Facultades y escuelas profesionales

Two to five days after injection of murine cDNAs encoding for p i mouse subunits, the oocytes were screened for expression of GABA^ receptors by the bath-application of GABA (0.1 pM to 1 mM). Under voltage clamp at a holding potential of -40 mV no change in the membrane currents and conductances were evoked even by high concentrations of GABA (1 mM; Fig. 4.1A). Interestingly, the resting membrane resistance was lower (0.05 to 0.25 Mfl) than normally seen with oocytes expressing heteromeric GABA^ receptors (0.5 to 2.5 MO). There were no visible morphological differences between the batches of oocytes indicating th a t structural damage induced by the expression of homomeric p i subunit receptors was unlikely. Application of the competitive GABA^ receptor antagonist, bicuculline at high concentrations (50 pM), also had no effect on the resting membrane currents and conductances (Fig. 4.1A). However, bath application of low concentrations of both zinc (1 pM) or picrotoxin (10 pM) restored the normal membrane resistance in these oocytes (Fig. 4.1A). Zinc has been shown to be a non­ competitive antagonist at the GABA^ receptor (Smart and Constanti, 1990; Smart, 1990). Zinc induced an outward current and conductance decrease which was also seen with the GABA-channel blocker, picrotoxin. Picrotoxin and zinc-inhibition curves were constructed for the decrease in the conductance induced by these antagonists (Fig. 4.2A,B). The IC50 for picrotoxin and zinc were estimated as 2.11 ± 0.6 and 0.23 ± 0.03 pM respectively indicating th at zinc has a greater affinity for this p i receptor subunit compared to picrotoxin. To establish the identity of the conductance being reduced by these inhibitors current-voltage relations were constructed. The I-V relationships for the unusual ion channel formed by the p i subunit displayed a slight degree of outward rectification with reversal potentials

Chapter four

estimated as -27.1 and -24.7 mV respectively which are close to the Cl reversal potential in these cells (Fig. 4.2A,B; Barish, 1983; Dascal, 1987).

The p i homomeric channels were not affected by some other GABA^ receptor agonists under study. GABA (500 pM), muscimol (500 pM) and isoguvacine (500 pM) failed to induce any change in the membrane potential or current suggesting th a t the binding site for these agonists is either occluded or not adopting the correct conformation in this homomeric receptor (Fig. 4.3A). Interestingly, the barbiturate, pentobarbitone, did induce an inward current associated with a conductance increase (Fig 4.3A). These results indicate th at the binding site for GABA and barbiturates are clearly different with only the barbiturate site existing in these homomeric p i subunits. Construction of an equilibrium dose-response curve for pentobarbitone produced a Hill coefficient and EC50 value of 1.9 ± 0.26 and 6.0 ± 0.34 pM respectively (Fig. 4.3B). The current-voltage relation for the pentobarbitone-induced conductance revealed a reversal potential of -15.5 mV which is again similar to the Cl' reversal potential in these cells (Fig. 4.3B).

Heteromeric GABA^ receptors contain a number of allosteric binding sites (for review see Burt and Kamatchi, 1991; Olsen and Tobin, 1990). To establish whether such binding sites also existed on the homomeric p i subunit, a number of GABA^ receptor allosteric modulators were applied to this receptor. Initially, both positive modulators, including the benzodiazepines and neurosteroids and negative modulators such as inverse agonists were tested. The unusual ion current was not affected by the benzodiazepines, flurazepam (10 pM) and midazolam (10 pM) or the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-p-carboline-3-carboxylate (DMCM; 10 pM; Fig. 4.4A). Neither the benzodiazepine antagonist, flumazenil (Ro 15- 1788; 10 pM; Fig. 4.4A), nor the neurosteroid pregnanolone (500 nM) had any effect on the current (Fig. 4.4B). Chlormethiazole has been previously shown to be a positive allosteric modulator of the GABA^ receptor (Harrison and

Chapter four

Simmonds, 1983; Hales and Lambert, 1992). Application of chlormethiazole (100 pM) had no effect on the current formed by the homomeric p i subunit (Fig. 4.4B). Propofol is a novel general anaesthetic which has been previously shown to be a potent enhancer of GABA responses (Hales and Lambert, 1991; Prince and Simmonds, 1992). Interestingly, propofol (100 pM) potentiated the membrane conductance which could be inhibited by the co-application of picrotoxin (10 pM; Fig 4.4B). Penicillin has been observed to reduce GABA- evoked chloride currents (Chow and Mathers, 1986; Twyman et oZ., 1992; Katayama et al., 1992). Application of 1 mM penicillin G induced an outward current and conductance decrease which could be further accentuated by the co-application of picrotoxin (1 pM) in an additive manner (Fig. 4.4C). This provided evidence th at penicillin G and picrotoxin were acting on the same unusual ion current formed by the homomeric p i subunit. Anthracene-9- carboxylic acid (A-9-C) has been previously shown to be a chloride channel blocker (Bowie and Smart, 1993). Bath-application of A-9-C (1 mM) had no effect on the membrane current or conductance (Fig. 4.4C).

Xenopus oocytes have been shown to express an endogenous gene for the

acetylcholine receptor a subunit which was thought to be able to co-assemble with acetylcholine receptor subunit proteins, synthesised after cRNA injection, to yield functional acetylcholine receptors (Buller and White, 1989). It was therefore conceivable th at the homomeric p i subunits could also co- assemble with a transcription product from the oocyte’s own genome, producing the novel ion current formed by the p i subunit. Therefore many control experiments were performed to determine th at the novel current seen in this study was a product solely of the p i subunit expression including: (i) injection of an unrelated reporter gene (Lac Z) to determine whether an injection of cDNA will produce the unusual ion current and (ii) the utilisation of actinomycin D which prevents any transcription of the host cells genome. To ascertain the effectiveness of actinomycin D on the prevention of transcribing DNA to mRNA, a control experiment was performed using cDNA

Chapter four

injections of a l p l subunits in the presence and absence of actinomycin D.

Thus the first approach was to inject the Lac Z gene into the Xenopus oocyte which encodes for the p-galactosidase protein (Bassford et al., 1978). This ensured the unusual ion current was not a product of expression from the oocyte’s own genome which was somehow induced to be expressed after injection of a volume (15 nl) of cDNA into the nucleus. Two to five days after the injection of the Lac Z gene the oocytes exhibited normal resting membrane resistances and bath-application of GABA (1 mM), zinc (10 pM), picrotoxin (50 pM) or pentobarbitone (50 pM) had no effect on the resting membrane current or conductance (Fig. 4.5A). It is therefore improbable th at the injection of cDNAs per se activates an endogenous gene within the oocyte which could be expressing the protein for the novel ion channel seen in this study. To further ascertain th at the expression of the ion channel was only from the injected p i subunit, oocytes were incubated in the presence of 50 pg/ml actinomycin D after injection with cRNA encoding for mouse pi subunits or cDNAs encoding for a l p l GABA^ receptors. As actinomycin D blocks transcription of DNA to mRNA, injection and subsequent expression of the products of cRNA should be unaffected by actinomycin D but expression of injected cDNA should be prevented. After two to five days incubation in actinomycin D, the cRNA injection of p i subunits expressed to give receptors with the same ion channel properties as those seen for cDNA injections of the p i subunit. GABA (500 pM) failed to affect the ion channel properties with both zinc (1 pM) and picrotoxin (50 pM) inducing an outward current and conductance decrease (Fig. 4.5B). Moreover, pentobarbitone (10 pM) induced an inward current and conductance increase (Fig. 4.5B). To ensure th a t actinomycin D was preventing transcription at this concentration, oocytes injected with a l p l cDNAs were also bathed in actinomycin D. Expression of a l p l subunits was not seen after incubation in actinomycin D for two to five days. GABA (10 to 500 pM) and picrotoxin (50 pM) failed to elicit any response (Fig. 4.5C). To ascertain th at this lack of expression was

Chapter four

due to the effects of actinomycin D and not due to poor expression of the cDNAs, oocytes from the same experiment were also incubated in the absence of actinomycin D. In this case, the injection of a l p l subunits for the GABA^ receptor led to the efficient formation of GABA-gated anion channels. GABA (10 and 100 pM) induced large inward currents and conductance increases (Fig. 4.5D). Application of 10 pM picrotoxin inhibited the response induced by GABA (10 pM; Fig. 4.5D). From these control experiments it is very likely th at the currents seen in this study arose from only the expression of homomeric mouse pi subunits and not from a product of the expression of the oocyte's own genome.

4.2.2 COMPARISON OF HOMOMERIC p i SUBUNIT GABA^ RECEPTORS FROM TWO DIFFERENT SPECIES

The unusual ionic current seen with expressed mouse p i homomeric GABA^ receptors is intriguing. Whether such an effect is related specifically to this mouse GABA^ subunit was studied by comparison with expressed homomeric bovine p i GABA^ receptor subunits.

Bath-application of GABA (100 pM) or muscimol (100 pM) induced a small inward current (Fig. 4.6A). The bovine p i homomeric receptors therefore appeared to have GABA-gating properties which were lacking in the mouse p i GABA^ subunit. The GABA response was unaffected by the benzodiazepine midazolam (50 pM) but was enhanced by 50 pM pentobarbitone (Fig. 4.6A). The GABA-induced response was completely inhibited by the GABA^ receptor antagonists bicuculline (10 pM), picrotoxin (10 pM) and zinc (10 pM; Fig. 4.6B). Addition of the chloride channel blocker A-9-C had no effect on the GABA-induced response (Fig. 4.6B). The responses induced by GABA on this homomeric bovine p i GABA^ receptor were shown to be dose-dependent with the application of increasing concentrations of GABA inducing larger responses (Fig. 4.6C). Construction of an equilibrium response curve revealed

Chapter four

a Hill coefficient and EC50 value of 0.8 ± 0.08 and 29.34 ±5.1 pM respectively (Fig. 4.7). Current-voltage relations for GABA revealed a reversal potential of -21.35 mV which is close to the Cl reversal potential in these cells (Fig. 4.7; Barish, 1983; Dascal, 1987). Interestingly, the I-V relationships for this GABA^ receptor revealed a saturation of the GABA-induced current at positive potentials possibly indicating a very inefficient expression of these single bovine subunits. Alternatively the expression could be efficient but the channels may have a small single channel conductance or possibly a relatively low channel opening frequency and/or short mean open or long mean closed times at depolarised membrane potentials.

4.3 DISCUSSION

This study represents an examination of the functional properties of homomeric p i GABA^ receptors and the apparent ability of a small number of amino acid residues to change the pharmacological profile of the same subunit from different species.

4.3.1 SPECIES DEPENDENCE OF THE PHARMACOLOGICAL PROFILE