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Journal of Physiology (1992), 447, pp. 587-625 With 15 figures Printed in Great Britain

A NOVEL MODULATORY BINDING SITE FOR ZINC ON THE GABAA RECEPTOR COMPLEX IN CULTURED RAT NEURONES

BY TREVOR G. SMART From The School of Pharmacy, Department of Pharmacology, 29-39 Brunswick Square, London WC1N lAX

(Received 16 April 1991) SUMMARY

1. The properties of y-aminobutyric acidA (GABAA) receptor-ion channel complexes and the interaction with the transition metal zinc, were studied on rat sympathetic and cerebellar neurones in dissociated culture using patch clamp recording techniques. 2. The antagonism of GABA-induced membrane currents by zinc on sympathetic neurones was subject to developmental influence. Using embryonic sympathetic neurones acutely cultured for 24-72 h, GABA responses were more depressed by zinc when compared to responses evoked on adult neurones cultured for the same period. For neurones developing in vivo, the percentage inhibition of GABA responses produced by zinc in embryonic neurones was estimated to decline by 50 % after 48-2 days following birth. 3. Embryonic sympathetic neurones maintained in culture for prolonged periods (40-50 days in vitro, DIV) became less sensitive to zinc when compared to neurones cultured for shorter periods (10-20 DIV). The decrease in the zinc inhibition for neurones maintained in vitro proceeded at an apparent rate of 0-55 % per day. 4. Activation of the GABA receptor by muscimol (0-2-2 (1M) was also antagonized by zinc (50-100 JtM). 5. Lowering the pH of the perfusing Krebs solution did not affect the inhibition of GABA responses by zinc on sympathetic neurones. 6. Modulation of the GABAA receptor by some benzodiazepines, a barbiturate, a steroid based on pregnanolone, or antagonists bicuculline and picrotoxinin, did not interfere with the antagonism exerted by zinc on sympathetic neurones. A novel binding site for zinc on the GABAA receptor is proposed. 7. Analysis of the GABA-activated current noise on sympathetic neurones revealed two kinetic components to the power spectra requiring a double Lorentzian fit. The time constant describing the fast component (p2, 2-1 ms) was unaffected by zinc, whereas the slow component time constant (T,l 21-7 ms) was slightly reduced to 17-1 ms. 8. The apparent single-channel conductance for GABA-activated ion channels was determined from the power spectra (yr = 22-7 pS) and also from the relationship between the mean GABA-induced inward current and the variance of the current (yv = 24 pS). Zinc (25-100 fM) did not affect the single-channel conductance. MS 9307

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9. Single GABA-activated ion channels were recorded from outside-out patches taken from the soma of large cerebellar neurones. Single GABA channels were capable of activation to multiple current amplitudes which were assessed into the following conductance levels: 8, 18, 23, 29 and 34 pS. The main conductance state was at 29 pS, but a minority of patches also exhibited predominant openings to the 18 pS state. Zinc did not affect the main single-channel conductance of 29 pS, but reduced the opening frequency. 10. Two exponential components were required to describe the frequency histogram for all open times, suggesting two different open channel states. Both the short (Tr01 0-76 ms) and long (r02, 4-3 ms) mean open time constants were unaffected by zinc (To, = 0-67 ms and T.2 = 51 ims) although the relative frequency of occurrence of long duration open states was decreased. 11. The closed time distributions were fitted by the sum of two to three exponentials suggesting the existence of multiple closed states. The mean closed time constants were designated short, intermediate and long accordingly. Zinc increased the mean long closed time constant and also the relative contribution of this component to the overall distribution. Zinc did not induce any rapid closure/ blocking events in the single GABA channel currents. 12. Channel openings were either discrete single events or occurred in bursts. Within each burst, successive openings were separated by brief closures or by transitions to other conductance levels. Burst duration distributions were fitted with the sum of three exponentials. The mean burst duration time constants were: TBI = 0 33 ms, 7B2 = 3-1 ms and TB3 = 26-4 ms. In zinc, only the long duration mean burst time constant was affected, TB3 = 18-5 ms. The relative contribution of the long bursts to the overall frequency histogram was also reduced by zinc. 13. The mean number of openings per burst decreased in zinc from 6-5 to 4. The frequency histogram for all intraburst open times was described by the sum of two exponentials. Ten micromolar zinc caused a small reduction in the mean time constant of the long intraburst open times without affecting the relative contribution of this component. 14. The mode of inhibition of GABA responses by zinc is complex. These results can be explained by proposing that zinc binds to a novel site on the receptor protein, probably in the un- or monoliganded state, and stabilizes the channel in the closed conformation. The location of the binding site could be within an area of 'high hydrophobicity contrast', possibly on or near the extracellular flanking zone of the putative transmembrane domain, M2, for the GABAA receptor. This site is apparently susceptible to developmental processes, rendering zinc less effective as a GABA antagonist in adult neurones. INTRODUCTION

The transition metal zinc is unevenly distributed throughout the central nervous system (CNS) being concentrated within nerve terminals principally in the telencephalon (Ibata & Otsuka, 1969; Friedman & Price, 1984; Perez-Clausell & Danscher, 1985; Holm, Andreasen, Danscher, Perez-Clausell & Nielsen, 1988). A number of different roles have been postulated for zinc in nerve terminals, including: acting as a 'packaging agent' for neurotransmitters within synaptic vesicles

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(Frederickson, 1989); influencing neuronal development (Dvergsten, Fosmire, Ollerich & Sandstead, 1983); a pathological role by interacting with the major excitatory and inhibitory amino acid receptors in the CNS (Peters, Koh & Choi, 1987; Westbrook & Mayer, 1987; Mayer, Vyklicky & Westbrook, 1989; Mayer & Vyklicky, 1989; Smart & Constanti, 1990), and a neuromodulatory role, influencing y-aminobutyric acid (GABA)-mediated synaptic transmission in the hippocampus (Xie & Smart, 1991). One intriguing aspect of the involvement of zinc in the function of the CNS concerns its interaction with the GABAA receptor. Young, mostly embryonic neurones possess GABAA receptors mediating chloride-dependent responses which are inhibited by zinc, whereas more mature adult neurones exhibit relatively zincresistant GABAA responses (Smart & Constanti, 1990; Smart, 1990a). The site of action for zinc on the neuronal GABAA receptor was not studied, but on invertebrate muscle, zinc antagonized GABA responses by probably binding to a histidine group contained in the putative GABA receptor structure (Smart & Constanti, 1982). The objectives of the present study were threefold: to examine the time course for the developmental change in zinc sensitivity of the GABAA receptor using cultured neurones; to locate a putative binding site for zinc on the allosteric GABAA receptor protein complex using combination studies with other ligands, and also to ascertain how zinc may inhibit the embryonic GABAA response using membrane current noise and single-channel current analysis. A preliminary report of part of this work has appeared (Smart, 1990b). METHODS

Cell culture Sympathetic neurones. Sprague-Dawley embryonic rats (embryonic day, E19-21) were removed from adult female rats previously anaesthetized by ether inhalation and killed by exsanguination. Superior cervical ganglia were removed from the embryos and pooled (ten to twelve ganglia) in icecold, unsupplemented Leibovitz's medium (L-15; GIBCO). Ganglia were freed from associated tissue, cut into two to three pieces and washed with cold L-15. Tissue pieces were incubated in Hank's Ca2+- and Mg2+-free (CMF) balanced salt solution containing 0-25 % w/v trypsin (GIBCO) and 80-100 units/ml collagenase type VII (Sigma) for 45 min at 37 °C in 95 % air-5 % CO2. After 15 min incubation, the tissue was briefly triturated (10 x ) using a wide-bore heat-polished Pasteur pipette. Enzymatic dissociation was terminated by adding growth medium and the tissue was mechanically dissociated with four polished Pasteur pipettes with decreasing orifice diameters. The tissue was triturated with each pipette 20 times, leaving the suspension to settle for 2-3 min, then removing and pooling the supernatant containing single cells. Fresh growth medium was added to the suspension of remaining tissue fragments prior to further mechanical dissociation. This procedure ensured complete dissociation of all the tissue. The pooled supernatant was centrifuged at 190 g for 10 min. The medium was replaced with fresh growth medium and the cells were resuspended by mild agitation. Cells were subsequently plated inside a small glass ring (Smart, 1987) positioned centrally on 35 mm dishes previously coated for 1-2 h with laminin at 20 ,ug/ml per dish (FLOW labs). Cells were plated at approximately one to two ganglia per dish and were grown in L-15 medium supplemented with 10% v/v fetal calf serum (GIBCO), 50 ng/ml 7S-nerve growth factor (mouse submaxillary gland, Calbiochem), 2 mM-glutamine (GIBCO), 0-6% w/v glucose (BDH), 0-19% w/v NaHCO3 (BDH), penicillin-G (100 units/ml) and streptomycin (100 ,ag/ml; GIBCO). Cells were incubated at 37 °C in an atmosphere of 95 % air-5 % C02. After 3-5 days in culture, Schwann and fibroblast cell proliferation was controlled by 10 jtM-cytosine arabinoside treatment for 24 h. Cultures were fed twice weekly with fresh growth medium. Postnatal (day P1-12) sympathetic ganglia were prepared in a similar manner to the embryonic neurones. Cultures of ganglion neurones from mature adult animals (P > 93, 200-300 g mass) required variation to the enzymatic dissociation procedure. Animals were anaesthetized by an

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intraperitoneal injection of urethane (1 5 mg/kg body weight). Six ganglia were dissected free from connective tissue sheaths, chopped and pooled in ice-cold L-15. The tissue pieces were dissociated with 5 mg/ml dispase II (Boehringer Mannheim), 1 mg/ml collagenase type IA (Sigma), 100 ,tg/ml DNase I (Sigma) and 20,u1 NaHCO3 solution (75% w/v, to maintain pH 7), in Hank's CMF solution. Ganglia were incubated for 2 h with mild pipetting (10 x ) using a wide-bore polished pipette after 30 and 90 min from the start of the incubation. Enzymatic dissociation was terminated with fresh growth medium. Mechanical dissociation and all subsequent procedures for producing adult neuronal cultures were identical to the embryonic cultures. Cell growth in the adult cultures was considerably slower compared to the embryonic cultures; cytosine arabinoside was used, if at all, only after 1-2 weeks in culture. Cerebellar neurones. Cerebellar cultures were prepared using similar methods to those reported previously (Moonen, Neale, MacDonald, Gibbs & Nelson, 1982; Cull-Candy & Usowicz, 1989a). Cerebellar cortices were isolated from E17-18 rats, carefully freed from meninges and pooled in icecooled minimum essential medium (MEM) containing 10% v/v fetal calf serum, 10% v/v horse serum, 2 mM-glutamine, 0-6 % w/v glucose and 100 units/ml penicillin-G and 100 ,ug/ml streptomycin (plating medium). Two to three cortices were chopped into slices and taken up into a 5 ml syringe with 1 ml plating medium. The tissue suspension was expelled through a sterile nylon mesh (pore diameter 210 ,um) into a 5 cm dish. Fresh plating medium was used to remove residual tissue left on the mesh. Washing continued until the suspension contained approximately one cerebellum per millilitre of washing solution. This produced a suspension of 'microexplants' and single cells which were plated onto 35 mm dishes previously coated with either astrocytes derived from cerebellar cultures, poly-L-lysine (Sigma, 70000-150000 regular molecular mass, 36 ,tg/ml) or collagen (Vitrogen, 3 mg/ml) and poly-L-lysine. Cells were incubated at 37 °C in 95 % air-5 % CO2 atmosphere and after 4 days the plating medium was replaced with a growth medium based on MEM supplemented with 10 % v/v horse serum, 2 mM-glutamine, 0-6 % w/v glucose, 100 units/ml penicillin-G, 100 ,ag/ml streptomycin and 20 mM-KCI. After 4-5 days in culture, cells were treated with 10 /LM-cytosine arabinoside for 24 h and cultures were fed twice weekly with growth medium. Large cerebellar neurones (diameter > 35 ,um) were used for electrophysiology after 12-38 days in culture and could be distinguished from granule cells on the basis of morphology using the size of the soma and type of dendritic arborization. These large 'macroneurones' would include Purkinje cells and also presumed inhibitory interneurones (Moonen et al. 1982; Neale, Moonen, MacDonald & Nelson, 1982; Gruol & Franklin, 1987; Cull-Candy & Usowicz, 1989a). Electrophysiology Experiments were performed using a List EPC7 amplifier with either whole-cell or outside-out patch configurations as described by Hamill, Marty, Neher, Sakmann & Sigworth (1981). Patch electrodes were formed from thin-walled borosilicate glass (Clark Electromedical, GC-150T) for whole-cell recording, and both thin- and thick-walled glass (GC-150) for single-channel recording. Patch pipettes, coated with Sylgard resin only for single-channel recording, were heat-polished and filled with a pipette solution containing (mM): KCl, 150; CaCl2, 0-279; MgCl2, 1; Na-EGTA, 05; HEPES, 5; pH 7 15. On some occasions, to facilitate single GABA channel recording the following pipette solution was used (mM): CsCl, 120; TEA-OH, 33; CaCl2, 1; MgCl2, 1; EGTA, 11; HEPES, 10; pH 7 1. Pipettes had resistances of 1-5 MQ for whole-cell recording and 10-15 MQ for singlechannel recording. The neurones were viewed under phase-contrast optics (Nikon, 40 x long working distance objective, 400-800 x overall magnification) and continuously superfused (10 ml/min) in the culture dish (vol 1 ml) with a Krebs solution containing (mM): NaCl, 140; KCl, 4-7; MgCl2, 1-2; CaCl2, 2; glucose 11; HEPES, 5; pH 7-4 at 30 'C. GABA antagonists and modulators were applied via the superfusate and GABA (50 /LM) was also applied via pressure ejection from a patch pipette positioned approximately 20-50 ,um from the cell soma. Ejection pressures varied from 50 to 90 kPa. Whole-cell voltage clamp current data were recorded on a Brush-Gould 2400S ink-jet pen recorder and single-channel currents were filtered at 10 kHz (-3 dB, 6-pole Bessel filter, 36 dB/octave) and stored on a Racal store 4DS FM tape-recorder (bandwidth DC-5 kHz). Fluctuation analysis of GABA-activated membrane currents GABA-induced membrane current noise was stored on FM tape and subsequently high-pass filtered at 0 5 Hz and low-pass filtered at 500 Hz (-3 dB, 8 pole, Butterworth). The filtered control and GABA-induced current noise signals were digitized at 1024 Hz (CED 1401 interface) and

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displayed in time segments of 500 ms (512 samples), enabling resolution of 2 Hz. All records were visually inspected for synaptic currents or artifacts and edited accordingly. The spectral density of each current noise record was measured within the bandwidth of the spectrum and a mean spectrum was calculated from at least sixteen to thirty-two separate spectra. The net mean power spectrum for GABA-induced noise was obtained by subtraction of the mean background spectrum in the absence of agonist. The net, one-sided agonist-induced power spectrum was then fitted, using a non-linear least-squares routine (Levenberg-Marquardt), with the sum of two Lorentzian components. The Lorentzian curve is defined by the equation S(f) = {SJ(0)/[ I + (f/f1)2]} + {S2(0)/[1 + (f/f2)2]}, where S(f) is the spectral density (pA2) at any frequency, f (Hz). S1(0) and S2(0) are the spectral densities at zero frequency and f1 and f2 represent the cut-off frequencies at which S(f ) has fallen to S(0)/2 for the two respective Lorentzian components. These frequencies can also be determined as time constants, rln where Tn = 1/(27Tfn) with n representing the nth component of the spectrum. The apparent single-channel current was estimated from the variance of the current calculated from the Lorentzian fit to the power spectra using i =

7T[S1(0)fi+S2(0)f2]/2Im,

where i represents single-channel current and Im the mean GABA-induced membrane current. The single-channel conductance (yr) was then determined from yS i/(VH- VGABA), where VH is the membrane holding potential and VGABA is the GABA reversal potential. Only steadystate GABA-induced membrane currents were used for spectral analysis. Most GABA responses did not desensitize, but when fading occurred it was usually restricted to 20-35 % of the maximum induced current and data were obtained only when the noise was stationary. A plot of Im against current variance was analysed to determined linearity and thus satisfy the criterion of low probability of channel opening during noise analysis. The single-channel current was determined from this relationship and used to estimate the apparent GABA single-channel conductance (yJ).

Single-channel current analysis Single GABA channel currents were analysed in outside-out membrane patches excised from neuronal somata. In most patches (> 90 %), more than one active GABA channel was discernable. Patches were accepted for kinetic analysis if there was apparently only one active channel, or the number of multiple channel openings never exceeded 2 % of all the detected openings. Singlechannel data were stored on FM tape (DC-5 kHz) and filtered at 1-3 kHz (-3 dB, 6-pole Bessel) prior to digitizing at 5-15 kHz (5-10 x the low-pass -3 dB filter frequency). Data were analysed using an 80386-based computer system. GABA channels exhibited a number of conductance states including openings to 8, 18, 23, 29 and 34 pS. The 8 pS state was quite rare and was not considered further in this analysis. The main conductance state which the channel opened to was usually the 29 pS state, although in some patches the 18 pS state frequently occurred. In all the patches included in this study, the main conductance state was 29 pS and openings to 18 pS were rejected prior to analysis. The other conductance states (23 and 34 PS) were closely clustered around the 29 pS state, and often difficult to resolve adequately in all patches. No attempt was made to edit these states.

Amplitude distribution Amplitude histograms were constructed from digitized points selected from current records designated as open channel states. The distributions were plotted as a percentage of the total recording time at a particular current level. The peak of the histogram was used to estimate the mean unitary current level subsequently used by the transition detection analysis for assembling all the channel open and closed times. Gaussian curves were fitted to the amplitude distributions defining the mean current, peak current and standard deviation using a non-linear least-squares routine. The Gaussian curve fit provided a more accurate estimate of the mean current level and the area under the curve was used to determine the percentage of time the channel resided in the open state. The single-channel conductance was calculated from the mean unitary current determined from the Gaussian curve fits, using a VGABA Of 0 mV adjusted by the patch electrode and extracellular chloride concentrations.

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Open and closed time durations Separate open and closed durations were measured using a 50 % threshold cursor to the main conductance state at 29 pS. The transition detection of open and closed events was used to form an idealized record of the digitized data. Individual open and closed idealized durations were measured and collated into frequency histograms for analysis by fitting exponential functions. The area under the exponential curve, the time constant and standard errors were determined using a Levenberg-Marquadt non-linear least-squares routine. The analysis of open and closed times has not distinguished between openings and transitions between the larger conductance states (23, 29 and 34 pS). Thus any brief openings and closures may not be distinguished from brief transitions between different conductance states of the channel. However, the clustering of conductance states around 29 pS indicates that this error is likely to be small since these transitions are not mistaken for closures given the small difference in channel current. Brief transitions to the 18 pS state from the main conductance state could be subject to possible misclassification as brief full closures, but since this subconductance state occurred less frequently and was eliminated as far as possible, the analysis of closed times was not corrected for possibly misclassified brief closures, which were assumed to represent full closures (Cull-Candy & Usowicz, 1989b; see Colquhoun & Sigworth, 1983). It is possible that the component described by the first exponential time constant used in the frequency histogram for all closed durations may contain brief closures and also brief transitions to the 18 pS state. All the open durations were measured and partially resolved open states were assumed to represent openings to the main conductance state of 29 pS. Since some patches at a low probability of opening for the GABA channel may still contain more than one active channel, the closed time distributions were analysed in two ways (see MacDonald, Rogers & Twyman, 1989 a). All the closed durations were included in the analysis between all the various open states of the channel, including multiple openings, and secondly, only those closures between transitions to the 29 pS, main conductance state were analysed with all other closures rejected. The time constants used to fit these distributions varied only slightly. All closures between transitions to the main conductance state have been used in this study. Burst duration analysis Bursts were defined by openings or groups of openings separated by closed periods equal to, or longer than, a critical gap duration, defined by tc (Colquhoun & Sigworth, 1983; Colquhoun & Sakmann, 1985). Successive openings which were separated by closures shorter than t, were defined as belonging within a burst and openings separated by closures equal to, or longer than, tc belonged to discrete bursts. tc was selected for each patch by using the method of Colquhoun & Sakmann (1985). This method aimed to make the proportion of misclassified closures, both between and within bursts, equal. tc was found by solving the equation 1-exp (-t,/r3) = exp (_ tc/'r2), where 73 and 72 represent the long and intermediate closed time constants in the distribution of all closed times. Both the short and intermediate closures were considered to represent closures occurring within bursts. In some patches, the level of channel activity was sufficiently high that precise determination of the long closed time constant proved difficult. In these circumstances, bursts were defined as a group of openings separated by closed periods greater than three times the mean closed time within bursts, calculated using T, and T2 determined from the closed time distributions (Neher, 1983). Bursts were analysed for their duration and also open times within a burst (intraburst open time). These data were collated into frequency distributions and fitted with multiexponential functions to determine the mean time constants, relative areas and standard deviations. Single-channel analyses were performed using software supplied by J. R. Dempster (University of Strathclyde, PAT V6 .2). RESULTS

Zinc modulation of GABA responses in embryonic neurones Whole-cell voltage clamp recordings from embryonic (day E21) rat sympathetic neurones were obtained at -55 mV holding potential. Pressure-applied GABA

ZINC AND GABAA RECEPTOR ION CHANNELS A

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Fig. 1. Effect of zinc on GABA and muscimol responses in cultured sympathetic and cerebellar neurones. Whole-cell inward membrane currents were evoked by pressure ejection (65 kPa (A), 80 kPa (B), 30 ms, 0 05 Hz; *) of GABA onto embryonic sympathetic neurones maintained in culture for 11 (A) and 107 (B) days in vitro (DIV). Bath-applied zinc (100 #M) antagonized GABA responses recorded from acutely cultured neurones (A), but was less active on neurones maintained in long-term culture (B). Neurones used in A and B belonged to cultures prepared from the same litter of animals. C, bath-applied muscimol (0-5 ,sm; open bar) was fourfold more potent than GABA on embryonic sympathetic neurones in evoking desensitizing inward membrane currents coupled with an increased current noise. Brief hyperpolarizing voltage commands (-30 mV, 1 s, 0 3 Hz) monitored the increased membrane conductance. Muscimolactivated responses were readily blocked by 50 /LM-zinc in young cultures (14 DIV, data obtained using different cultures to A and B). D, embryonic large cerebellar neurones also responded to bath-applied GABA (2 /M; filled bar) with an inward desensitizing current, a conductance increase (hyperpolarizing commands, -10 mV, 500 ms, 0 3 Hz) and an increased membrane current noise. Zinc (100,M) reversibly inhibited these responses in cultures maintained for 30 DIV. All cells were held at -55 mV holding potential. Current calibrations in A, B and C, 100 pA; D, 200 pA. Time calibrations in A and B, 0-5 s; C and D, 20s.

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induced an inward membrane current response (IGABA) which did not wane in amplitude during subsequent applications (Fig. IA). The superfusion of zinc (50-300 /UM) onto embryonic neurones, maintained in culture for 11 days (11 days in vitro, DIV), produced a reversible inhibition of the GABA response. If the period in A

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40 60 120 80 100 140 Total development time (days) Fig. 2. Dependence of zinc inhibition on neuronal age. A, histogram of the percentage inhibition of GABA-induced responses by zinc on sympathetic neurones cultured from embryonic (E21, O), young postnatal (P3, *) and adult (P > 90, 0) animals. Cells were whole-cell voltage clamped following 1-3 days in culture. Bars represent means+s.E.M. from eight embryonic, six postnatal and sixteen adult neurones. B, the percentage inhibition of individual GABA responses by zinc were measured for embryonic (E21, [I), postnatal (P3, *) and adult (P93, 0) neurones maintained in culture for different periods, denoted by the 'total development time' (see text for details; n = 103 20

determinations from 65 neurones).

tissue culture was considerably extended from 11 to 107 DIV for neurones from the same batch of cultures, the GABA responses were slightly reduced in amplitude and became less sensitive to zinc inhibition and quite frequently insensitive (n = 5

ZINC AND GABAA RECEPTOR ION CHANNELS 595 neurones) (Fig. 1B). Zinc also inhibited responses evoked by muscimol (0-2-2 /tM) on the GABAA receptor. By comparing equivalent responses, muscimol (0 5 /LM) was considered to be at least fourfold more potent than GABA (2 ,tM) at inducing an inward membrane current and associated conductance increase on neurones grown for 14 DIV. These responses were reversibly inhibited by 50,uM-zinc (Fig. 1C). TABLE 1. Analysis of the time dependence for zinc inhibition of GABA responses in embryonic and adult sympathetic neurones in vitro Time spent Maximum % in vitro inhibition Slope (% N at DIV = 0 per day) t P Neuronal age (DIV) < 0005 -0-55 -2-81 0-50 70 Embryonic (E21) 38 -0-006 -0 0097 > 0 5 Adult (P > 93) 31 0-20 26-8 These data were calculated from separate linear regression analyses of seventy-nine determinations (N) of zinc inhibiting GABA responses on embryonic and adult neurones. Cells were maintained for variable periods of time in vitro. Analyses were performed on some of the data shown in Fig. 2B. The maximum inhibition is calculated from the regression analysis at day 21 for embryonic neurones and day 104 for adult neurones, i.e. age of the neurones at the time of culturing when DIV = 0. t values were determined either from the correlation coefficient, or by testing whether the slopes of the lines were significantly different to zero at the 5% level using a two-tailed t test.

Cultured embryonic neurones were also prepared from rat cerebellum and maintained for up to 36 DIV. On large cerebellar neurones, GABA (2 /,M) induced desensitizing inward currents and conductance increases which were inhibited by 100 /tM-zinc (Fig. 1D). Zinc inhibition is dependent on neuronal development The differential sensitivity of GABA responses recorded from embryonic and adult neurones to inhibition by zinc was examined by culturing neurones from donor animals at different ages, specifically chosen to allow for different in vivo periods of development. This should reveal whether zinc inhibition was related to the stage of neuronal development. Sympathetic neurones were more suited to this study since the tissue culture conditions were reasonably standardized for both embryonic and adult cells, negating any effect of culture conditions on the inhibitory action of zinc (see Methods). Neurones were used for electrophysiology within 1-3 DIV. The relationship between the donor animal age and the percentage inhibition of IGABA with zinc revealed a consistent antagonism of GABA responses induced on embryonic (E21) neurones, compared to adult neurones (postnatal days, P > 93), where the mean percentage inhibition was considerably reduced with a larger spread of individual values (Fig. 2A). In many adult neurones, zinc failed to cause any inhibition of the GABA response (n = 15). Young postnatal neurones (P3) possessed GABA receptors which were more variable, exhibiting either 'embryonic-type' or 'adult-type' sensitivity to zinc. Although only three donor animal ages were tested (n = 30 neurones), the decline in sensitivity to zinc could be fitted by a single exponential function with a time constant of 69-5 days. The initial percentage inhibition of IGABA produced by zinc on

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embryonic neurones was estimated to decline in vivo with neuronal development by

50°% in 48-2 days (half-decay time). The apparent reliance of zinc antagonism on the developmental age of sympathetic neurones could also be demonstrated by incubating dissociated neurones in vitro for different periods of time, presumably allowing development to proceed partly under culture conditions. The parameter 'total development time' was derived from the sum of the donor animal age, used to prepare the cultures (defining conception as day 0, i.e. E21 = 21 days and P3 = 24 days), and the time neurones spent in vitro (DIV) before experimentation (Fig. 2B). The relationship between total development time and the zinc-induced inhibition produced a much greater scatter of data points which is mostly due to the different times individual neurones have spent in vitro. The decline in the zinc inhibition of GABA responses on neurones maintained in culture for long periods was more evident with embryonic neurones compared to adult cells (Fig. 2B). The apparent rate of decline in zinc inhibition was estimated separately for both embryonic and adult neurones, assuming a linear relationship and using linear regression analyses (Table 1). This indicated, for embryonic neurones, that the inhibition declined in vitro at a slow rate, estimated at 0 55 % per day, and predicted that these cells would have to be maintained in culture for approximately 148 days to achieve complete resistance to zinc inhibition. Interestingly, the sensitivity of adult neuronal GABA responses to zinc did not significantly decrease with the time spent in vitro, suggesting that whatever process was responsible for rendering the GABA receptor less sensitive to zinc in embryonic neurones, at this later stage of development it was probably inoperative. However, it should be observed that the relative time spent in vitro for adult neurones was considerably less (maximum 20 DIV) when compared to embryonic neurones.

External pH does not affect zinc-induced inhibition Following the consistent action of zinc as a GABA antagonist on embryonic neurones, these cells were used to examine the location of a putative binding site on the GABAA receptor complex. Previous intracellular studies on crustacean muscle GABA receptors suggested that zinc antagonized GABA responses by binding to imidazole groups in histidine residues in the receptor structure (Smart & Constanti, 1982). These histidine residues were also titratable by varying the external pH of the bathing medium and H+ competed with zinc for a common binding site. A possible Fig. 3. A, pressure-ejected GABA (69-6 kPa, 50 ms, 0-025 Hz) induced inward membrane currents on sympathetic neurones (E21) that were readily antagonized by bath-applied zinc (between the filled arrows). In this and the following figures, the chart recorder speed was transiently increased during the GABA responses, and selected GABA currents in control, during zinc and following recovery are shown on an expanded time base. The time calibrations refer to those portions of the records illustrating the GABA responses. B, in the same neurone, decreasing the external pH to 6-18 (open arrow) induced a small inward current and also reduced the GABA-evoked membrane current (dashed line indicates the zero-current level). Re-application of zinc (filled arrows) still inhibited the GABA response at a holding potential of -55 mV. C, histogram of the percentage inhibition of the GABA response (IGABA) by zinc at control pH (7 4) and at a mean pH of 6-28 (pH range 6-18-6A46; n = 4 cells). Bars represent means + S.E.M. Records in A and B form part of a continuous recording.

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100

/iM-Zn2+

500

+Pentobarbitone

+Pentobarbitone / Zn2+

Pentobarbitone recovery

C

m

I'D 0 c

0

.0 c

a)

0)

C)

(a) C.) CL

Fig. 4. For legend

see

facing

ms

page.

ZINC AND GABAA RECEPTOR ION CHANNELS

599

binding site for zinc on the embryonic neuronal GABAA receptor complex might also involve histidine residues, which are present on the presumed extracellular domains of the o- ,- and y-polypeptide subunits comprising the GABAA receptor. Pressureejected GABA induced inward membrane currents which were antagonized by 100 /tM-zinc at pH 7 4 (Fig. 3A). By reducing the pH to 6418, effectively raising the H+ concentration, a small persistent inward membrane current was induced and 'GABA was also reduced. At pH 618, zinc was still an effective and reversible antagonist of IGABA (Fig. 3B) producing an equivalent percentage inhibition of the GABA response compared to pH 7-4 (Fig. 3C; n = 4). Zinc does not affect barbiturate, benzodiazepine or steroid modulation of IGABA If the zinc binding site on the GABAA receptor was not sensitive to H+, other possible locations included the variety of allosteric sites sensitive to barbiturates, benzodiazepines and steroids. It is conceivable that zinc may act at one of these sites as a GABA antagonist in an analogous manner to the benzodiazepine inverse agonists (Jensen & Lambert, 1984). Combinations of zinc with individual allosteric modulators of GABA receptor function were used to examine the likelihood of a common binding site. GABA responses were inhibited with 100 1am-zinc and allowed to recover to the control level (Fig. 4A) before being enhanced by 50 pM-bath-applied pentobarbitone. In the presence of pentobarbitone, reapplication of zinc produced a comparable level of inhibition of 'GABA (Fig. 4B). The zinc-induced inhibition was unaffected by prior enhancement of the GABA response with pentobarbitone (Fig. 4C; n = 5). The lack of interaction between zinc and pentobarbitone was not due to the order of application of these agents, since an initial application of pentobarbitone induced a reversible enhancement of IGABA (Fig. 5A) which was unaffected when pentobarbitone was reapplied after the GABA responses were inhibited by zinc (Fig. 5B and C; n = 4). This implied discrete binding sites for zinc and barbiturates on the GABAA receptor complex. In similar experiments, zinc was also used in combination with the benzodiazepines chlorazepate or flurazepam. Chlorazepate (20 /tM) produced a small enhancement (30 %) in IGABA which could be reversed providing the exposure to chlorazepate was limited to not more than a few minutes (Fig. 6A). In the same neurone, after prior inhibition of the GABA response by 100 /tM-zinc, chlorazepate still modulated IGABA producing an equal percentage enhancement (Fig. 6B). Zinc did not apparently interfere with benzodiazepine binding to the GABAA receptor (Fig. 6C). Similar results were obtained with 1-10 ,am-flurazepam. These combination experiments were also performed with different concentrations of pentobarbitone (50-100 /tM) and chlorazepate (1-10 yM) without revealing any interaction between these agents and zinc. Fig. 4. GABA responses (75-8 kPa, 50 ms, 0 03 Hz) were reversibly inhibited by 100 /tMzinc (A; filled arrows) on embryonic sympathetic neurones voltage clamped at -55 mV. After recovery from zinc, bath application of pentobarbitone (50 aM) to the same cell (B; open arrow) enhanced the GABA response which was inhibited by subsequent coapplication of 100 /LM-zinc. C, histogram of the percentage inhibition induced by zinc in the absence (O) and presence (0) of pentobarbitone (n = 5).

T. G. SMART

600 A

80.7 kPa GABA

-55 mV

0

-

50 pA

2s

A 50 uM-pentobarbitone w~~~~~~~~~W

1

9

500 ms

+Pentobarbitone

Control

Recovery

80.7 kPa GABA

B

0

-55 mV

A

A

V

2s

50 /iM-pentobarbitone

100 pM-Zn2+

500 ms

Zn2+ C

Zn2+/pentobarbitone

200 m

0

150

0 c 0 '._

._4-o

s:c 100 0 co

0) 50 20

0)

0 Flnc2+

Fig. 5. For legend see facing page.

Zn2+ recovery

RECEPTOR ION CHANTNELS 601 The GABAA receptor protein complex contains an additional sensitivity to steroidal agents based on metabolites of progesterone (Lambert, Peters, Sturgess & Hales, 1990; Simmonds, 1991). 5-,J-Pregnan-3-a-ol-20-one (pregnanolone; 250nm) produced a reversible enhancement of pressure-applied GABA responses (61 %). In embryonic neurones, zinc alone produced a 64 % inhibition of IGABA' compared to a 62 % inhibition of GABA responses initially enhanced with pregnanolone (n = 3). Furthermore, both picrotoxinin (5 /tM) and bicuculline (5 ftM) are established antagonists acting at the GABAA receptor by binding to discrete sites (Simmonds, 1982), but neither agent affected the zinc-induced inhibition (data not shown; n = 4). ZINC AND

GABAA

GABA-evoked current noise The mechanism of inhibition of GABA-evoked membrane currents by zinc was analysed using embryonic neurones. Unusually for vertebrate neurones, it was difficult to routinely observe GABA single-channel currents in embryonic sympathetic neurones using either cell-attached or excised membrane patches taken from cells which had been maintained in culture for longer than 24 h. In this study cultures were usually maintained for longer than 1 DIV, therefore the elementary properties of GABA-operated ion channels and the effect of zinc were initially studied using fluctuation analysis. In some neurones, perfusion of GABA induced inward membrane currents displaying an initial peak which subsequently desensitized to a maintained steady state within 30 s. The steady-state current was approximately 65-80 % of the peak current. Desensitization was also apparent by a reduction in the variance of the GABA-induced current noise. In other neurones, the GABA-activated membrane current attained a steady state in the apparent absence of any initial desensitization and the current noise variance was also maintained during continued GABA application (Figs 7A and 8A). Fluctuation analysis of GABA-evoked current noise Neurones were selected according to the time spent in culture to produce GABA responses either highly, or only slightly, sensitive to zinc inhibition (cf. Figs 7 A and 8A). Any synaptic currents present during the recordings were either edited from the noise records, or experiments were performed in 10 JM-hexamethonium to block spontaneous nicotinic acetylcholine receptor-mediated excitatory postsynaptic currents. The spectral parameters of current noise in either hexamethonium-treated or untreated cells were similar, and therefore all records were pooled for analysis. Fig. 5. A, pressure-ejected GABA (80-7 kPa, 25 ms, 0 03 Hz) evoked responses which were enhanced by 50 ,#M-pentobarbitone (open arrows) on embryonic sympathetic neurones. Following a recovery from pentobarbitone, 100 ,uM-zinc was applied (filled arrow) causing an inhibition of IGABA (B). When the inhibition attained a steady state, 50/SMpentobarbitone was reapplied in zinc producing a similar enhancement of the GABA response compared to that observed in A (B; open arrows). C, histogram of the percentage enhancement of IGABA induced by pentobarbitone in the absence (El) and presence (1) of 100 ,uM-zinc (n = 4 cells). Both the records in A and B form part of a continuous chart recording. Holding potential -55 mV.

T. G. SMART

60'2 A

78-6 kPa GABA

1100 pA A 20

2s

V

gaM-chlorazepate * | Ff >~~rC'

500 ms

Control B

+Chlorazepate

W+10

78.6 kPa GABA

-554~_A 0

.A

J v

A

A

20

100 pM-Zn2,

V

\

pM-chlorazepate

500 ms

+Zn2+ /chlorazepate

Zn2+ C

50

0

ra)C

40 a)

0 0

coca

30'

C C

m

C5 < 20 0 C

a)

100 v" \\\N S\

\\\E\\\\\

Chlorazepate Chlorazepate/Zn 2+ Fig. 6. For legend see facing page.

fj100 8 pA 2s

ZINC AND GABAA RECEPTOR ION CHANNELS

603

Estimates of the elementary properties of sympathetic GABA-activated channels were made at -55 mV using bath application of GABA (2-5 ftM). Power spectra require two Lorentzian components The power density spectra for GABA-induced noise in the absence and presence of 50 guM-zinc were fitted by the sum of two Lorentzian components with slow (zr) and fast (T2) time constants determined from the respective cut-off frequencies (fn; Fig. 7B). For the control spectrum in Fig. 7B, r1 = 2541 ms and r2 = 3-7 ms and in the presence of zinc, r1 = 18-7 ms and r2 = 2-5 ms. Zinc caused a tenfold reduction in the power of the GABA spectrum, but the effect on the time constants was much smaller. In older sympathetic neurones (45 DIV), the GABA response was less sensitive to zinc (Fig. 8A) and the current noise power was reduced by only twofold. The power density spectra were still fitted by the sum of two Lorentzian components (Fig. 8B) with time constants for control of r1 = 18-4 ms and r2 = 1-38 ms, and in the presence of zinc, r1 = 14-7 ms and -r2 = 1-6 ms.

Single-channel conductance from noise analysis The apparent single GABA channel current, i, was estimated directly from the power spectrum. For the spectra in Fig. 7B, single-channel conductances (y8) of 20-1 pS in control and 23-4 pS in 50 guM-zinc were obtained. The single-channel current and elementary conductance could also be calculated from

YV = [var (I)/Im(VH-VGABA)]. Under conditions where the channel opening probability is low, i.e. at low agonist concentrations, a linear relationship is predicted between the variance of the current noise (var(I)) and the mean agonist-induced membrane current, Imn as observed in Fig. 7C. The slope of this relationship determined the apparent single-channel current, which for the control GABA-evoked channel current was - 1-29 pA, and in zinc - 1-19 pA (Fig. 7 C), producing single GABA channel conductances of 22-76 pS (control) and 23-28 pS (in zinc) using a mean reversal potential (VGABA) of 0+2 mV (n = 8). The single-channel conductances determined from the spectrum for older neurones (Fig. 8B) were 22-67 pS (control) and 24-4 pS (in zinc). The single-channel current was also estimated from the variance-mean membrane current plot (Fig. 8 C) yielding for the control GABA current - 1'23 pA, and in the presence of zinc -1t24 pA, with corresponding single-channel conductances of 22-34 pS (control) and 22-48 pS (in zinc). Fig. 6. Zinc does not affect benzodiazepine-induced enhancement of IGABA. A, GABA responses (78-6 kPa, 40 ms, 0 03 Hz) were evoked on embryonic sympathetic neurones and enhanced in a reversible manner by 20 /LM-chlorazepate (open arrows). Following a 10 min wash (W + 10) to recover from chlorazepate treatment, IGABA was inhibited by 100 ,uM-zinc (B, filled arrow) and still enhanced by subsequent co-application of chlorazepate. Holding potential -55 mV. C, histogram of the percentage enhancement of IGABA by chlorazepate alone (U1) and in the presence of zinc (C); n = 5. GABA currents in A and B form part of a continuous chart recording.

604

T.

A

G.

SMART +50 .Mm-Zn2+

Control

-55 mV

I0 pA

_-

A 2.5

V

V

A

mM-GABA

2.5

jLm-GABA

50 pA II

10 s

B

10

2.5 MM-GABA

1= a) C.,

0.1 -

0.

15 la

0.01 .

+50 ,uM-Zn2+

.D

0.1.

Co

C

00) C.)

0)

0. 0.001

500

Frequency (Hz)

Fig. 7. For legend

see

facing

page.

ZINC AND GABAA RECEPTOR ION CHANNELS c

605

2.5 piM-GABA 300

-

CL

Slope =-1.29 pA

0