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¹ Department of Molecular pharmacology and physiology, College of Medicine, University of South Florida, Tampa, FL, 33612-4799, USA

	Abstract

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The onset of motor learning in rats coincides with exclusive expression of GABA_A receptors containing ₆ and subunits in the granule neurons of the cerebellum. This development temporally correlates with the presence of a spontaneously active chloride current through ₆-containing GABA_A receptors, known as tonic inhibition. Here we report that the coexpression of ₆, ₂, and subunits produced receptor–channels which possessed two distinct and separable states of agonist affinity, one exhibiting micromolar and the other nanomolar affinities for GABA. The high-affinity state was associated with a significant level of spontaneous channel activity. Increasing the level of expression or the ratio of ₂ to ₆ and subunits increased the prevalence of the high-affinity state. Comparative studies of ₆₂, ₁₂, ₆₂₂, ₁₂₂ and ₄₂ receptors under equivalent levels of expression demonstrated that the significant level of spontaneous channel activity is uniquely attributable to ₆₂ receptors. The pharmacology of spontaneous channel activity arising from ₆₂ receptor expression corresponded to that of tonic inhibition. For example, GABA_A receptor antagonists, including furosemide, blocked the spontaneous current. Further, the neuroactive steroid 5-THDOC and classical glycine receptor agonists -alanine and taurine directly activated ₆₂ receptors with high potency. Specific mutation within the GABA-dependent activation domain (^Y157F) impaired both low- and high-affinity components of GABA agonist activity in ₆^Y157F receptors, but did not attenuate the spontaneous current. In comparison, a mutation located between the second and third transmembrane segments of the subunit (^R287M) significantly diminished the nanomolar component and the spontaneous activity. The possibility that the high affinity state of the ₆₂ receptor modulates the granule neuron activity as well as potential mechanisms affecting its expression are discussed.

(Received 26 March 2007; accepted after revision 28 March 2007; first published online 29 March 2007)
Corresponding author J. Amin: University of South Florida, College of Medicine, Department of Pharmacology and Therapeutics, MDC box 9, 12901 Bruce B. Downs Blvd. Tampa, FL 33612, USA. Email: jamin{at}health.usf.edu

	Introduction

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Ligand-gated ion channels comprise a large family of membrane-embedded receptors which play a central role in neuronal transmission. The binding of a neurotransmitter to the receptor domain of this class of membrane proteins opens an integrated ion channel, allowing selected ions to permeate. Such receptor–channels are either excitatory or inhibitory in nature, modulating the frequency of action potential initiation. The most widely expressed inhibitory class of ligand-gated ion channels are -aminobutyric acid type A (GABA_A) receptors. Assembled from a diverse range of subunits termed _(1-6),_(1-3), _(1-3), , and , each subtype of GABA_A receptor exhibits unique kinetics, agonist affinity and pharmacology (Hevers & Luddens, 1998; Sieghart & Sperk, 2002; Wallner et al. 2003; Hanchar et al. 2005).

Cerebellar granule neurons are central to the control of information flow through the cerebellar cortex and are postulated to play a fundamental role in motor learning activity (Marr, 1969; Tyrrell & Willshaw, 1992; Thompson & Stephenson, 1994; Mellor et al. 1998). These neurons evince unique anatomical characteristics and GABA_A subunit expression that temporally coincide with the learning and development of motor skills. During the first postnatal week in rats, granule cells start migrating towards the inner granule layer, where they progressively begin to express both ₆ and GABA_A subunits (Laurie et al. 1992; Persohn et al. 1992; Wisden et al. 1996). The ₆ subunit mRNA becomes detectable approximately 1 week after birth and is followed by the ₆-dependent expression of the subunit (Laurie et al. 1992; Jones et al. 1997; Nusser et al. 1999). The expression of ₆ and subunits gradually increases throughout postnatal development, reaching their highest levels in adulthood (Laurie et al. 1992; Persohn et al. 1992; Jechlinger et al. 1998). This exclusive expression paradigm makes receptors containing ₆ and subunits the predominant GABA_A receptor subtype expressed within cerebellar granule neurons in adulthood (Quirk et al. 1994; Nusser et al. 1999; Tretter et al. 2001). The temporal expression of ₆ and subunits within the granule neurons correlates with the development of a spontaneous chloride current known as tonic inhibition (Kaneda et al. 1995; Brickley et al. 1996; Wall & Usowicz, 1997; Hamann et al. 2002). The spillover or diffusion of GABA from synaptic events is thought to activate the ₆-containing GABA_A receptors resulting in a tonic inhibition (Isaacson et al. 1993; Rossi & Hamann, 1998; Hamann et al. 2002; Mody & Pearce, 2004; Semyanov et al. 2004; Farrant & Nusser, 2005). Studies in animal models are gradually establishing the importance of tonic inhibition in the regulation of motor activity (Thompson et al. 1998; Chiu et al. 2005). For example, GABA transporter type 1 (GAT1) knockout mice display various neuronal deficits, including tremor and ataxia, that may arise due to a significant increase in the level of tonic chloride conductance within the cerebellar granule neurons (Chiu et al. 2005).

To simulate the temporal coexpression of ₆ and subunits within granule neurons, we investigated the characteristics of ₆ receptors under different levels and conditions of expression. The structure–function relationship of ₆₂ receptors was further examined using mutations of conserved residues within the ₂ or subunit.

	Methods

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Oocyte preparation

The Xenopus laevis frogs were anaesthetized by bathing in a solution containing 0.1% MS222 (Tricaine methane sulphonate, Sigma-Alderich, St Louis, MO, USA). Before ovariectomy, the state of anaesthesia was assessed by pinching the toe of the frog. After surgery, the frog was killed by decapitation according to a protocol approved by the Institutional Animal Care and Use Committee. Oocytes were placed in a calcium-free oocyte Ringer solution (calcium-free OR2; 83.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, 1 mM Na₂HPO₄ and 5 mM Hepes, pH 7.5) plus 0.3% collagenase A (Roche Applied Science, Indianapolis, IN, USA) for approximately 1 h. Stage V and VI oocytes were isolated and maintained by incubating in OR2 (82.5 mM NaCl, 1 mM CaCl₂, 2.5 mM KCl, 1 mM MgCl₂, 2 mM sodium pyruvate, 1 mM Na₂HPO₄, 50 U ml^–1 penicillin, 50 U ml^–1 streptomycin and 5 mM Hepes, pH 7.5) with 2% horse serum at 18°C.

Quantification of complementary RNAs (cRNA) and oocyte injections

The procedure for in vitro transcription of cRNA have been previously described (Walters et al. 2000). The quality of cRNA was determined by electrophoresis on a 1% formaldehyde-containing agarose gel. cRNA concentrations were measured spectrophotometrically. For most experiments, we tested two preparations of cRNAs for each subunit.

Micropipettes for injecting cRNA were fabricated using a Sutter P87 horizontal puller (Sutter Instruments Co., Novato, CA, USA) and, to ensure uniformity of size, the tip of each micropipette was cut with microscissors under 45x magnification next to a control-cut needle. Using a Picospritzer II (General Valve Corporation, Fairfield, NJ, USA), cRNA subunits reconstituted in diethylpyrocarbonate-treated water were injected into Xenopus laevis oocytes at a ratio of 1 : 1₂ : 1.8(₂ or ). The cRNA combinations were injected in amounts of 1.5–3 ng, 5–7 ng, and 8–12 ng per oocyte to produce, respectively, low, intermediate and high levels of expression. For comparison of the different GABA_A subtypes, we coinjected 5–7 ng of cRNA (intermediate expression level) for each combination (₆₂, ₁₂, ₆₂₂, ₁₂₂ and ₄₂), using two sets of cRNA-mixture preparations and two batches of oocytes.

Drug preparations

Forusemide, bicuculline and picrotoxinin were purchased from Sigma-Alderich Corp. (St Louis, MO, USA). Allotetrahydrodeoxycorticosterone (5-THODC) was obtained from Steraloids, Inc (Newport, RI, USA). Forusemide, bicuculline, picrotoxinin and 5-THODC were dissolved in dimethylsulfoxide at their respective stock solution concentrations of 100, 40, 100 and 20 mM. The test solutions were made by diluting the stock solutions in the recording OR2 solution (mM: NaCl, 82.5; KCl, 2.5; Hepes, 5; CaCl₂, 1; MgCl₂, 1; pH 7.5). The highest concentration of the vehicle solution (0.5% of DMSO) did not significantly alter the level of ₆₂ recptors activity.

Electrophysiology

Three to four days after injection, oocytes were placed on a mesh within a small perfusing volume chamber (75 µl), with t_1/2 and clearance times of approximately 3 and 10 s, respectively. For complete description of the drug application system see Walters et al. (2000).

We used a two-electrode voltage-clamp amplifier (Turbo TEC-05 npi, Adams and List, Westbury, NY, USA) to record currents in response to the application of drugs. Recording microelectrodes were fabricated with a Narishige PP-83 puller (Narishige, Japan) and filled with 3 M KCl. We used electrodes with input resistances of 0.7–1.6 M. Membrane potential was clamped to –70 mV. Data were visualized on a TA-240 chart recorder (Gould Instrument System, Valley View, CA, USA) during the experiments and stored online using Pulse Fit.

Measurement of the spontaneous current and statistical analysis

High concentrations of GABA (mM) do not evoke a current in mock-injected oocytes indicating an absence of endogenous GABA_A receptors. Upon impaling an oocyte with a pair of electrodes, the oocyte initially displayed a leak current (holding potential = –70 mV). This leak current did not reverse at –30 mV (the predicted reversal potential for chloride in oocytes under these conditions) and within 4–5 min reduced to < 30 nA. If waiting time following the impalement was longer (10–15 min), the leak current would have gradually decreased to a value of a few nanoamps, suggesting that the initial random leak may result from an incomplete sealing of the membrane around the electrode. In experiments where the time allocated for recording from each oocyte were short (4–5 min, due to large number of oocytes tested in one day), the averaged control leak current measured from the mock-injected oocytes was subtracted from the data and such corrections are noted in Results. However, in most experiments, the wait-time was more than 10 min and thus the mock-injected oocytes did not show any significant leak current. It is also important to note that spontaneous currents arising from ₆₂ receptors (in most experiments) are at least an order of magnitude higher than any control leak current recorded (the range of averages of leak current in control cells in different experiments was 14–23 nA where the wait time before measurement was < 5 min).

The analysis of variance (ANOVA one-way) and Fisher's LSD multiple comparison test were used for statistical investigation. All statistical calculations are presented as means ± standard error of the mean.

Data analysis

The EC₅₀ and Hill coefficients for the agonists were estimated by fitting the data from concentration–response relationships to the Hill equation according to the following formula (Sigma plot 2000 or Origin 6.0):

(1)

Alternatively, the data were fitted with a sum of two Hill equations (Origin, 6.0)

(2)

where I is the peak current at a given concentration of agonist (A), I_max is the maximum current, EC₅₀ is the concentration of agonist yielding a half-maximal current, and n_H is the Hill coefficient.

In the high or low expression condition for the wild-type ₆₂ receptors, where one component dominates, the fit of the data points to the sum of two Hill equations using Origin software does not give a satisfactory result (confidence > 0.95). Even in cases when the fitting was successful, the obtained double fit for most low and high expression experiments did not closely follow the experimental data point obtained at high or low concentration ranges, respectively. We postulate that under low or high expression conditions, the apparent desensitization/inactivation of the high affinity component may hinder reliable fitting with the sum of two Hill equations.

To quantify the inhibitory effect of antagonists, the data were fitted to the following equation

(3)

where I is the peak current at a given concentration of antagonist (An), I_max is the level of spontaneous current in the absence of antagonist, IC₅₀ is the concentration of antagonist inhibiting half of the spontaneous current, and n_H is the slope.

	Results

Top Abstract Introduction Methods Results Discussion References

 
₆₂ receptors exhibit distinct agonist affinity states

cRNA of rat wild-type ₆, ₂ and subunits (Bernard et al. 1998) was injected into Xenopus laevis oocytes in increments of 1.5–3, 5–7 and 8–12 ng per oocyte to produce low, intermediate and high levels of expression, respectively (at a ratio of 1₆ : 1₂ : 1.8). Three to four days after injection, GABA-activated currents were recorded using a GABA concentration range from 0.0002 to 300 µM. Figure 1 shows the current traces and GABA concentration–response relationships for three representative oocytes with low, intermediate and high levels of expression of ₆₂ receptors. At low expression levels (Fig. 1, filled circles; holding potential –70 mV), the GABA concentration–response relationship yielded an EC₅₀ (a concentration eliciting half-maximal current) of 1.77 µM and a Hill coefficient (n_H) of 0.49 (GABA maximal current; GABA I_max = 206 nA). At intermediate expression levels (Fig. 1, open circles), a spontaneous current became apparent (shaded area; 80 nA; GABA I_max = 328 nA) which reversed at –33 mV (reversal potential = –28.70 ± 1.64 mV, range –22 to –35 mV; n = 15) in accordance with the predicted chloride reversal potential (Taleb & Betz, 1994). Fitting of the data points from this oocyte with a single Hill equation yielded an EC₅₀ of 0.28 µM and an n_H of 0.41, representing a more than 5-fold increase in GABA sensitivity as compared to the low expression data set. At high expression levels, the spontaneous chloride current increased to over 200 nA (Fig. 1, filled triangles; 269 ± 61 nA, n = 15). The presence of a larger spontaneous current (200 nA) was concomitant with a further increase in overall GABA sensitivity (EC₅₀ = 0.01 µM, n_H = 0.53) and desensitization at GABA concentrations greater than the EC₅₀ value (the group data for GABA EC₅₀, n_H and maximum values for the low, intermediate and high expression are given in Table 1). Collectively, the range of GABA EC₅₀ values derived from different levels of expression ranged from 0.01 to 4.86 µM, with an n_H of 0.5–0.7 (n = 24).

View larger version (27K): [in this window] [in a new window]   Figure 1.  62 receptors exhibit spontaneous activity and two separable state of agonist affinities A, representative GABA current traces for high (top), intermediate (middle) and low (bottom) expression conditions with oocytes clamped at –70 mV. The dotted line indicates the zero-current level; the shaded area represents the spontaneous activity. The thick lines above the current traces represent the duration of GABA application. The duration of agonist application decreased with increasing concentration of agonist since the currents reached steady state (peaked) more rapidly at higher concentrations of agonist. B, plot of GABA concentration–response relationships including the spontaneous currents. All data were fitted with a single Hill equation. C, concentration–response relationship of the normalized GABA currents. The EC50, nH and maxima parameters for the group data for high, intermediate and low expression conditions are shown in Table 1. The continuous line shows the plot of the fit of a sum of two Hill equations to the data points from an oocyte with intermediate expression level demonstrating the presence of two components with different sensitivities to GABA. The dashed line shows the overall plot of the fit (group data) of sum of two Hill equations to the GABA concentration–response data points for the 62 receptor at median expression. D, a representative current trace for consecutive application of 0.03–20 µM picrotoxinin on an oocyte with high expression of 62 recptor. The thick lines below the current traces represent the duration of antagonist application. The arrow indicates the start of the picrotoxinin application at the concentration shown below it. E, the concentration–response relationship for picrotoxinin block of the spontaneous current form 62 receptor. Picrotoxinin inhibited greater than 90% of the spontaneous current with an IC50 of approximately 0.2 µM. F, the effect of 20 µM picrotoxinin application on the leak current (20 nA) from a mock-injected oocyte (see methods).

View this table: [in this window] [in a new window]   Table 1.  Parameters obtained from fitting the Hill equation to GABA, -alanine, taurine, I4AA, and 5-THDOC data

Reversal potential measurements indicate that a chloride conductance underlies the spontaneous current but do not demonstrate that the current is mediated by ₆₂ receptors (since there are also endogenous chloride channels present within oocytes). Picrotoxinin, a specific pore blocker of GABA_A receptors, was used to determine whether the spontaneous chloride current originates from ₆₂ receptor expression. Figure 1D and E shows the picrotoxinin-induced current traces and concentration–response relationship from an oocyte expressing high levels of ₆₂ receptor. Seven incremental concentrations of picrotoxinin (from 0.03 to 20 µM) were applied to oocytes expressing ₆₂ subunits to construct a concentration–response relationship. Concentrations were applied incrementally because picrotoxinin possesses a high-affinity binding component for the ₆₂ receptor that is resilient to complete wash out. Picrotoxinin blocked the spontaneous current arising from ₆₂ receptors with both high efficacy and potency. The IC₅₀ value for the picrotoxinin action was approximately 0.2 µM, with picrotoxinin, at 20 µM, blocking more than 90% of the spontaneous current (for all parameters, see Table 2). Figure 1F shows a control in which picrotoxinin is added to a mock-injected oocyte displaying 20 nA of leak current. Picrotoxinin (20 µM) did not attenuate the control leak current. These experiments demonstrate that the spontaneous current observed following expression of ₆, ₂ and cRNAs originates from GABA_A receptors.

GABA_A antagonists block the spontaneous activity arising from ₆₂ receptors

We examined the effects of several specific GABA_A antagonists on the spontaneous current arising from ₆₂ receptors. Among the antagonists tested, furosemide proved to be a specific antagonist for GABA_A receptors containing the ₆ subunit (Korpi et al. 1995; Korpi & Luddens, 1997). Furosemide was added at 2, 8, 20, 50, 200 and 500 µM concentrations to oocytes expressing ₆₂ receptors. Figure 2A shows the corresponding current traces and the concentration–response relationship for inhibition of the spontaneous current. Furosemide inhibited 76% of the spontaneous current with an IC₅₀ of 12.3 µM (see Table 2).

View larger version (22K): [in this window] [in a new window]   Figure 2.  Furosemide, bicuculline, gabazine and Zn2+ inhibit the spontaneous current arising from 62 receptors A, furosemide blocked the spontaneous activity of 62 receptors. Current traces and the concentration–response relationship for furosemide-dependent inhibition of the spontaneous current arising from an oocyte with a high level of expression of 62 receptors. The dotted line indicates the zero-current level; the shaded area represents the spontaneous activity. The thick lines below the current traces represent the duration of antagonist application. B, bicuculline and gabazine inhibited the spontaneous activity of 62 receptors. Current traces representing bicuculline (5 µM) and gabazine (5 µM) inhibitory action on the spontaneous activity. C, the representative current traces and concentration–response relationship for Zn2+ block of the spontaneous current arising from 62 receptors.

Zinc inhibits GABAergic responses within neurons and is postulated to function as an endogenous modulator of ion channels in the CNS (Legendre & Westbrook, 1991; Smart, 1992; Dunne et al. 2002; Smart et al. 2004). We tested the effect of Zn²⁺ on the spontaneous current arising from ₆₂ receptor expression. Figure 2C shows representative current traces and the concentration–response relationship of the Zn²⁺-mediated inhibition of the spontaneous current (IC₅₀ of 1.42 µM; Table 2).

Thus a range of established GABA_A antagonists, including furosemide, bicuculline, gabazine, picrotoxinin and Zn²⁺ all inhibit spontaneous activity arising from the expression of ₆₂ receptors.

Expression of a functional receptor–channel requires ₆, ₂, and subunits

The presence of two components in the ₆₂ GABA concentration–response relationship suggests the coexistence of at least two distinct and separable populations of ion channels. We tested the capacity of ₆ and ₂, ₂ and , and ₆ and , as well as that of ₂ alone to express ligand-gated ion channels by injecting these cRNA combinations into oocytes at quantities that yield a high level of expression for the ₆₂ receptor (8–12 ng per oocyte). Four days post-injection, oocytes were tested for the presence of spontaneous activity and GABA-dependent activity using GABA concentrations of up to 500 µM. These subunit combinations yielded neither functional receptor–channels, nor a spontaneous current (n = 46). At significantly greater quantities of cRNA (20–30 ng per oocyte), ₂ or ₂ and ₆ yielded receptor–channels which displayed spontaneous channel activity. However, the resulting ₂ or ₆₂ receptors exhibited a markedly reduced GABA maximal current (< 30 nA for ₂ n = 15 and < 150 nA for ₆₂ n = 30). Moreover, even at expression levels of 20–30 ng of cRNA per oocyte (4-fold the quantities used for intermediate expression of ₆₂), neither ₂ nor ₆₂ receptors produced the magnitude of spontaneous current activity observed in ₆₂ receptors (data not shown). Together these results suggest that neither the ₂ nor the ₆₂ receptors contribute significantly to the observed channel activity arising from the expression of ₆₂ receptors.

Spontaneous channel activity is a unique property of the ₆₂ receptor

Using the intermediate expression protocol (5–7 ng of cRNA), we tested expression of _122S(Short), ₁₂, ₄₂, _622S and ₆₂ subunit combinations to determine if these receptors produced spontaneous channel activity. For each oocyte expressing a given subtype of GABA_A receptor, we measured the magnitude of the spontaneous current as well as the GABA- and pentobarbital-induced maximal current 4 days post-injection. Figure 3A shows the background leak-subtracted magnitudes of spontaneous current for the aforementioned GABA_A subunit combinations (see Methods and Table 3). The spontaneous current activity recorded from ₆₂ receptors was significantly higher than that for any other GABA_A receptors (ANOVA one-way analysis F ratio = 15.76; P < 0.001). Fisher's LSD multiple comparison test also showed that the magnitudes of spontaneous currents arising from ₆₂ receptors were different from those of other GABA_A receptors (P < 0.05). These experiments demonstrated that the significant level of spontaneous channel activity is a property unique to ₆₂ receptors.

View larger version (12K): [in this window] [in a new window]   Figure 3.  Comparison of the spontaneous currents, GABA-induced maxima and pentobarbital relative maxima to GABA for 62, 62, 42, 12, and 12 receptors under equivalent expression conditions A, comparison of the spontaneous currents for 62, 622, 42, 122 and 12 receptors under equivalent expression conditions. The 62 receptor exhibited a significantly higher spontaneous current than did the other GABAA receptors. B, comparison of the GABA-induced maximal current for 62, 622, 42, 122 and 12 receptors under equivalent expression conditions. GABA maximal current was determined from experiments in A using GABA concentrations 20–50 times the respective EC50 value. GABA had the lowest efficacy (maximal) for 62 receptors. C, comparison of the relative maximal current of pentobarbital to GABA for the GABAA receptor subtypes. Pentobarbital maximal current was determined from experiments in A using 1 mM concentration. Pentobarbital exhibited a significantly higher efficacy than did GABA for 62 receptors.

GABA exhibits a low efficacy for ₆₂ receptors

The maximal GABA-induced current for each subunit combination tested (_122S, ₁₂, ₄₂, _622S and ₆₂) was determined in the preceding experiments (where the spontaneous currents at intermediate expression levels were compared). GABA concentrations were used at 20–50 times the respective EC₅₀ values (for EC₅₀ values, see Table 1). A comparison of the maximal GABA-evoked currents for the five GABA_A receptor subtypes is shown in Fig. 3B (for current values see Table 3). The maximal GABA-evoked current for ₆₂ receptors was only 16–28% of that of the other subunit combinations tested. These data reveal that the GABA-sensitive component of ₆₂ receptors is significantly smaller than that seen for other GABA_A receptor subunit combinations. A comparison of the spontaneous current relative to the total current (maximal GABA-induced plus the spontaneous current) demonstrates that for ₆₂ receptors the spontaneous activity represented approximately 23% of the total attainable current.

Pentobarbital, an intravenous anaesthetic, is a potent modulator of GABA_A receptors and at high concentrations can directly activate them. Previous studies have shown that the GABA-dependent and pentobarbital-dependent activation domains are distinct, given that pentobarbital can activate a mutated GABA_A receptor whose GABA-dependent activation domain is impaired (Amin & Weiss, 1993; Amin, 1999). We also determined the maximal pentobarbital-induced current for each GABA_A receptor subtype within the preceding experiments at intermediate expression levels (on the same oocytes where the spontaneous activity and the GABA maxima were determined). Figure 3C shows the maximal current induced by 1 mM pentobarbital relative to that induced by GABA (pentobarbital I_max/GABA I_max x 100) for the GABA_A receptor subtypes tested (see also Table 3). For all GABA_A receptors tested, excepting ₆₂ receptors, pentobarbital produced similar or lower maximal current than GABA. The pentobarbital-evoked maximal current was more than three times greater than that induced by GABA for ₆₂ receptors and was similar in magnitude to the GABA maximal current for other GABA_A receptors. Thus, pentobarbital is markedly more efficacious than GABA and acts as a full agonist for the ₆₂ receptor when compared to GABA.

₆₁ and ₆₃ receptors also exhibit the high-affinity state

Cerebellar granule neurons express high levels of ₁, ₂, ₆, , ₂ and ₃ subunits in the adult rats (Laurie et al. 1992; Persohn et al. 1992; Wisden et al. 1996; Jechlinger et al. 1998). The ₆ and subunits in combination with either ₁, ₂ or ₃ cRNAs (at intermediate expression levels) were coinjected into oocytes and the maximal GABA-induced current (100 µM) and the extent of the spontaneous activity for each receptor subtype was measured to determine whether the high-affinity state and the spontaneous activity of the ₆₂ receptor depend upon the subtype of subunit (Fig. 4A). GABA induced a similar maximal current for ₆₁, ₆₂ and ₆₃ receptors. Further, all three _61-3 receptors displayed high levels of spontaneous activity, indicating the presence of the high-affinity state. The level of spontaneous activity was greater for ₆₁ and ₆₃ than for ₆₂ receptors, suggesting that the ₆₁ and ₆₃ receptors may exhibit a higher propensity to assemble into the high-affinity state. Next, we determined a picrotoxinin concentration–response relationship (0.03–20 µM) to establish whether the spontaneous activity indeed arises from ₆₁ and ₆₃ receptors (Fig. 4B and C). Similar to the ₆₂ receptor, picrotoxinin blocked the spontaneous current arising from ₆₁ and ₆₃ receptors with high potency (IC₅₀ of 0.3, see Table 2) and efficacy (90% block at 20 µM). These experiments demonstrated that either the ₁, ₂ or ₃ subunit may coassemble with ₆ and subunits to express spontaneously active receptor–channels in the high-affinity state.

View larger version (11K): [in this window] [in a new window]   Figure 4.  Expression of the high-affinity state of 62 receptors is independent of the isoform of the subunit GABA induced similar maximal current for 61, 62, and 63 receptors (filled bars) with high levels of spontaneous activity indicating the presence of the high-affinity state (open bars). B and C, the concentration–response relationship for picrotoxinin block of the spontaneous current from 61 and 63 receptors.

Potency and efficacy of different GABA and glycine agonists upon ₆₂ receptors

We compared the maximal induced current evoked by GABA with that of two other established GABA agonists, trans-4-aminocrotonic acid (TACA) and imidazole-4-acetic acid (I4AA), in the ₆₂ receptor (at intermediate expression levels). Previous studies have established that TACA is a full agonist and I4AA as a partial agonist in GABA_A receptors (Woodward et al. 1993; Chebib & Johnston, 1999; Mortensen et al. 2004). Figure 5A shows the maximal current induced by I4AA (1100 µM 200 x EC₅₀) and TACA (500 µM 200 x EC₅₀) relative to that of saturating concentrations of GABA (300 µM). For I4AA, the relative maximal current to that of evoked by GABA was 0.59 ± 0.06 (n = 9). In comparison, maximal currents evoked by TACA were consistently larger than that those evoked by GABA (1.22 ± 0.03, n = 5) demonstrating that for ₆₂ receptors both GABA and I4AA behave as partial agonists relative to TACA.

View larger version (19K): [in this window] [in a new window]   Figure 5.  Comparison of the I4AA and TACA maximal-induced currents as well as -alanine and taurine concentration-response relationship for 62 receptors A, comparison of the I4AA and TACA maximal-induced currents relative to GABA for 62 receptors. B, concentration–response relationship for I4AA and the fit of a sum of two Hill equations to these data points. The 62 receptors response to I4AA concentrations exhibited two components with marked difference in apparent affinity. C, the -alanine concentration–response relationships for oocytes with high (), intermediate () and low () 62 expression. The dashed line shows the plot of the fit (average from group data) of sum of two Hill equations to the -alanine concentration–response data point for wild-type 62 receptor at intermediate expression condition. D, taurine concentration–response relationships for 62 receptors at low (), intermediate (), or high () expression condition. The dashed line shows the plot of the fit (average from group data) using a sum of two Hill equations to the taurine data points for the 62 receptor at intermediate expression condition.

Both -alanine and taurine are classical glycine receptor agonists (Kuhse et al. 1993) and may act as neurotransmitters within the CNS. Using a range of -alanine concentrations from 0.05 to 10 000 µM, we determined the efficacy and potency of this agonist in oocytes expressing low, intermediate and high levels of ₆₂ receptors (displaying 0, 70 and 190 nA of spontaneous current, respectively; Fig. 5C). For an oocyte with a low level of expression (filled circles), the EC₅₀ for -alanine agonist was 0.5 mM (the group data for -alanine are shown in Table 1). With increasing expression, the sensitivity of the ₆₂ receptor to -alanine increased concomitantly with the high spontaneous activity (see open circles and filled triangles), reducing the EC₅₀ value to approximately 1 µM (single fit to the Hill equation). The fit of the -alanine data from the ₆₂ intermediate expression to the sum of two Hill equations yielded EC₅₀ values of 1.16 and 594.66 µM, respectively, for the high and the low affinity components (see Table 1). A comparison of the overall GABA and -alanine maximal currents demonstrated that -alanine was as efficacious as GABA for ₆₂ receptors (I_max -alanine at 10 000 µM/I_max GABA at 300 µM = 1.08 ± 0.03, n = 7).

Figure 5D shows taurine concentration–response relationships for three sets of oocytes with low (filled circles), intermediate (open circles) and high (filled triangles) levels of ₆₂ expression (dispalying 40, 80 and 140 nA of spontaneous current, respectively; the group data for taurine are shown in Table 1). Fitting of the data points to the sum of two Hill equations yielded EC₅₀ values of 5 and 807 µM for the high- and low-affinity components, respectively (Table 1). Taurine behaved as a partial agonist for ₆₂ receptors as compared to GABA or -alanine. The relative efficacy of taurine (50 000 µM) to -alanine (10 000 µM) at near saturating concentrations was 0.69 ± 0.04 (n = 7).

The level of ₂ subunit expression determines the apparent affinity of ₆₂ receptors

We injected the ₆, ₂ and cRNA into oocytes in the ratios of 1₆ : 0.1₂ : 1.8, 1₆ : 0.3₂ : 1.8, or in the control 1₆ : 1₂ : 1.8. The amount of injected cRNA was 5–7 ng of cRNA (intermediate expression) except for 1₆ : 0.1₂ : 1.8 where 8–12 ng (high expression) of cRNA was injected. A GABA concentration–response relationship was constructed as shown in Fig. 6A and B. At the 0.1₂ ratio (1₆ : 0.1₂ : 1.8; filled squares), the ₆₂ receptors were predominantly present in the low-affinity state and displayed three notable properties: (1) the resulting ₆₂ receptors were insensitive to GABA concentrations below 0.02 µM (EC₅₀ of 1.3 µM; Table 1); (2) after removal of GABA, the current's return to baseline was satisfied by a fit to a single exponential; and (3) these receptor–channels showed no discernible spontaneous activity. With an increase in the ₂ ratio (0.3₂, filled circles), GABA-induced currents were detected at concentrations as low as 0.002 µM (EC₅₀ of 1.5 µM; single fit, see Table 1). Moreover, the higher sensitivity to GABA was concomitant with an appearance of spontaneous channel activity (shaded area) and a shallower Hill coefficient than for the 0.1 ₂ ratio. Further, current decay, following agonist washout at higher concentrations followed a multiexponential decay (analysis not shown). At the control ratio of 1₆ : 1₂ : 1.8 (filled triangles), both high and low affinity components were readily discernible. The appearance of the high-affinity component thus coincided with a significant level of spontaneous channel activity with the current decay following removal of the agonist exhibiting a multiexponential paradigm. In experiments with the ₂ cRNA at a ratio 2–4 times higher (e.g. 1₆ : 2 or 4₂ : 1.8; data not shown), the resulting ₆₂ receptors were predominantly present in the high-affinity state, similar to that observed under condition of high expression (see also Fig. 1).

View larger version (23K): [in this window] [in a new window]   Figure 6.  The effect of change in 2 subunit ratio on the expression of 62 and 122 receptors A, the current traces of receptor–channels from the expression of 16 : 0.12 : 1.8, 16 : 0.32 : 1.8, or control 16 : 12 : 1.8 ratios. The dotted line indicates the zero-current level; the shaded area represents the spontaneous activity. The thick lines above the current traces represent the duration of GABA application. B, the GABA concentration–response relationship for the three tested ratios of 6, 2, and subunits. The data derived from 16 : 0.12 : 1.8 and 16 : 0.32 : 1.8 were fitted with a single Hill equation, while the data for 16 : 12 : 1.8 were fitted with a sum of two Hill equations. C, the concentration–response relationship for 11 : 0.082 : 1.82S, 11 : 0.42 : 1.82S and control11 : 12 : 1.82S subunit combination.

With the increase in the ratio of ₂ cRNA, both ₆₂ and _122S receptors showed increases in their apparent affinity for GABA. However, the magnitude of the shift in GABA sensitivity to lower concentrations was 12-fold for the _122S receptor in comparison to more than 400-fold for the ₆₂ receptor. The increase in GABA sensitivity was concomitant with the appearance of spontaneous channel activity within the ₆₂ receptor, but not for the _122S receptor.

5-THDOC directly activates ₆₂ receptors

Neuroactive steroids are metabolites of the principal sex and stress steroid hormones and represent a large class of endogenous compounds active within the CNS (Belelli & Lambert, 2005). One such metabolite, 5-THDOC, is a potent modulator of GABA_A receptors (Puia et al. 1994), and is a highly hydrophobic compound that mediates its actions through a mechanism that is distinct from that of GABA binding (Morris & Amin, 2004). We examined the direct action of 5-THDOC upon ₆₂ receptors at a range of concentrations. Figure 7 shows 5-THDOC-induced current traces and the concentration–response relationship for ₆₂ receptors (at intermediate- and high-expression conditions). Concentrations as low as 30 nM of 5-THDOC augmented the spontaneous current. For direct activation by 5-THDOC, EC₅₀ and n_H values of 0.89 µM and 0.97 were derived, respectively (see Table 1). The 5-THDOC-induced maximal current at 20 µM was similar in magnitude to that of GABA. Thus, 5-THDOC, previously known for its modulatory action on GABA_A receptors, can directly activate ₆₂ receptors with high potency.