the res idues 257 to 261 us ing the re-

agents pCMBS a n d MTSEA were pub l i shed previously

(Xu et al., 1995) a n d are i n c l u d e d in Figs. 5 a n d 9 for

comple teness .

periment. Solutions of the mercurials were prepared each day and stored on ice until use. Solutions of iodoacetate were prepared daily and kept on ice in the dark until use.

Statistics

M E T H O D S

Oligonucleotide-mediated Mutagenesis

The cDNA's encoding the rat cq and ~2 subunits in the pBlue- script SK(-) plasmid (Stratagene Corp., La Jolla, CA) were obtained from Dr. P. Seeburg (Shivers et al., 1989; Ymer et al., 1989), and the 131 subunit in the pBlnescript SK vector from Dr. A. Tobin (Khrestchatisky et al., 1989). The Altered-sites mutagenesis procedure (Promega Corp., Madison, WI) was used to substi- tute cysteine residues, one at a time, in the cq subunit as previously described (Xu et al., 1995). Mutations were confirmed by DNA sequencing.

Preparation of mRNA and Oocytes

The in vitro mRNA transcription and the preparation and injec- tion of Xenopus oocytes was performed as described previously (Xu et al., 1995). Oocytes were injected with 10 ng of mRNA encoding the cq, [31, and 3'2 subunits in a 1:1:1 ratio.

Reagents

MTSEA + and MTSES- were synthesized as described previously (Stauffer and Karlin, 1994). pCMBS , pCMB-, and iodoacetate were obtained from Sigma Chemical Co. (St. Louis, MO).

Electrophysiology

GABA-induced currents were recorded from individual oocytes under two-electrode voltage clamp, at a holding potential of -80 inV. Electrodes were filled with 3 M KCI and had a resistance of <2 Mf~. The ground electrode was connected to the bath via a 3 M KCl/Agar bridge. Data was acquired and analyzed on a 486/ 33 MHz computer using a TEV-200 amplifier (Dagan Instru- ments, Minneapolis, MN), a TL1-125 DMA data interface (Axon Instruments, Foster City, CA) and software written using the Ax- obasic language (Axon Instruments). The oocyte was perfused at 5 ml/min with Ca2+-fi'ee Frog Ringer solution (CFFR) (115 mM NaC1, 2.5 mM KC1, 1.8 mM MgC12, 10 mM HEPES pH 7.5 with NaOH) at room temperature. The recording chamber had a volume of ~0.25 ml.

Experimental Protocol

We tested the susceptibility of wild-type and mutant GABAA receptors to a 1-min application of the snlfhydryl reagents applied in the presence or in the absence of 100 p,M GABA. The follow- ing sequence of perfusing solutions was used: 100 p,M GABA (10 s), CFFR (3 min), 100 p.M GABA (10 s), CFFR (3 min), sulfhydryl reagent _+ 100 IzM GABA (1 min), CFFR (3 min), 100 p~M GABA (10 s), CFFR (3 inin) and 100 p.M GABA (10 s). The average of the two peak currents before the sulfhydryl reagent was applied was compared with the average of the two peak currents after. The fractional effect was taken as [1 - (IGAsA. ~/IGABA, b~*o,-~) ]"

The sulfhydryl reagents were applied in CFFR solution, except for iodoacetate which was applied in NaCl-free CFFR. Solutions of the MTS reagents were prepared immediately before each ex-

Data in Figs. 4 to 9 are presented as the means - SE. Significance was determined by one-way analysis of variance using Duncan's post-hoc test (p < 0.05) (SPSS-PC).

R E S U L T S

Using si te-directed mutagenes i s we subst i tu ted cysteine, o n e at a t ime, for 26 consecut ive residues (eqGlu250 to eqAsn275) in a n d f l ank ing the M2 m e m b r a n e - s p a n n i n g

segment . The m u t a n t eq subuni t s were expressed in Xe- nopus oocytes with wild type [3~ a n d ~/2 subuni ts . O n e to two days after mRNA in jec t ion GABA- i n d u c e d cur ren t s

were el ici ted for all of the mutan t s . The average magni - tude of the peak, inward cur ren t s i n d u c e d by 100 ~M

GABA (V = - 8 0 mV) was - 1 . 4 3 -+ 0.4 OA for wild type a nd for the m u t a n t s r a n g e d be tween - 0 . 1 6 _+ 0.03 ~A

for P253C a n d - 1 . 8 + 0.2 p ~ for $272C (Fig. 2). In the absence of sulfhydryl reagents , the GABA-induced cur-

rents of wild-type a n d all of the mu t a n t s were stable for the d u r a t i o n of a typical expe r imen t , ~ 3 0 rain.

For oocytes express ing wild-type GABAA recep to r (def ined as recep tor fo rmed by coexpress ion of the eq,

[3~, a n d ~/2 subuni t s ) , a 1-min app l ica t ion of e i the r 0.5 mM pCMBS, 0.1 mM pCMB, 10 mM MTSES, 100 mM

iodoaceta te or 2.5 mM MTSEA e i ther in the p resence

N275C R274C A273C S272C 1271C i

$270C i L269C T268C T267C M266C T265C L264C V263C T262C T261 C V260C G259C F258C V257C T256C R255C A254C P253C V252C $251C E250C

WT

. . . . , . . . . , . . . . i . . . . i

m

/

I

i m

I

I

m D

I

D

i i

I

. . . . , . . . . i . . . . j . . . . j

0 5OO 1 0 0 0 1500 2000

CURRENT (nA)

FIGURE 2. The average peak current induced by 100 IxM GABA for oocytes expressing wild type or cysteine mutant GABAA receptors. Each bar represents the average current (V= -80 mV) and SEM for six oocytes.

197 Xu AND AXABAS

of 100 ~M GABA or in its absence caused a < 1 0 % change in the subsequent GABA-induced currents (Figs. 3 and 4). Changes o f similar magn i tude were observed after l-rain applications o f GABA alone (data no t shown). 100 mM iodoaceta te reversibly r e duc e d the peak cur- ren t du r ing coappl ica t ion with GABA (Fig. 3 C); coappl icat ion o f the o the r sulfhydryl reagents had no obvi- ous reversible effects on the GABA-induced currents (Fig. 3).

A

B

p C M B S ~

G A B A ~ ~

1 min p C M B

G A B A

V V V V V 1--Jin 23~ Reactions with pCMBS

A 1-min appl icat ion o f 0.5 mM pCMBS, a d d e d in the presence o f GABA, irreversibly inhibi ted the subsequen t GABA-induced currents o f the mutan ts V257C, T261C, L264C, T265C, T268C, I271C, and $272C significantly m o r e than wild type a (Fig. 5 A). It is impor- tant to no te that in the presence o f GABA the channels u n d e r g o transitions between the open , desensit ized and closed states; we c a n n o t distinguish in which state (s) react ion is occu r r ing in the presence o f GABA.

A 1-min appl icat ion o f 0.5 mM pCMBS, a d d e d in the absence o f GABA, irreversibly inhibi ted the subsequent GABA-induced currents o f the mutants V257C, L264C, T265C, T268C, and $272C significantly m o r e than wild type and significantly potent ia ted the subsequent GABA- induced currents o f the m u t a n t N275C (Fig. 5 B). GABA al tered the accessibility o f three o f the mutan ts for react ion with pCMBS: the accessibility o f N 2 7 5 C was r educed in the presence o f GABA and the accessibilities o f T 2 6 1 C and I271C were increased in the presence o f GABA.

C I A

G A B A

~ ~ ~ ~ . 7 ~ j ~ ~minlg0nA

D M T S E S ~

G A B A ~

E M T S E A ~

G A B A - -

V V V V ~ / ' ~ l_~min 2~176

Fmu~E 3. Two-electrode voltage clamp recordings demonstrat- ing that 1-min applications of the sulfhydryl reagents have no irreversible effects on the wild-type GABAA receptors. The panels show the application of (A) 0.5 rnM pCMBS, (B) 0.1 mM pCMB, (C) 100 mM iodoacetate, (D) 10 mM MTSES, and (E) 2.5 mM MTSEA +. A 1-min application of 100 I~M GABA precedes and follows the applications of the sulfhydryl reagents. Holding potential -80 mV.

Reactions with pCMB

A 1-min appl icat ion o f 0.1 mM pCMB, a d d e d in the p resence o f GABA, irreversibly inhibi ted the subse-

1. We identify engineered cysteine residues that reacted with the sulfhydryl reagents if exposure to the sulfhydryl reagents irreversibly altered the subsequent GABA-induced currents of a mutant significantly more than the subsequent currents of wild type. For mutants where the effects of modification are large it is easy to identify residues that have reacted, however, where the effects of modification are small we depend on statistical analysis to identify residues that have reacted with the sulfhydryl reagents. We determine the statistical significance by the oneway analysis of variance using Duncan's post-hoc test to identify the mutants that are significantly different from wild type. There are, however, a variety of post-hoc tests that dif- fer in stringency and, therefore, in which mutants they identify as be- ing different from wild type. For example, analysis of the effect of a l-rain application of pCMBS in the presence of GABA by the least significant difference test, a less stringent test than Duncan's, indicates that in addition to the seven mutants identified by Duncan's test, N275C is also significantly different; analysis of the same data by the Student-Newman-Keuls test, a more stringent post-hoc test, identifies the same mutants as Duncan's test. Therefore, for those mutants where the effects of modification are small, the stringency of the post- hoc test selected determines which mutants we consider to be significantly altered by the sulfhydryl reagents.

q u e n t GABA-induced currents o f the mutan ts V257C, T261C, L264C, T265C, T268C, I271C, and $272C significantly m o r e than wild type (Fig. 6 A).

A 1-min appl icat ion o f 0.1 mM pCMB, a d d e d in the absence o f GABA, irreversibly inhibi ted the subsequent GABA-induced currents o f the mutan ts L264C, T265C, T268C, and $272C significantly m o r e than wild type (Fig. 6 B). As with pCMBS, a 1-min appl icat ion o f 0.1 mM pCMB, a d d e d in the absence o f GABA, significantly po ten t ia ted the subsequen t GABA-induced currents o f the m u t a n t N275C (Fig. 6 B). Coappl ica t ion o f pCMB with GABA al tered the accessibility o f four o f the residues: the accessibility o f N275C was decreased in the presence o f GABA and the accessibilities o fV257C, T261C, and I271C, were increased in the presence o f GABA.

Reactions with Iodoacetate

A l-rain appl icat ion o f 100 mM iodoacetate , a d d e d in the p resence o f GABA, irreversibly al tered the subsequen t GABA-induced currents o f four o f the mutants : the currents o f the m u t a n t T262C were po ten t ia ted

198 Residues Lining the Channel of the GABA A Receptor

M T S E A

MTSEA + G

MTSES

MTSES + G

IA

I A + G

pCMB

pCMB + G

pCMBS

pCMBS + G

-10 0 10

EFFECT (%)

FIGURE 4. The mean irreversible effects of a l-rain application of the sulflaydryl reagents, applied either in the presence or in the absence of 100 IxM GABA, to oocytes expressing wild-type (~[3C/2) GABAA receptors. The reagents were applied at the fol- lowing concentrations 0.5 mM pCMBS, 0.1 mM pCMB, 100 mM iodoacetate, 10 mM MTSES, and 2.5 mM MTSEA, Application of reagent in the presence of GABA is indicated by + G at the end of the reagent's name. The means, SEM's and number of oocytes tested are shown. Note that the range of the x-axis scale is much smaller than in Figs. 5 to9.

a n d t h e c u r r e n t s o f t h e m u t a n t s L264C, T 2 6 8 C , a n d

I 2 7 1 C w e r e i n h i b i t e d (Fig. 7 A).

A 1-min a p p l i c a t i o n o f 100 m M i o d o a c e t a t e , a d d e d in

t h e a b s e n c e o f GABA, i r r eve r s ib ly a l t e r e d t h e subse-

)CMBS + GABA

A '4 ~ ' N275C = ~t~ R274C

H A273C $272C

4 ~ 1271C 4 ~ $270C 4 H L269C

~ T268C ~ T267C

~ ~266C T265C L264C V263C T262C T261 C

4 ~ V260C ,, }1 G259C 4 ~ F258C

4 ~ V257C , ~ T256C

~ R255C 4 ~ A254C

I~ P253C 4 ~ V252C

IN~ $251C ~ ~ E250C

4 il MMT i

-75 -50 -25 0 25

EFFECT (%)

pCMBS - ' ~ B :

3 H

3

5

4 NI~ 4 N ~ 4 ~

4 N N

4 N N 4 ~

-75 -50 -25 0 25

EFFECT (%)

)CMB + GABA

A s N

o ~

= 4

3 H~

3 ' U

3 H '= H 3

3 I 3 ~4 e ~

-75 -50 -25 25

EFFECT (%)

pCMB

N275C a e R274C A273C ~ H $272C 4 1271C 4

$270C ~ L269C , ~ T268C T267C 3 VI266C ~ T265C 4 L264C 7 V263C = T262C 5 T261C eH V260C 4 3259C 4 F258C 3 f V257C 6 H T256C ~ R255C 3 A2.54C 6

P253C 3 ~{ V252C 3 $251C 3 E250C 3

WT 6

-75 -50 -25 0 25

EFFECT (%)

FIGURE 6. The irreversible effect of a 1-min application of 0.1 mM pCMB, applied in the presence (A) or in the absence (B) of 100 IxM GABA, on the subsequent GABA-induced currents of wild type and mutant GABAA receptors. Solid bars indicate mutants for which the effect was statistically significantly different than the effect on wild type. Negative change indicates inhibition and positive change indicates potentiation of the subsequent GABA-induced currents. The means, SEM's and number of oocytes tested are shown.

q u e n t G A B A - i n d u c e d c u r r e n t s o f f ive o f t h e m u t a n t s

s ign i f i can t ly m o r e t h a n wi ld type. T h e s u b s e q u e n t cur -

r en t s o f t h e m u t a n t s T 2 6 1 C , T 2 6 5 C , a n d N 2 7 5 C w e r e

p o t e n t i a t e d a n d t h e c u r r e n t s o f t h e m u t a n t s T 2 6 8 C a n d

I 2 7 1 C w e r e i n h i b i t e d (Fig. 7 B). 2 C o a p p l i c a t i o n o f

G A B A a l t e r e d t h e access ib i l i ty o f f ive o f t h e m u t a n t s to

r e a c t i o n wi th i o d o a c e t a t e : t h e access ib i l i ty o f T 2 6 1 C ,

T 2 6 5 C , a n d N 2 7 5 C was d e c r e a s e d by c o a p p l i c a t i o n o f

G A B A a n d t h e access ib i l i ty o f L 2 6 4 C was i n c r e a s e d by

c o a p p l i c a t i o n o f GABA.

Reactions with MTSES

A 1-min a p p l i c a t i o n o f 10 m M M T S E S , a p p l i e d in t h e

p r e s e n c e o f GABA, i n h i b i t e d t h e s u b s e q u e n t GABA-

i n d u c e d c u r r e n t s o f t h e m u t a n t V 2 5 7 C a n d p o t e n t i a t e d

FIGURE 5. The irreversible effect of a 1-min application of 0.5 mM pCMBS, applied in the presence (A) or in the absence (B) of 100 I~M GABA, on the subsequent GABA-induced currents of wild- type and mutant GABA A receptors. Solid bars indicate mutants for which the effect was statistically significantly different than the effect on wild type by a one way analysis of variance by Duncan's cri- teria. Negative change indicates inhibition and positive change indicates potentiation of the subsequent GABA-induced currents. The means, SEM's and number of oocytes tested are shown.

2. For the mutant T262C, iodoacetate applied either in the presence or absence of GABA potentiated the subsequent GABA-induced current by 20%. Using Duncan's post-hoc test, only the effect in the presence of GABA was significant, however, by the less stringent least significant difference test the effects in the presence and in the absence of GABA are significant (see footnote 1). Despite the difference in the significance by Duncan's test we do not believe that the accessibility of T262C is reduced in the absence of GABA. Similar consider- ations apply to the effects of MTSEA on this mutant.

199 Xu AND AKABAS

M T S E S - + G A B A

}A ' 4 I-

4 ~

e @ 3 31{ /

4 II

1 2

B ~

4

4 I -

4 I

4

4 4H

-50-25 0 25 5O

EFFECT (%)

I O D O A C E T A T E

, , , ,

N275C B '4 R274C A273C 4 S272C 4 H 1271C 4 ~

S270C e L269C ~ H T268C 3 i T267C e rv1266C 1= ~- T265C , L264C 4 F V263C 4 H T262C 8 T261C e V260C 4 G259C 4 F258C e I ~ V257C 6 I~ T256C e I~ R255C 5 I~ A254C ~ P253C 4

V252C 3 [ ] $251C 4 H E250C 4

WT 6 [~ , , T ,

-50-25 0 25 50

EFFECT (%)

FI(;URE 7. The irreversible effect of a l-rain application of 100 mM iodoacetate, applied in the presence (A) or in the absence (B) of 100 IxM GABA, on the subsequent GABA-induced currents of wild type and nmtant GABAA receptors. Solid bars indicate mutants for which the effect was statistically significantly different than the effect on wild type. Negative change indicates inhibition and positive change indicates potentiation of the subsequent GABA-induced currents. The means, SEM's and number of oocytes tested are shown.

MTSES" - ~ - f ,

A : 8 I -

4 E e E

~ E 4 N

3 ~ N

: I 4 ~ 4 ~

4 tt

4 6

-75-50-25 0 25 50

N275C R274C A273C S272C 1271C

S270C L269C T268C T267C M266C T265C L264C V263C T262C T261 C V260C G259C F258C V257C T256C R255C A254C P253C V252C $251C E250C

WT

I O D O A C E T A T E + G A B A

-75-50-25 0 25 50

EFFECT (%) EFFECT (%)

FIGURE 8. The irreversible effect of a 1-min application of 10 mM MTSES, applied in the presence (A) or in the absence (B) of 100 b~M GABA, on the subsequent GABA-induced currents of wild type and mutant GABA a receptors. Solid bars indicate mutants for which the effect was statistically significantly different than the el: fect on wild type. Negative change indicates inhibition and positive change indicates potentiation of the subsequent GABA-induced currents. The means, SEM's and number of oocytes tested are shown.

the cu r r en t s o f the m u t a n t N275C (Fig. 8 A). A 1-min a p p l i c a t i o n o f MTSES, a p p l i e d in the absence o f GABA, only a l t e r ed the s u b s e q u e n t cu r ren t s o f the m u t a n t N275C; r e su l t ing in p o t e n t i a t i o n (Fig. 8 B). MTSES ap- p l i ed e i t he r in the p r e s e n c e o r in the absence o f GABA h a d n o effect o n the o t h e r mutan ts .

Reactions with the Cationic Reagent MTSEA

A 1-min a p p l i c a t i o n o f the posi t ively c h a r g e d MTSEA (2.5 raM), a p p l i e d in the p r e s e n c e o f GABA, irreversibly a l t e r ed the cu r r en t s o f six o f the mutan ts : the currents o f T261C, T262C, T265C, T268C, a n d N275C were p o t e n t i a t e d a n d the c u r r e n t o f the m u t a n t I271C was i n h i b i t e d (Fig. 9 A).

A 1-min a p p l i c a t i o n o f MTSEA, a p p l i e d in the absence o f GABA, i r revers ib ly a l t e r ed the s u b s e q u e n t GABA- induced cu r r en t s o f t h r e e o f the mutan ts . T h e cu r r en t s o f the m u t a n t s T261C a n d N275C were p o t e n - t ia ted a n d the c u r r e n t o f the m u t a n t I271C was inhib- i ted s ignif icant ly m o r e t han wild type (Fig. 9 B). Thus , c o a p p l i c a t i o n with GABA i n c r e a s e d the accessibi l i ty o f T265C a n d T268C for r e a c t i o n with MTSEA. 9

D I S C U S S I O N

Residues Exposed in the Channel Lumen

T h e GABA- induced cu r r en t s o f 9 ou t o f 26 cysteine- subs t i tu t ion m u t a n t s in a n d f l ank ing the M2 m e m - b r a n e - s p a n n i n g s e g m e n t were i r revers ibly a l t e r ed by ex t r ace l lu l a r a p p l i c a t i o n o f c h a r g e d , sulfhydryl-specif ic r eagen t s (Table I) . We infer f rom the i r revers ib le al ter- a t ion o f the GABA- induced c u r r e n t t ha t the sulfhydryl r eagen t s r e a c t e d with an e n g i n e e r e d cysteine. For sev- era l reasons we assume tha t these r eagen t s will only react with sulfhydryls tha t a re on the water access ible surface o f the p ro t e in . First, pCMBS was essent ial ly impe r - m e a b l e t h r o u g h the e ry th rocy te m e m b r a n e d u r i n g a 90-rain i ncuba t i on , a l t h o u g h pCMB was p e r m e a b l e (VanSteveninck , W e e d , a n d Roths te in , 1965). Second , the ra te o f r e a c t i o n o f the MTS reagen t s with an ionized th io la te a n i o n is 5 • 109 t imes faster t han the ra te o f r e a c t i o n with the u n i o n i z e d th iol (Rober t s e t al., 1986) a n d the ra te o f r eac t i on o f the mercu r i a l s with an i on i z e d th io la te a n i o n is 2 • 10 '~ t imes faster t han the ra te with the u n i o n i z e d th io l (Has ino f f e t al., 1971); only sulfhydryls e x p o s e d to wate r will ion ize to a signifi- can t ex tent . Thus , we infer tha t e n g i n e e r e d cysteine


M T S E A * + G A B A

N275C R274C A273C S272C 1271C

$270C L269C T268C T267C M266C T265C L264C V263C T262C T261C V260C

I~ ~259C F258C V257C T256C R255C

,~ A254C e I~ P253C ,= 14 V252C 3 H $251C 5 I~ E250C o w r

M T S E A *

4 4 4

S 3 I 4 4

4 4} - 5 5~

-25 0 25 50 75 -25 0 25 50 75

EFFECT (%) EFFECT (%)

FIGURE 9. The irreversible effect of a 1-min application of the positively charged MTSEA (2.5 mM), applied in the presence (A) or in the absence (B) of 100 p~M GABA, on the subsequent GABA- induced currents of wild type and mutant GABAA receptors. Solid bars indicate mutants for which the effect was statistically signifi- candy different than the effect on wild type. Negative change indicates inhibition and positive change indicates potentiation of the subsequent GABA-induced currents. The means, SEM's and number of oocytes tested are shown.

residues that react with these sulfhydryl reagents are o n

the water accessible surface of the p ro te in . Fur ther - more , we assume that the only water-accessible surface for residues in m e m b r a n e - s p a n n i n g segments is the surface exposed in the ion c h a n n e l l u m e n . We infer

that the s t ruc ture of each m u t a n t is s imilar to the structure of wild type because we observed GABA-induced

cur ren t s in oocytes express ing each of the 26 cysteine- subs t i tu t ion mutan ts . There fore , for those m u t a n t s that

reac ted with the sulfhydryl reagents , we infe r that the

c o r r e s p o n d i n g wild-type res idues atVa1257, cqThr261, eqThr262, (XlLeU264, (x]Thr265, a1Thr268, eqIle271, cqSer272, a n d (x1Asn275 are exposed in the c h a n n e l lu- m e n f o r m i n g the l i n ing of the ion c o n d u c t i n g pathway.

O u r in fe rence that a res idue is exposed in the channel if it reacts with the sulfhydryl reagents is i n d e p e n -

d e n t of whe the r the reac t ion results in po t en t i a t i on or i n h i b i t i o n of the s u b s e q u e n t GABA-induced currents .

E luc ida t ion of the de ta i led mechan i sms for these effects will r equ i re the d e t e r m i n a t i o n of the effect of

modi f i ca t ion on single c h a n n e l c o n d u c t a n c e a n d o p e n

probabil i ty. Nevertheless, the observat ion that reac t ion of an indiv idual r eagen t gave i n h i b i t i o n for some mutants a n d p o t e n t i a t i o n for others (Table I) implies that

the effects of modi f i ca t ion are n o t solely due to local electrostatic or steric effects.

It is t e m p t i n g to assume that the residues for which

the sulfhydryl reagents had n o effect are n o t exposed in the c h a n n e l l u m e n . A l though this is the likely expla- n a t i o n it may no t be correct in all cases. Residues that

are exposed in the c h a n n e l may no t react due to local steric factors such as those that l imit the ability of MTSES to react with mos t of the c h a n n e l - l i n i n g resi-

dues. Alternatively, because we have m e a s u r e d the ef-

fects o f modi f i ca t ion us ing macroscopic cu r ren t s it is possible that, for example , modi f i ca t ion mi gh t increase o p e n probabi l i ty bu t decease s ing le -channe l conductance or vice versa resu l t ing in an u n a l t e r e d macro-

scopic cur ren t . alVa1257 was the mos t cytoplasmic m u t a n t accessible

to the reagents appl ied extracellularly; we do n o t know whe the r the reagents can pene t r a t e b e y o n d this po in t in the channe l . MTSES is i m p e r m e a b l e t h rough the channe l ; we were u n a b l e to measure outward cur ren t s

when the 115 mM NaCI in the ext race l lu lar ba th was re-

T A B L E I

Summary of the Cysteine-substitution Mutants and the Sul~hydryl Reagents with Which They Reacted*

pCMBS pCMBS/G pCMB pCMB/G IA IA/G MTSES M E S E S / G M T S E A MTSEA/G

N275 + + + + + + $ 2 7 2 . . . . I 2 7 1 . . . . . . T 2 6 8 . . . . . . + T 2 6 5 . . . . + + L 2 6 4 . . . . . T262 + + T261 - - + + + V 2 5 7 . . . .

*These results are abstracted from Figs. 3 to 7. Only the mutants that reacted with the sulfhydryl reagents are shown. Abbreviations: +, covalent modification caused potentiation of the subsequent GABA-induced current; - , covalent modification caused inhibition of the subsequent GABA-induced currents; blank indicates no effect;/G indicates the sulfhydryl reagent was applied in the presence of GABA; IA is iodoacetate.

201 Xv AND AKABAS

placed by 115 mM NaMTSES (data not shown). Consis- tent with this, isethionate, which also contains a sul- fonate, was not measurably pe rm ean t (Bormann et al., 1987). The narrow region of the channel is likely to be at or below alVa1257 because picrotoxin, a channel blocker that is roughly spherical with a d iameter of 9 ,~ appears to bind in the channel at the level of alVa1257 (Xu et al., 1995). Fur thermore , the residues aligned with alVa1257 in the acetylcholine receptor are thought to be at the narrow point in the channel (Vil- larroel et al., 1991; Imoto et al., 1991; Cohen et al., 1992). Thus, the extracellularly applied reagents may not be able to reach the mutants, E250C to T256C, that are more cytoplasmic than aaVa1257 (Fig. 10). Alterna- tively, reduced glutathione, an anion, may be able to enter the cytoplasmic end of this anion-selective channel and protect or regenerate the free sulfhydryl of exposed residues in the region f rom 250 to 256. Experi- ments utilizing patch clamp recording will be necessary to resolve these issues.

The functional effects of mutagenesis have been studied for several GABAA receptor M2 segment residues. In Drosophila, cyclodiene insecticides and picrotoxin bind to the GABAA receptor and inhibit GABA- induced currents. A point muta t ion of Ala to Ser in the residue which aligns with eqVa1257, a residue exposed in the channel, confers resistance to these agents (french- Constant et al., 1993). This muta t ion in the Drosophila GABAA receptor had a small effect on single channel conductance, a fivefold stabilization of the open state and a ninefold reduct ion in the rate of desensitization (Zhang et al., 1994). Mutation to Phe, in the rat GABA A receptor, of the residues at the positions aligned with a1Thr261, an exposed residue, in ei ther the a, [3, or ~/ subunits also abolished picrotoxin sensitivity (Gurley et al., 1995). We have shown that coapplication of picrotoxin protected the mutan t a1V257C f rom modification by pCMBS but coapplication of picrotoxin did not protect the mutan t a1T261C f rom modification by

pCMBS or MTSEA (Xu et al., 1995). Picrotoxin also protected the mutan t alV257C f rom modification by MTSES applied in the presence of GABA (data not shown). Based on these results we inferred that picrotoxin binds in the channel lumen at the level of alVa1257 (Xu et al., 1995). The binding of the anticon- vulsant drug loreclezole is dependen t on the residue in the [3 subunit that aligns with oqSer270, a residue that is not exposed in the channel. Loreclezole binds when there is an Asn at this position in the [3 subunit, as there is in the [32 and [33 subunits but the affinity is 300-fold lower when there is a Ser at this position as occurs in the [31 subunit (Wingrove et al., 1994). In the homologous glycine receptor, muta t ion of residues in the M2 segment altered single channel conductance (Bor- mann et al., 1993).

Secondary Structure of the M2 Membrane-spanning Segment

When the residues in and flanking the M2 segment are plotted on an 0~ helical net or wheel representat ion the exposed residues, except a lThr262, lie on one side of the helix within an arc of 120 ~ (Fig. 10 B) and form a stripe extending down the side of the helix (Fig. 10 A). Approximately 110 ~ of arc would be exposed on an helix if the channel were lined by five a helices ar- ranged parallel to the channel axis; this is consistent with the p resumed pentamer ic structure of the GABAA receptor (Unwin, 1993; Nayeem et al., 1994). The exposure of a lThr262 indicates that the helix may be dis- torted in this region, a l though we cannot exclude the possibility that the muta t ion distorts the structure of the T262C mutant . Interestingly, using the same approach a similar distortion of the a helical pat tern was observed in the aligned region of the M2 segment of the acetylcholine receptor (Akabas et al., 1994a). In a 9-~ resolution structure of the acetylcholine receptor, Unwin (1993) no ted that the channel-lining helix was kinked in the middle; assuming that homologous pro-

A

$270 ~ ' ' jB..~ - ~

M266 _.~:~'~BL264 T265m."

V260 T267

$272 A25~2~5 ' ~

F258 L269

�9 SUSCEPTIBLE TO SULFHYDRYL REAGENTS 0 NOT SUSCEPTIBLE TO SULFHYDRYL REAGENTS

T256 R274

V252 $270

259

26A273 T262

FIGURE 10. Ot helical projections of the residues in and flanking the M2 membrane spanning segment of the rat a~ subunit. The mutants that reacted with the sulfhydryl reagents are indicated with solid black squares. The nonreactive mutants are indicated by open circles. (A) is a helical net projection. The x-axis represents the circumfer- ence of the helix. The y-axis represents distance along the helix, the extracellular end is at the top. (B) is a helical wheel representation.

202 Residues Lining the Channel of the GABAA Receptor

teins have similar three-dimensional structures, the kink may correspond to the region of cx1Thr262.

Charge-selectivity Filter

The ability of the positively charged MTSEA to react with cysteines substituted for six of the channel lining residues (Fig. 9 and Table I) suggests that cations can enter the extracellular end of the GABAA receptor channel at least to the level of 0qThr261.3 This may im- ply that the charge selectivity filter that distinguishes between anions and cations is at least as cytoplasmic as cxlThr261. Alternatively, because the channel may not be ideally anion selective (the upper limit of potassium permeability relative to chloride is 0.05 [Bormann et al., 1987]) the ability of MTSEA to react might be due to the small, but possibly finite cation permeability. Complementary to these results is our finding in the cation-selective acetylcholine receptor that the extracellular end of the channel is also accessible to anionic sulfhydryl reagents (Akabas et al., 1994a).

Of the nine residues exposed in the channel, none are charged, six are polar and three are hydrophobic. This suggests that the electrostatic interactions between permeat ing anions and the channel wall are likely to be charge-dipole interactions rather than charge-charge interactions. The anion permeability sequence reported for the GABA A receptor is SCN > I > Br > C1, with the reverse conductance sequence (Bormann et al., 1987). This is consistent with a weak interaction between anion binding sites in the channel and the permeat ing anions (Wright and Diamond, 1977).

Position of the Channel Gate

The ability of the charged sulfhydryl reagents to pene- trate from the extracellular end of the channel to the level of 0qVa1257 in the absence of GABA, i.e., in the closed state of the channel, suggests that the channel lumen is patent in the closed state and that the position of the gate is at least as cytoplasmic as oqVa1257.

To infer the position of the gate we must assume that in the closed state the sulfhydryl reagents reach the channel-lining cysteines by entering the extracellular end of the channel and moving down the channel. Is it possible that the reagents reach some of the more cytoplasmic residues in the closed state by an alternative pathway, such as through the lipid bilayer to the cytoplasm and then in the cytoplasmic end of the channel? Although these reagents are largely ionized at pH 7.5,

3. To rule out the possibility that MTSEA might be entering the channel in its uncharged form we examined the ability of the permanently positively charged MTS-ethyltrimethylammonium (MTSET) to react with T261C. MTSET reacts with T261C in the presence of GABA, in- dicating that positively charged ions can enter the anion-selective channel of the GABAA receptor (Horenstein and Akabas, unpub- lished observation).

all will have some finite concentrat ion of their union- ized form which might be permeable through the lipid bilayer. The membrane permeability of pCMBS is very low (VanSteveninck et al., 1965) and it has been used to distinguish the sidedness of exposure of endogenous cysteine residues in a bacterial anion transporter (Yan and Maloney, 1993). Fur thermore, if the reagents did reach the cytoplasm they would most likely be scav- enged by the relatively high concentrat ion of free sulfhydryls in the cytoplasm before they could enter the channel. Because the spontaneous open probabilities of our mutants are unknown, we cannot preclude the possibility that in the absence of GABA the sulfhydryl reagents can reach the channel lining cysteines during brief spontaneous openings of the channel. By measur- ing the rates of reaction of the sulfhydryl reagents with channel-lining cysteines in the presence and absence of GABA we can set a limit on the spontaneous open probability that would be necessary to explain our results.

Our inference that the gate is near the cytoplasmic end of the channel is consistent with the results of similar experiments on the homologous acetylcholine receptor (Akabas et al., 1994a).

GABA-induced Changes in the Reaction of Channel-lining Residues with the Sulfhydryl Reagents

All of the channel-lining residues reacted with at least one of the sulfhydryl reagents in both the presence and in the absence of GABA (see footnote 2 regarding T262C) (Table I). Thus, the same residues are exposed in the channel lumen in the presence and in the absence of GABA. In the absence of GABA the channel is mainly in the closed state, however, as noted above, in the presence of GABA the channel fluctuates between the open, desensitized and closed states; we cannot at present distinguish in which of these state(s) reaction with the sulfhydryl reagents is occurring. Nevertheless, GABA-induced conformational changes do not appear to involve a significant rotation of the channel-lining M2 segments.

Coapplication of GABA altered the ability of at least one of the sulfhydryl reagents to react with eight of the nine channel-lining residues (Table I). GABA-induced changes in the channel might effect the reaction of the sulfhydryl reagents with channel-lining cysteine residues in several ways. First, GABA-induced structural changes might alter local steric factors that influence the ability of a particular reagent to orient itself in or- der to react. Second, opening the channel gate may alter the electrostatic profile within the channel and this may change the residence time of ions at different positions along the channel; this might also alter the extent of ionization of the cysteine which would effect the reaction.

203 Xu AND AKABAS

Differences between the Sulfhydryl Reagents

A n in t e r e s t i ng aspec t o f these e x p e r i m e n t s is tha t no s ingle r e a g e n t r e a c t e d with all o f the e x p o s e d res idues; pCMBS a n d pCMB r e a c t e d with e igh t o f n ine , i odoace - tate r e ac t ed with seven o u t o f n ine , MTSES r e a c t e d with two o u t o f n ine , a n d the ca t ion ic r eagen t , MTSEA, r e a c t e d with six o u t o f n ine (Table I) . pCMBS, the larg- est o f the reagen ts , r e a c t e d with all o f the e x p o s e d residues e x c e p t T262C; given tha t pCMBS is fair ly r ig id it is poss ib le tha t s ter ic fac tors l imi t its access to T262C. T h e sma l l e r an ion ic r eagen t , i odoace t a t e , r e a c t e d with a subse t o f the r e s idues access ib le to the mercur ia l s , but , in add i t i on , it also r e a c t e d with T262C. T h e lack o f reac t ion o f i o d o a c e t a t e with V257C a n d $272C m i g h t be d u e to the s lower ra te o f r e a c t i o n o f i o d o a c e t a t e with sulfhydryls c o m p a r e d to the mercu r i a l s a n d p e r h a p s the r e s i d e n c e t ime o f i o d o a c e t a t e in the r eg ion o f these res idues is insuf f ic ien t to al low reac t ion .

Given tha t the chemis t ry o f the MTS derivat ives is s imilar , the lack o f MTSES r eac t i on with five mutan ts , T261C, T262C, T265C, T268C, a n d I271C, tha t r e a c t e d with MTSEA is surpr is ing . T h e MTS derivat ives were used at c o n c e n t r a t i o n s tha t give s imi lar ra tes o f reac- t ion with smal l sulfhydryls in f ree so lu t ion (Stauffer a n d Karl in, 1994). MTSES is l a rge r t han MTSEA by 1 to 2 ,~, b u t is s imi lar in size to pCMBS, t h e r e f o r e size does n o t s eem to be the crucia l issue. MTSES is con s ide r a b ly

m o r e f lex ib le than pCMBS; the b e n z e n e r i ng in pCMBS a n d pCMB is r igid, t he r e by k e e p i n g the anionic g r o u p away f rom the react ive m e r c u r i a l g roup , however , the su l fona te g r o u p o f MTSES can ro ta te to a pos i t i on a d j a c e n t to the react ive t h io su l fona t e e n d o f the molecu le . In this c o n f o r m a t i o n the react ivi ty with the negat ive ly c h a r g e d th io la te o f the e n g i n e e r e d cys- t e ine may be r e d u c e d ; why this c o n f o r m a t i o n o f MTSES s h o u l d p r e d o m i n a t e in the c h a n n e l is u n c l e a r a n d o t h e r factors may be i m p o r t a n t .

Comparison with the Acetylcholine Receptor

T h e s c a n n i n g cyste ine accessibi l i ty m e t h o d has also b e e n a p p l i e d to the M2 s e g m e n t o f the a s u b u n i t o f the ace ty lcho l ine (ACh) r e c e p t o r (Akabas et al., 1994a). Ten residues between the ACh recep to r residues aGlu241 a n d o~Glu262 are e x p o s e d in the channe l . Seven o f the n ine re s idues tha t we have shown are e x p o s e d in the GABAA r e c e p t o r c h a n n e l a l ign with res idues e x p o s e d in the ACh r e c e p t o r channe l . T h e ACh r e c e p t o r residues tha t a l ign with the GABAa res idues 0qThr262 a n d a l S e r 2 7 2 are n o t e x p o s e d in the ACh r e c e p t o r channe l . Thus , a l t h o u g h the s t ruc tures o f these h o m o l o g o u s p r o t e i n s a re s imi lar t h e r e a re d i f fe rences . F u r t h e r s tudy o f these d i f fe rences may h e l p us to u n d e r s t a n d the m e c h a n i s m s o f cha rge selectivity a n d ion c o n d u c t i o n in the l i gand -ga t ed ion channe l s .

We thank Drs. Peter Seeburg and Allan Tobin for gifts of the GABAA receptor subunit cDNAs, Gilda Salazar-Jimenez for preparing and injecting Xenopus oocytes, Celeste DeMarco for technical assistance and Drs. Jonathan Javitch and Arthur Karlin for helpful discussions and comments on this manuscript.

This work was supported by NIH NS30808 and a Grant-in-Aid from the American Heart Association to M. H. Akabas. M. H. Akabas is the recipient of an Established Scientist Award from the New York City Af- filiate of the American Heart Association and a Klingenstein Fellowship Award in the Neurosciences. M. Xu was supported in part by NIH Training Grant NS07258.

Original version received 24 August 1995 and accepted version received 3 November 1995.

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205 X u AND AKABAS

identification of channel-lining residues in the m2 membrane-spanning segment of the gabaa

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