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¹ Departamento de Bioquímica y Biología Molecular, Edificio Santiago Gascón, Campus del Cristo, Universidad de Oviedo, E-33006, Oviedo, Asturias, Spain

	Abstract

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The identity of the G-protein coupling thyrotropin-releasing hormone (TRH) receptors to rat ether-à-go-go related gene (r-ERG) K⁺ channel modulation was studied in situ using perforated-patch clamped adenohypophysial GH₃ cells and dominant-negative variants (G-QL/DN) of G-protein subunits. Expression of dominant-negative G_q/11 that minimizes the TRH-induced Ca²⁺ signal had no effect on r-ERG current inhibition elicited by the hormone. In contrast, the introduction of dominant-negative variants of G₁₃ and the small G-protein Rho caused a significant loss of the inhibitory effect of TRH on r-ERG. A strong reduction of this TRH effect was also obtained in cells expressing either dominant-negative G_s or transducin subunits, an agent known to sequester free G-protein ß dimers. As a further indication of specificity of the dominant-negative effects, only the dominant-negative variants of G₁₃ and Rho (but not G_s-QL/DN or G_t) were able to reduce the TRH-induced shifts of human ERG (HERG) activation voltage dependence in HEK293 cells permanently expressing HERG channels and TRH receptors. Our results demonstrate that whereas the TRH receptor uses a G_q/11 protein for transducing the Ca²⁺ signal during the initial response to TRH, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in pituitary cells. They also identify G_s (or a G_s-like protein) and G₁₃ as important contributors to the hormonal effect in these cells and suggest that ß dimers released from these proteins may participate in modulation of ERG currents triggered by TRH.

(Received 28 February 2005; accepted after revision 12 May 2005; first published online 19 May 2005)
Corresponding author F. Barros: Departamento de Bioquímica y Biología Molecular, Edificio Santiago Gascón, Campus del Cristo, Universidad de Oviedo, E-33006, Oviedo, Asturias, Spain. Email: fbarros{at}correo.uniovi.es

	Introduction

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Regulation of ether-à-go-go related gene (ERG) K⁺ channel activity by the hypothalamic neuropeptide thyrotropin-releasing hormone (TRH) constitutes an essential point of hormonal control of electrical activity, and hence of intracellular Ca²⁺ levels ([Ca²⁺]_i) and the secretory response in anterior pituitary cells (Barros et al. 1994, 1997; Weinsberg et al. 1997; Bauer, 1998; Bauer et al. 1998, 1999). The human ERG (HERG) channel has been also recognized as an important determinant of action potential characteristics in heart muscle and its inhibition by inherited mutations or a panoply of cardiac- and noncardiac-related prescribed drugs has been associated with an increased risk of cardiac arrhythmia and sudden death (Chiang & Roden, 2000; Keating & Sanguinetti, 2001; Redfern et al. 2003; Finlayson et al. 2004). Interestingly, the observation that arrhythmogenic syncopes are usually associated with physical, emotional or auditory stress suggests a link between hormonal (e.g. adrenergic) stimulation and cardiac ion channel function including HERG (Thomas et al. 2004). Furthermore, ERG channels also seem to play a key role setting the electrical behaviour of other cell types including neurones and glial, chromaffin, pancreatic ß and tumour cells (Zhou et al. 1998a; Emmi et al. 2000; Rosati et al. 2000; Gullo et al. 2003; Sacco et al. 2003; Lastraioli et al. 2004). Nevertheless, unlike the relatively well known molecular and kinetic characteristics of ERG channels and in spite of their physiological and pathological relevance, many of their molecular mechanisms of regulation by different physiological agents remain unclear.

In native lactotrophs and clonal GH adenohypophysial cells, endogenous ERG currents are inhibited by activation of the G protein-coupled TRH receptor (TRH-R; Bauer et al. 1990, 1994; Barros et al. 1992, 1993; Schäfer et al. 1999; Schledermann et al. 2001). The TRH-R is coupled to a G-protein of the G_q/11 family (reviewed in Gershengorn & Osman, 1996) resulting in phospholipase C (PLC) activation and generation of myo-inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5-bisphosphate (PIP₂). It is also known that TRH is able to activate several PKC isozymes in GH₃ cells (Kiley et al. 1991; Akita et al. 1994). However, TRH-induced inhibition of ERG current in these cells does not depend on PKC or PKA activation (Bauer et al. 1990, 1994; Barros et al. 1992, 1993; Schäfer et al. 1999; Schledermann et al. 2001). Whether G_q/11 protein transduction, typically linked in many cells (including adenohypophysial cells) to Ca²⁺ signalling, plays a role in ERG channel regulation remains controversial. Thus, a pathway for ERG regulation by TRH involving a G₁₃- and Rho-mediating signalling cascade has been described in whole-cell voltage-clamped GH₄C₁ cells (Storey et al. 2002), but the relative importance of this transduction pathway as compared with the ‘classical’ G_q/11-mediated TRH-induced signalling was not assessed. Furthermore, G_q/11 but not G_i/o or G₁₃ has been recently shown to mediate muscarinic inhibition of ERG currents in tsA-201 cells coexpressing rat ERG1 channels and M1 muscarinic receptors (Hirdes et al. 2004). In adenohypophysial cells, the G_q/11-mediated coupling of TRH-R to PIP₂ hydrolysis leads to an initial elevation of [Ca²⁺]_i via IP₃ that mediates a peak of secretion associated with a transient hyperpolarization of the cell membrane due to activation of Ca²⁺-dependent K⁺ channels (Gómez-Varela et al. 2003b). However, normal inhibition of ERG channel activity in response to TRH has been observed in individual cells in which the initial Ca²⁺ response is totally absent (Barros et al. 1991, 1992, 1994). Although the PKC branch of the PLC signalling cascade is necessary for reversal of the TRH-induced ERG inhibition in GH₃ cells (Gómez-Varela et al. 2003b) and PIP₂ depletion could lead to HERG current reduction in HEK293 cells (Bian et al. 2001), the TRH-induced inhibition of the GH₃ cell r-ERG current also takes place after blockade of PIP₂ consumption with a PLC inhibitor (Gómez-Varela et al. 2003b). These results and the reported modification of the TRH effects on r-ERG in cholera toxin-treated GH₃ cells (Barros et al. 1994; Bauer et al. 1994) open the possibility that, at least in adenohypophysial cells, a transduction cascade(s) involving either a G_s-like protein or a G₁₃- and Rho-based pathway, couples the TRH-R to endogenous ERG channel inhibition.

In this report we use double mutants (G-QL/DN) of G-protein subunits able to act as dominant-negative inhibitors against specific G-proteins (Yu et al. 2000) to explore the specificity of TRH-R coupling to G-proteins for ERG K⁺ channel inhibition in GH₃ rat anterior pituitary cells. Our results demonstrate that whereas the TRH-R certainly uses a G_q/11 protein for transducing the Ca²⁺ signal during the initial response to the hormone, this G-protein is not involved in the TRH-induced inhibition of ERG currents. Dominant-negative variants of G₁₃ and Rho, but not of G_q/11, are able to significantly reduce the inhibitory effect of TRH on ERG. Furthermore, a prominent reduction is observed upon introduction of dominant-negative G_s. Interestingly, a strong reduction of the TRH-induced inhibition is also observed in cells overexpressing transducin subunits (G_t), an agent known to sequester free G-protein ß dimers (Crespo et al. 1994; Faure et al. 1994; Palomero et al. 1998). The specificity of the dominant-negative and G_t effects is demonstrated by their failure to modify the Ca²⁺ response in the same cells. Furthermore, dominant-negative G_q/11 (but not G_t or dominant-negative G_s, G₁₃ and Rho) was able to reduce TRH-induced release of Ca²⁺ from intracellular stores in HEK293 cells permanently expressing HERG channels and TRH-Rs. In these cells, however, only the dominant-negative forms of G₁₃ and Rho (but not G_t or dominant-negative G_s) were able to antagonize the modifications in activation voltage dependence induced by TRH on HERG currents. On the other hand, only G_t and dominant-negative G_q/11 expression reduced the TRH-induced HERG current inibition at positive voltages. Apart from emphasizing the specificity of the different dominant-negative constructs, this also suggests that the cellular background and/or the channel isoform may influence the transduction mechanism(s) involved in hormonal regulation of ERG.

	Methods

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Plasmids and chemicals

The original plasmid containing the cDNA for the HERG channel was a generous gift of Dr E. Wanke (University of Milan, Italy). pEGFP-N3 plasmid was obtained from Clontech. pcDNA3.1 plasmids containing dominant-negative forms of G_q (G_q-Q209L/D277N), G₁₃ (G₁₃-Q266L/D294N), G_s (G_s-Q227L/D295N) and RhoA (RhoA-T19N 3xHA-tagged-NH2) were obtained from Guthrie (Guthrie cDNA Resource Center; currently transferred to University Missouri-Rolla cDNA Resource Center, Rolla, MO, USA). G_t was cloned in pcDNA3 as an EcoRI/XhoI fragment transferred from pcDNAI (provided by Dr J. S. Gutkind, N. I. of Dental Research, N.I.H., Bethesda, MD, USA). TRH and nystatin were purchased from Sigma. E-4031 and anti-HERG polyclonal antibodies were from Alomone Laboratories; Fura-2 and Fura-2/AM were from Molecular Probes.

GH₃ cell culture and transfection

GH₃ rat anterior pituitary cells (ATCC-CCL 82.1) were plated in 35-mm diameter tissue culture plastic dishes containing sterile glass coverslips coated with poly L-lysine and grown at 37°C in a humidified atmosphere of 95% air and 5% CO₂. The culture medium consisted of a 1: 1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture (Sigma) supplemented with 100 U ml^–1 penicillin, 1.1 mg ml^–1 streptomycin and a serum mixture of 15% horse serum and 2.5% fetal bovine serum. The coverslips constituted the bottom of a small recording chamber (0.2–0.3 ml) that was continuously perfused with saline at a rate of about 1 ml min^–1. Cells trypsinized 24 h prior to transfection lying in poly-L-lysine-coated coverslips were transiently transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Unless otherwise indicated, 5.0 µg of plasmids containing the different constructs and pEGFP-N3 codifying green fluorescent protein (eGFP) as a marker for transfection in a 10: 1 ratio were used. The mixture of 5.5 µg of total DNA and Lipofectamine was incubated in serum-free medium for 20 min and added to the plates containing the cells in serum-containing medium without antibiotics. Recordings were performed 24–48 h after transfection.

Generation and isolation of permanently transfected HEK 293 cell clones

HERG channel cDNA was subcloned into HindIII/BamHI sites of the pcDNA3 vector (Invitrogen). Monolayer cultures (50% confluent) of human embryonic kidney cells (HEK293; ATCC CRL-1573) were transfected with this construct using Lipofectamine (Gibco). Three days after transfection, the cells were trypsinized and diluted in a medium containing 1 mg ml^–1 geneticin. Subsequently they were cultured until cell colonies were visible. Individual colonies were picked with cloning cylinders and tested for HERG currents. A clone named H36 was selected for further transfection. Cells of clone H36 were cotransfected with plasmid pcDNA3.1/Hygro(+) (Invitrogen) containing the cDNA for the TRH-R (de la Peña et al. 1992) inserted between the HindIII/XbaI sites of the vector. Hygromycin B (150 µg ml^–1) was used to select H36 clones coexpressing HERG channels and TRH-Rs. We chose for further work a clone named HEK-H36/T1 showing: (a) robust HERG currents under voltage-clamp, (b) reproducible calcium responses when perfused with TRH after loading the cells with the fluorescent Ca²⁺ indicator Fura-2, and (c) a predominant level of a 155 kDa band of HERG protein, corresponding to the mature and more glycosylated HERG likely to be located in the plasma membrane (Zhou et al. 1998b; Petrecca et al. 1999), immunodetected in cell extracts with HERG-specific antibodies. Cells were grown at 37°C in a humidified atmosphere of 95% air and 5% CO₂. HEK 293 cells were cultured in the same medium as GH₃ cells supplemented with 100 U ml^–1 penicillin, 0.1 mg ml^–1 streptomycin and 10% fetal bovine serum. HEK-H36/T1 cells were maintained in the presence of 1 mg ml^–1 geneticin sulphate and 150 µg ml^–1 hygromycin B (Gibco) and plated on the poly-L-lysine-coated coverslips for recording. Transient transfection of HEK-H36/T1 cells was performed following the procedures indicated above for the GH₃ cells.

Electrophysiological recordings, solutions and data analysis

Current recordings were performed at room temperature with the perforated-patch variant of the patch-clamp technique as previously described (Barros et al. 1991, 1992, 1994, 1997; Gómez-Varela et al. 2003b). Electrodes were fabricated from borosilicate or kimax disposable micropipettes (Boralex, Rochester Scientific, Rochester, NY; Fisherbrand, Fisher Scientific, Pittsburg, PA or Kimble glass Inc., Vineland, NJ, USA). Electrode resistance amounted 2–5 M when filled with the pipette solution containing (mM): 65 KCl, 30 K₂SO₄, 10 NaCl, 1 MgCl₂, 50 sucrose and 10 Hepes (pH 7.4 with KOH). The tip of the pipette was initially filled with nystatin-free solution and the remainder of the pipette was back-filled with the same solution also containing 250 µg ml^–1 nystatin, added from a stock of 50 mg ml^–1 nystatin freshly dissolved in dimethylsulphoxide. These solutions were sonicated just before use. The course of perforation was followed by monitoring the progress of capacitive transients under voltage-clamp mode, setting the pipette voltage at a value of –70 mV. Access resistance, as estimated from the capacitive compensation circuitry on the amplifier, reached 10–30 M within 5–20 min after the seal was made. Solution junction potentials were nulled before seal formation. Once patch permeabilization reached the indicated levels, the extracellular solution was changed as indicated and the cell was voltage-clamped at the desired holding potential. An EPC-7 patch-clamp amplifier (HEKA Elektronic, Lambrecht, Germany) was used to record membrane currents. Stimulation, data acquisition and analysis were carried out using Pulse and PulseFit software (HEKA Elektronic) running on Macintosh computers. Current records were sampled every 1 ms and digitally filtered at 500 Hz. r-ERG current data are shown without correction for leakage and capacitative transients. A P/n method was used for leak and capacitive current subtraction of the HERG recordings in HEK-H36/T1 cells. Further data processing was performed with PulseFit and Igor Pro (WaveMetrics, Lake Oswego, OR, USA).

The standard extracellular saline used for perforation and monitoring [Ca²⁺]_i contained (mM): 137 NaCl, 4 KCl, 1.8 CaCl₂, 1 MgCl₂, 10 glucose, and 10 Hepes (pH 7.4 with NaOH). Recordings of r-ERG currents in GH₃ cells were performed after changing the extracellular medium to high-K⁺, Ca²⁺-free solution once permeabilization of the patches had been completed. This solution contained (mM): 140 KCl, 4 MgCl₂, 10 EGTA and 10 Hepes titrated to pH 7.4 with KOH. Inward currents were studied during hyperpolarization pulses to –100 mV from a holding potential of –10 mV. The hyperpolarization pulses were preceded by a 100 ms ramp from 0 to –50 mV that can yield an estimation of the membrane conductance within this voltage range and would tend to potentiate the otherwise voltage-dependent effect of TRH (Bauer et al. 1990; Barros et al. 1992, 1994, 1997). To prevent variations due to differences in deactivation rates from cell to cell, the magnitude of the inward currents was estimated with the PulseFit software as the total inward charge computed between cursors located at 0.5 and 100% duration of the hyperpolarization pulses. HERG currents were recorded in HEK-H36/T1 cells in standard extracellular saline following the pulse protocols indicated on the figures. Kinetic parameters of activation and deactivation were obtained as previously described (Barros et al. 1998; Viloria et al. 2000). The voltage dependence of current activation was assessed using standard tail current analysis. Tail current magnitudes normalized to maximum were fitted with a Boltzmann function:

where V is the test potential, V is the half-activation voltage, and k is the slope factor. Steady-state voltage dependencies of activation were obtained as previously described (Viloria et al. 2000) applying depolarization pulses of variable magnitude and up to 10 s duration from two holding potentials: +40 mV to hold the channels fully open and –80/–100 mV to hold them fully closed. The position of the Boltzmann curves under true steady-state conditions was estimated as an extrapolated mean from the curves obtained at both holding potentials to ensure that they were a function exclusively of depolarization pulse characteristics, regardless of the previous (open or closed) state of the channels. The time course of activation was monitored using an indirect envelope of tail current protocol (Viloria et al. 2000), varying the duration of the depolarization pulse and following the variation in the magnitude of the tail currents recorded after going back to a negative voltage. The rates of deactivation were determined from negative-amplitude biexponential fits to the decaying phase of tail currents. The first cursor of the fitting window was advanced to the end of the initial hook due to the recovery of inactivation.

Intracellular calcium measurements

Measurements of intracellular Ca²⁺ concentrations ([Ca²⁺]_i) were performed in cells platted in poly-L-lysine-coated coverslips as indicated above. In this case the coverslips were transferred to wells containing standard extracellular saline plus 5 µM Fura-2/AM (Molecular Probes) and loaded with the dye for about 60 min at room temperature. After loading with Fura-2, cells were washed with saline to remove non-hydrolysed Fura-2/AM and left for another 30 min before recording to facilitate AM hydrolysis by cellular esterases. Fluorescence measurements were performed in a Axiovert 100 microscope equipped with a Plan-NeoFluar 40x/0.75 objective and epifluorescence accessories (Carl Zeiss), attached to a fluorescence imaging system (TILL-Photonics GmbH, Martinsried, Germany). Control of the monochromator (Polychrome IV) and the 12-bit cooled CCD camera (IMAGO) was performed using TILLvisION imaging software. Cells were excited through a dichroic mirror reflecting less than 395 nm light. Fluorescence signals were filtered through a 410 nm long-pass filter. Cycles of sample excitation were repeated every 500 ms consisting in 10/20 ms periods of irradiation with 340, 360 and 380 nm light. The ratio of the emission intensities (340 nm/380 nm) was used as a measure for changes in intracellular Ca²⁺. When eGFP-transfected cells were used, a correction for eGFP fluorescence due to residual eGFP excitation at 340–380 nm was performed. Using eGFP-containing cells without Fura-2 we estimated previously that 13% of the fluorescence recovered above 510 nm (using a GFP filter set with a 500 nm dichroic and a 510 nm long-pass filter) following eGFP excitation at 488 nm is also present upon eGFP excitation at 380 nm using the conventional Fura-2 set-up. Less than 1% was recovered when eGFP was excited at 340 nm. Subsequently, the eGFP-derived fluorescence at 380 nm was estimated in every individual cell loaded with Fura-2 from the (13%) fluorescence intensity at 488 nm (a wavelength at which Fura-2 is not excited). This amount was subtracted from the total fluorescence at 380 nm to isolate the Fura-2-specific signal. Ca²⁺ concentrations were estimated from the 340 nm/380 nm fluorescence ratio by comparison with Fura-2 standards (Barros et al. 1994).

Statistics

Data values given in the text and in figures with error bars represent the mean ± S.E.M. for the number of indicated cells. Comparison between data groups was at first performed by parametric Student's unpaired t test (2-tailed) or ANOVA. Due to dispersion of the data in individual cells after some treatments (e.g. G₁₃-QL/DN and RhoA T/N transfections in Fig. 4), non-homogeneous variances (as evidenced after a Bartlett's test) were sometimes obtained. Therefore, alternate Welch's test assuming Gaussian populations with unequal S.D.s and a non-parametric Wilcoxon or Mann-Whitney test that does not make any assumption about the scatter of the data were also used to evaluate significance of mean differences between cell populations. For a posteriori comparison of two specific samples a Bonferroni or a Dunn test for multiple comparisons was also used following one-way ANOVA or Kruskal-Wallis non-parametric ANOVA tests, respectively. In all cases, P-values < 0.05 were considered as indicative of statistical significance.

View larger version (22K): [in this window] [in a new window]   Figure 4.  Percentage inhibition of r-ERG currents induced by TRH in GH3 cells expressing dominant-negative G subunits and Gt Current inhibitions were estimated from total inward charge as described in Fig. 3. Data from individual cells averaged on the bars are shown as open circles. Values from cells transfected with dominant negative variants of Gq, G13, RhoA and Gs, or transducin subunits (Gt) are shown. Data from cells transfected with vector pcDNA3B (Control 3B) and from untransfected cells (NO transf.) are also shown for comparison. Significant variations among population medians as evidenced by Mann-Whitney test or ANOVA and post hoc multiple comparison test are indicated.

	Results

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Validation of the dominant-negative strategy using G_q-QL/DN and the TRH-induced Ca²⁺ response of the GH₃ cells

To examine the transduction pathway leading to ERG inhibition upon TRH stimulation, we expressed xanthine nucleotide binding mutants of different G-protein subunits. These mutants carrying a double mutation (leucine and asparagine substituting for a glutamine and an aspartate, respectively) possess a lowered affinity by guanine nucleotides and an enhanced affinity by xanthine nucleotides that make them form stable and specific complexes with cognate receptors and compete with endogenous wild-type G-proteins (Yu et al. 2000). We first probed the effectivity of this approach using dominant-negative G_q (G_q-Q209L/D277N) and exploring the initial (Phase 1) TRH-dependent response of the GH₃ cells. This phase corresponds to a transient release of stored Ca²⁺ into the cytosol due to production of IP₃ by PLC-catalysed PIP₂ hydrolysis, leading to an initial and transient hyperpolarization of the cell membrane by activation of Ca²⁺-dependent K⁺ channels (Gómez-Varela et al. 2003b). We reasoned that blockade of this transduction pathway with G_q-QL/DN should minimize all the cellular responses that characterize this phase such as (i) the transient membrane hyperpolarization, (ii) the transient increase in potassium currents that determine this hyperpolarization, and (iii) the transient [Ca²⁺]_i increase that activates the Ca²⁺-dependent K⁺ currents. As shown in Fig. 1A the transient hyperpolarization of the cell membrane induced by TRH in perforated-patch current-clamped GH₃ cells is nearly abolished in cells expressing G_q-QL/DN (see averaged voltage traces in the insets). Interestingly, as shown in the individual cell recordings illustrated in Fig. 1A, the increase in the rate of production of action potentials (Phase 2 of hormone action) that follows the initial hyperpolarization was maintained in the presence of the dominant-negative. As a second validation of the dominant-negative effect, we studied the appearance of membrane currents immediately after TRH addition in patch-perforated voltage-clamped GH₃ cells bathed in high-K⁺ Ca²⁺-free solution. Figure 1B shows that the current increases induced by TRH were absent in cells expressing G_q-QL/DN. Thus, these increases represent G_q-mediated PLC activation and activation of K⁺ currents by Ca²⁺ released from intracellular stores via IP₃, because they were determined without extracellular Ca²⁺. Furthermore, the effect of G_q-QL/DN was specific since the TRH-induced currents remained unaltered in cells expressing the dominant-negative subunit of G₁₃, a G-protein predictably unrelated to the PLC–IP₃ Ca²⁺ signalling pathway.

View larger version (32K): [in this window] [in a new window]   Figure 1.  Blockade by dominant-negative Gq-QL/DN of the initial phase 1 of response induced by TRH in GH3 cells A, effect of dominant-negative Gq on TRH-induced Phase 1 of hyperpolarization. Representative recordings of membrane potential are shown in two cells either expressing the dominant-negative form of Gq (lower trace) or not (upper trace). Application of 1 µM TRH is marked with a horizontal line on top of the traces. Note the maintenance of Phase 2 of increased electrical activity in the Gq-QL/DN-transfected cell. Traces averaged point by point from several cells are shown in the insets. Continuous current traces averaged point by point and their corresponding S.E.M. are shown. In this cases, traces were synchronized to the time of TRH addition as indicated with an arrow. B, effect of dominant-negative Gq and G13 on Ca2+-dependent K+ currents elicited in GH3 cells by TRH during the initial Phase 1 of response. Continuous current traces averaged point by point and their corresponding S.E.M. are shown for cells transfected with vector pcDNA3B (Control 3B), or with plasmids codifying the dominant-negative mutants of Gq (Gq-QL/DN) and G13 (G13-QL/DN). Traces were synchronized to the time of 1 µM TRH addition as indicated with an arrow. High-K+, low-Ca2+ extracellular solution and a potential of –30 mV were used for better detection of inward K+ currents activated by Ca2+ released from intracellular stores via IP3.

The aforementioned results strongly differ from those obtained with cells transfected with eGFP and a plasmid coding for G_q-QL/DN (Fig. 2A). In this case, TRH raised the basal [Ca²⁺]_i level of 73 ± 9 nM to 141 ± 14 nM in the 26 cells showing no detectable eGFP expression. In contrast, only a peak value of 61 ± 5 nM [Ca²⁺]_i from a basal level of 49 ± 6 nM was obtained upon TRH addition in the 11 cells of the same field showing a clear eGFP fluorescence. It is also important to note that in this case only 3 of the 11 cells expressing eGFP showed any significant increase in [Ca²⁺]_i as compared with the 18 in which [Ca²⁺]_i was clearly increased from 26 cells not expressing the transfection marker. Furthermore, whereas only a modest [Ca²⁺]_i increase from 61 ± 6 to 94 ± 4 nM was observed in those three cells expressing eGFP, a prominent initial peak of Ca²⁺ that raised [Ca²⁺]_i from 73 ± 9 to 172 ± 7 nM was obtained in the 18 cells in which eGFP (and hence G_q-QL/DN) expression was not detectable. Analogous results were obtained in two additional experiments. This demonstrates that (i) a much lower percentage of cells expressing the dominant-negative variant of G_q respond to TRH, and (ii) even in the few cells expressing G_q-QL/DN that show a detectable response to the hormone, the magnitude of the [Ca²⁺]_i increase is clearly smaller than that of cells in which the transfection marker (and hence the dominant-negative G_q) has not been expressed.

View larger version (31K): [in this window] [in a new window]   Figure 2.  Effect of dominant-negative G subunits or Gt expression on Ca2+ liberation from GH3 cell intracellular stores in response to TRH The time course of variations in [Ca2+]i levels is shown for Fura-2 loaded cells from a microscope field transfected with Gq-QL/DN (A), Gs-QL/DN (B), G13-QL/DN (C), or Gt (D). Addition of 1 µM TRH is indicated by horizontal lines on top of the traces. Continuous traces averaged point by point and their corresponding S.E.M. are shown. Averaged basal levels are signalled by dashed lines. Data correspond to cells showing fluorescence of the transfection marker (eGFP+, left) or not (eGFP–, right). Variations of [Ca2+]i levels in the cell subpopulations showing a detectable peak Ca2+ increase after visual inspection are illustrated. In all cases the number of averaged cells with respect to the total number of cells present in the field is boxed. Data from the whole cell population present in the microscope field are shown in the insets of panel A. Averaged values from all data points before TRH addition and that of the initial maximum are indicated. Analogous results were obtained in two additional experiments.

Specificity of dominant-negative G subunits on TRH-induced Ca²⁺ response

The results presented above indicate that expression of the dominant-negative form of G_q constitutes an efficient way to suppress the Gq-dependent initial Ca²⁺ response to TRH of the GH₃ cells. Whereas coupling of endogenous and heterologously expressed TRH-Rs to Gq for activation of PLC-mediated PIP₂ hydrolysis has been widely documented, it has been also reported that in GH cells the TRH-R can interact with a number of different G-proteins that may include G₁₃, G_i and G_s or a G_s-like protein (Storey et al. 2002; reviewed by Gershengorn & Osman, 1996). To check the possible specificity of the dominant-negatives we also studied the Ca²⁺ response in cells expressing the QL/DN variants of G_s and G₁₃. The elevations of [Ca²⁺]_i in response to TRH remained the same in cells expressing either G_s-QL/DN (Fig. 2B) or G₁₃-QL/DN (Fig. 2C) as compared with cells from the same microscope field lacking eGFP expression. Similar results were obtained in cells expressing transducin subunits (Fig. 2D; see also below). Furthermore, as in control cells transfected with pcDNA3B (see above) or in untransfected cells (not shown), most of the cells expressing dominant-negatives of G_s or G₁₃ showed significant increases in peak [Ca²⁺]_i. This indicates that whereas coupling of TRH-R to G_q/11 is indispensable for the TRH-evoked Ca²⁺ response, coupling to a G-protein of the G_s or G₁₃ type is not required for this effect.

Effect of different dominant-negative G subunits on TRH-induced inhibition of endogenous r-ERG currents in GH₃ cells

To investigate the transduction cascade linking the TRH-R to r-ERG channel inhibition we always used perforated-patch conditions to minimize cell dialysis and to preserve intact the intracellular components necessary for the hormonal response. To isolate the r-ERG current present in GH₃ cells and to quantify its inhibition by TRH, high-K⁺ low-Ca²⁺ extracellular solutions and established voltage protocols were used. Thus, due to the fast inactivation of ERG channels at depolarized potentials and the presence of several outwardly rectifying voltage- and calcium-dependent K⁺ currents in GH₃ cells, currents were studied during hyperpolarization pulses to –100 mV from a holding potential of –10 mV (see Methods). High-K⁺ low-Ca²⁺ extracellular solutions were also used to increase the amplitude of the inwardly rectifying r-ERG currents and to reduce Ca²⁺ currents and activation of Ca²⁺-dependent K⁺ currents (Bauer et al. 1990, 1999; Barros et al. 1992, 1997; Weinsberg et al. 1997). Furthermore, since under these conditions the TRH-induced current inhibition is almost exclusively exerted on ERG currents, 5 µM of the ERG-specific blocker E-4031 (a concentration that totally blocks the r-ERG current) was added at the end of the experiments to subtract the E-4031-insensitive currents from the initial ones and compare the difference between the TRH- and E-4031-blocked currents in every individual cell (see Gómez-Varela et al. 2003b). Representative examples of TRH effects on r-ERG currents in cells transfected with different G-protein subunit variants are shown in Fig. 3. Only successfully expressing cells identified by their eGFP fluorescence were used for recording. As shown in Figs 3A and 4, the inhibitory effect of TRH on the E-4031-sensitive current was not modified by the transfection procedure. Thus, a value of 76.8 ± 2.7% (n = 20) was obtained in cells transfected with eGFP and pcDNA3B plasmid lacking any G-protein coding insert, as compared with the 78.6 ± 5.4% (n = 8) inhibition obtained in cells showing no detectable eGFP expression. Similar inhibitions have been previously reported under identical conditions using untransfected cells (Gómez-Varela et al. 2003b). Most importantly, the TRH-induced inhibition was the same in cells expressing dominant-negative G_q-QL/DN (74.8 ± 6.3%, n = 6). It is important to note that failure to significantly modify the TRH-induced inhibition of r-ERG was not due to lack of efficient expression of G_q-QL/DN, since the early increases in Ca²⁺-dependent K⁺ currents induced by TRH (a Gq-dependent and PLC/IP₃/Ca²⁺-related effect, see above) were absent in the same cells (Fig. 3B). Apart from adding further support to specificity of the G_q-QL/DN for the Ca²⁺ response, this indicates that whereas the TRH receptor certainly uses a G_q/11 protein for transduction of the Ca²⁺ signal during the initial response to the hormone, this G-protein is not involved in the TRH-induced inhibition of endogenous r-ERG currents in GH₃ cells.

View larger version (32K): [in this window] [in a new window]   Figure 3.  Effect of dominant-negative G subunits and Gt on TRH-induced inhibition of endogenous r-ERG currents in GH3 cells The time course of relative r-ERG current reduction by TRH and E-4031 is shown for six different cells transfected with vector pcDNA3B (A), or with plasmids encoding the dominant-negative mutants of Gq (B), G13 (C), RhoA (D) and Gs (E), or the subunit of transducin (F). Currents were recorded during 1 s hyperpolarizing pulses to –100 mV from a holding potential of –10 mV. The hyperpolarization step was preceded by a 100 ms ramp from 0 to –50 mV (Gómez-Varela et al. 2003b). Current estimations were performed from total inward charge during the hyperpolarization steps at –100 mV as described in Methods. Averaged charge values before any addition and those corresponding to the minimum following addition of 5 µM E-4031 were considered as 0 and 100%, respectively. Filled circles correspond to the current traces shown above. The first one or two data points following addition of TRH, when total inward currents become transiently enhanced by activation of Ca2+-dependent K+ channels due to massive liberation of Ca2+ from intracellular stores, have been deleted for clarity. Perfusion of 1 µM TRH and addition of E-4031 to the recording chamber are indicated. Values for the data in the absence and presence of TRH are indicated by horizontal dashed lines in the time courses. A continuous membrane current recording at a holding potential of –30 mV around the time of hormone addition is shown in B and marked with a thick arrow. Note the total absence of transient Ca2+-dependent K+ currents following TRH addition in the same cell showing a clear reduction of r-ERG currents. Similar results were obtained in the five additional cells expressing Gq-QL/DN.

It has been shown that the activation of G₁₃- (and G_q-) dependent pathways is able to signal to different effectors through the small G-protein RhoA (Seasholtz et al. 1999; Sah et al. 2000; Dutt et al. 2002; Vogt et al. 2003). Expression of the dominant-negative RhoA-T19N also significantly attenuated r-ERG modulation by TRH, leaving a mean inhibition of 53.4 ± 6.3% (n = 11, P < 0.01 versus control, Welch and Mann-Whitney tests; Figs 3D and 4). Again, a considerable dispersion of the data was obtained in this case. Nevertheless, these data confirm previous results in GH₄C₁ cells and suggest that a transduction cascade involving G₁₃ (but not G_q) and Rho may participate in the inhibition of ERG channels by TRH.

Previous results in GH₃ cells showed a modification of the TRH-induced effects on r-ERG in cells treated with the G_s-modifying agent cholera toxin (Barros et al. 1994; Bauer et al. 1994). Although a coupling of TRH-R to G_s with subsequent activation of adenylyl cyclase has been reported in GH₃ cells, evidence against (i) stimulation of the enzyme by TRH and (ii) specific down-regulation of G_s following long-term expositions of GH₃ cells to TRH has been obtained also (reviewed in Gershengorn & Osman, 1996). Furthermore, a cholera toxin-dependent degradation of G_s has been demonstrated in GH₃ cells (Chang & Bourne, 1989), but it is also known that cholera toxin treatment can cause a significant reduction in the number of TRH-Rs (Yajima et al. 1988). This prompted us to check whether antagonization of the Gs pathway with dominant-negative G_s could cause any effect on the TRH-induced r-ERG current reductions. As shown in Figs 3E and 4, a prominent reduction of the inhibitory effect of TRH on r-ERG was observed in the presence of dominant-negative G_s-QL/DN. In this case, mean inhibition amounted only to 33 ± 3.9% (n = 10, P < 0.0001 versus control, Student's t and Mann-Whitney tests). This value is also significantly smaller than that obtained in the presence of RhoA-T19N (P < 0.02, Mann-Whitney test). It is important to note that in the presence of dominant-negative G_s not only is the reduction of the TRH-induced inhibition the biggest observed, but also the dispersion of the data is notably reduced (Fig. 4). This supports the conclusion that G_s plays a crucial role on the inhibitory effects of TRH in GH₃ cells.

Effect of transducin subunit expression on the TRH-induced inhibition of r-ERG in GH₃ cells

It has been recognized that receptors that activate G₁₃ also couple to G_q and G₁₁. It is also well known that G₁₃ and G_q/11 signals induce Rho activation and subsequent cellular responses by inducing GDP–GTP exchange in Rho guanine nucleotide exchange factors in response to extracellular stimuli (Seasholtz et al. 1999; Sah et al. 2000; Dutt et al. 2002; Vogt et al. 2003). Thus it seemed surprising that dominant-negative G_s (but not G_q), G₁₃ and Rho share antagonism on the TRH-induced effects on r-ERG. To check the possibility that reduction of TRH-induced inhibition involves a component common to G_s and G₁₃ but not available in heterotrimeric G_q, we studied the consequences of expressing transducin subunits (G_t), a scavenger of ß dimers after they are released from G-proteins by receptor stimulation (Crespo et al. 1994; Faure et al. 1994; Palomero et al. 1998). The inhibition of r-ERG by TRH was strongly attenuated by G_t (33.5 ± 4.4%; n = 14, P < 0.0001 versus control, Student's t and Mann-Whitney tests; Figs 3F and 4) up to levels equivalent to those observed with dominant-negative G_s. This effect of G_t was totally specific, since both the percentage of cells showing a detectable increase in peak [Ca²⁺]_i and the magnitude of the TRH-induced Ca²⁺ response remained the same regardless of the presence or the absence of G_t (Fig. 2D). As discussed below, this suggests that free ß subunits released from G_s (and perhaps shared by G₁₃) heterotrimers may be responsible for the TRH-induced inhibition of endogenous r-ERG currents in GH₃ cells.

Generation of a HEK293 cell clone permanently expressing HERG channels and TRH receptors and characterization of the TRH response

Unlike the strong and quite reproducible current inhibitions induced by TRH on the endogenous ERG current, only modest effects of the hormone have been reported on other kinetic characteristics of ERG either endogenous or heterologously expressed in GH₃ cells (Barros et al. 1992; Bauer et al. 1998; Schledermann et al. 2001). This fact and the sometimes variable effect of dominant negatives in the individual cells prevented us from reaching any consistent conclusion about the way in which different G proteins could be affecting other current parameters in these cells.

HEK293 cells permanently expressing HERG constitute an interesting and widely used model system to study easily the biochemical and electrophysiological properties of the channel (Zhou et al. 1998b). For this reason, we initially isolated several cell clones expressing HERG. Subsequently, they were cotransfected with a plasmid containing the TRH-R cDNA (de la Peña et al. 1992). Single colonies were selected and tested for both HERG current under voltage-clamp and TRH-induced Ca²⁺ responses with the fluorescent Ca²⁺ indicator Fura-2 (see Methods). A cell line named HEK-H36/T1 showing high HERG current density and also systematic Ca²⁺ increases when exposed to TRH was used for the experiments reported here. As shown in Fig. 7, the HEK-H36/T1 cells showed voltage-activated K⁺ currents typical of HERG under the perforated-patch conditions chosen to maintain as intact as possible the hormonal responses. Apart from HERG currents, we frequently detected also other outward K⁺ currents during the depolarization steps. The magnitude and the degree of inactivation of these currents during the depolarization pulses were highly variable. Nevertheless, their contribution was negligible along the tail current time course, as evidenced by their absence after treating the cells with the specific HERG inhibitor E-4031 (Fig. 5A). Thus only HERG current characteristics would be referred to by limiting the analysis to tail currents. Addition of TRH to HEK-H36/T1 cells loaded with Fura-2 triggered a transient increase in cytoplasmic Ca²⁺ that slowly declined to basal values upon hormone washout. On the average, the amplitude of this Ca²⁺ response was equivalent to that elicited in the same cells by carbachol (data not shown) probably acting through the endogenous muscarinic receptors of HEK293 cells. The TRH-induced Ca²⁺ responses were absent in the cell clones containing HERG channels but not coexpressing TRH-Rs.

View larger version (28K): [in this window] [in a new window]   Figure 7.  Effect of dominant-negative G subunits and Gt expression on Ca2+ liberation from HEK-H36/T1 cell intracellular stores in response to TRH Protocols and data evaluation were performed as described in Fig. 2. Only data from the cell subpopulations showing a visually detectable peak Ca2+ increase are plotted. In all cases the number of averaged cells respect to the total number of cells present in the field is boxed. Analogous results were obtained in two additional experiments.

View larger version (26K): [in this window] [in a new window]   Figure 5.  Effect of TRH on HERG current activation in HEK-H36/T1 cells A, effect of TRH on voltage dependence of HERG current availability. The voltage dependence of activation was studied in the absence (Control) or the presence of 100 nM TRH (TRH) by varying the amplitude of a 1 s prepulse from a holding potential of –80 mV, and measuring the magnitude of the peak tail current at a constant repolarizing voltage of –50 mV. Membrane currents recorded in the presence of TRH plus 1 µM E-4031 (E-4031) are also shown for comparison. Superimposed current traces for depolarizing steps between –50 and +40 mV (Control) or –40 and +60 mV (TRH and E-4031) are shown on the left. Control and TRH traces obtained after subtracting the E-4031-resistant currents are also shown on the right. Pulses were applied once every 20 s. Current traces have been subtracted for leak. Recording of currents in the presence of TRH started 2 min after introducing the neuropeptide in the recording chamber. Averaged fractional activation curves obtained before and after addition of TRH are shown at the bottom. Normalized outward tail current magnitudes at the peak are plotted versus test pulse potential (Em). Data normalized to maximum of tail current magnitude without TRH are presented. The continuous lines correspond to Boltzmann curves h(V) = Imax(1/(1 + exp(V – V)/k)), that best fitted the data. The relative position of the V values and their numeric magnitude are indicated in the graph. B, effect of TRH on HERG current activation rate. The time course of voltage-dependent activation was studied by varying the duration of a depolarizing prepulse and measuring the magnitude of the peak tail current at a constant repolarizing voltage of –50 mV. Families of currents at a depolarizing potential of +40 mV are shown for a cell before (Control) and after adding 100 nM TRH (TRH). Current traces corresponding to depolarization steps of 0, 20, 40, 80, 160, 320, 640, 1280, 2560 and 5120 ms are shown superimposed. The dependence of activation rates on depolarization membrane potential is shown at the bottom. The magnitude of the peak tail current upon repolarization was determined from recordings as shown at the top. The time necessary to attain half-maximum current magnitude at different depolarization potentials is plotted in the absence or the presence of TRH.

Due to the slow activation and deactivation kinetics of HERG channels, no steady-state conditions would be expected within 1 s depolarizations. A possible cause of the shift in activation voltage dependence could be a displacement of the V values to more depolarized potentials due to a slower activation rate leading to a less steady-state condition (Schönherr et al. 1999; Viloria et al. 2000). In fact, it is known that the activation rate of HERG channels in Xenopus oocytes is slowed by activation of coexpressed TRH-Rs (Barros et al. 1998). For this reason, we also tested the effects of TRH on HERG activation rates in HEK-H36/T1 cells using an indirect envelope of tail currents protocol (Barros et al. 1998; Viloria et al. 2000). As shown in Fig. 5B, the time required to attain a half-maximum current magnitude was increased by TRH near an order of magnitude between +40 and +60 mV. Thus the difference in the inflection potentials of the I–V curves could be due, at least in part, to the different activation rates around the V values. Nevertheless, an 18 mV shift from –22.5 ± 2.2 to –4.1 ± 2.8 mV (n = 6) was also detected in response to TRH when the I–V curves were generated from tail currents following long depolarization steps of 10 s duration (Fig. 6A). Finally, the position of the curves under real steady-state was extrapolated as the mean of those obtained from hyperpolarized (–80 mV) and depolarized (+40 mV) holding potentials (Schönherr et al. 1999; Viloria et al. 2000). In this case a 15 mV shift in V (from –25.0 to –10.0 mV, n = 2) was obtained (Fig. 6B). Altogether, this suggests that two distinct effects on activation contribute to the shifts when studied with 1 s short depolarization steps: a genuine shift of the voltage dependence of activation and a marked slowing of activation rates moving the channels further away from steady-state conditions.

View larger version (31K): [in this window] [in a new window]   Figure 6.  Effect of TRH on HERG activation voltage dependence under steady-state conditions A, effect of TRH on activation voltage dependence studied using long depolarization steps of 10 s. Tail currents upon repolarization to –50 mV were obtained following 10 s depolarizations to different voltages as indicated at the top. Only the end of the depolarizing steps and the tail currents at –50 mV are shown for clarity. Traces correspond to a cell before (Control) and 3 min after introduction of 100 nM TRH (TRH). Averaged I–V curves before and after the hormonal treatments are shown at the bottom. The magnitude of the peak tail current upon repolarization was determined from recordings as shown at the top and normalized to maximum of tail current magnitude without TRH. B, effect of TRH on HERG steady-state voltage dependence of activation. Steady-state voltage dependence of activation was studied following the protocols shown in the two upper panels, with a prepulse of varying magnitude and a duration ranging from 1 to 10 s, followed by a pulse test to –50 mV. Holding potentials of +40 mV to keep the channels fully open and –80 mV to hold them fully closed were used as indicated. Families of currents during the test pulse for Control and TRH-treated cells following 10 s prepulses to different potentials are shown. Fractional activation curves before and after TRH treatment are shown at the bottom. Open and filled symbols correspond to data obtained from –80 and +40 mV holding voltages, respectively. Data obtained at prepulse duration of 1 s (circles), 5 s (squares), and 10 s (triangles) are plotted. In every curve current values were normalized to tail current magnitude in response to the more depolarized potential of the prepulse series. The dashed lines represent the deduced position of the activation curves under steady-state conditions obtained as a mean of those corresponding to both holding voltages and prepulses of 10 s duration. The V values for the steady-state curves are indicated by arrows in the graphs.

The clear effects of TRH on HERG opening and closing contrast with the almost complete absence of hormone-induced modifications on inactivation properties. Thus only a slight but non-significant acceleration of inactivation rates was observed upon addition of TRH. Furthermore, both control and TRH-treated cells showed identical rates of inactivation recovery when the time course of the initial current increase in response to hyperpolarizing pulses was compared (not shown).

Effect of dominant-negative G subunits and G_t on TRH-induced Ca²⁺ responses and modifications of HERG I–V curves in HEK-H36/T1 cells

The influence of the dominant-negatives and G_t expression on the hormonal effects in HEK-H36/T1 cells was studied following procedures analogous to those described for endogenous channels in adenohypophysial GH₃ cells. Again, dominant-negative G_q, but not G_t or dominant-negative G_s, G₁₃ and RhoA, reduced TRH-induced increases of cytoplasmic Ca²⁺ in the HEK-H36/T1 cells (Fig. 7). The modulation of HERG channels by TRH in HEK-H36/T1 cells was not changed by the transfection procedure used for dominant-negative expression (Control in Fig. 8). Surprisingly, the TRH-induced HERG current inhibition at positive voltages was not significantly altered in the presence of dominant-negative G₁₃, RhoA or G_s, but was strongly reduced by G_q-QL/DN or G_t expression (Fig. 8A and C). On the other hand, the positive shifts in activation voltage dependence were reduced by RhoA-TN and nearly abolished by G₁₃-QL/DN (Fig. 8A and B). Although the shift in the I–V curves was almost absent in G_q-QL/DN-transfected cells, the basal position of the curves before adding hormone was clearly displaced to positive voltages in the cells expressing the dominant-negative form of G_q (Fig. 8A). This complicates the interpretation of the dominant-negative G_q influence on the hormonal effect in the I–V relation. Finally, expression of the ß dimer scavenger G_t left unaltered the TRH-induced voltage dependence shift in 10 of 14 tested cells, but blocked completely the reductions in maximal tail current magnitude triggered by the hormone. These results not only indicate a variation of the dominant-negative effects in different cellular and/or channel background, but also that these effects are differently manifested as a function of the channel parameter being considered.

View larger version (29K): [in this window] [in a new window]   Figure 8.  Effect of dominant-negative G subunits and Gt on TRH-induced modifications of HERG I–V curves in HEK-H36/T1 cells A, effect of G subunit expression on HERG activation voltage dependence. Voltage dependence of activation was studied in the absence (open symbols) or the presence (filled symbols) of 1 µM TRH by varying the magnitude of a 1 s depolarizing pulse as detailed in the legend of Fig. 5A. Data normalized to maximum of tail current magnitude without TRH are shown. V values obtained from the Boltzmann curves that best fitted the averaged data (continuous lines) and the number of recorded cells are indicated in the graphs. B, comparison of TRH-induced shifts on activation voltage dependence in cells expressing different G subunits. Data from the same cells used to generate the averaged I–V curves shown in panel A were used. Values of TRH-induced shifts from the different individual cells were averaged and are shown in the histogram. C, effect of G subunit expression on TRH-induced inhibition of HERG currents at positive voltages. Data from the same cells used to generate the averaged I–V curves shown in panel A were used. Values of TRH-induced reductions in maximal currents from the different individual cells were averaged and are shown in the histogram. Statistically significant variations versus controls in Student's t or Mann-Whitney tests are indicated in B and C.

	Discussion

Top Abstract Introduction Methods Results Discussion References

In principle, the observed specificity of the dominant-negatives seems rather surprising according to the proposed mechanism for negative dominance in the G-QL/DN mutants. Introduction of the QL/DN mutations shifts nucleotide binding specificity from guanine nucleotides to xanthine nucleotides. Because the low concentrations of xanthine nucleotides in vivo, essentially nucleotide-free G-QL/DN proteins would exist in cells, and these would form stable complexes with cognate receptors and inhibit them by competing with endogenous wild-type G proteins (Yu et al. 2000). Accordingly, it could be expected that all signalling pathways lying on a given receptor became inhibited by any G-QL/DN variant interacting with it. Our results demonstrate that this is not the case for the TRH-R. The presence of a heterogeneous population of binding sites for TRH in GH₃ cells has been previously reported (Gautvik & Lystad, 1981) and two TRH-R isoforms derived from the same gene by differential splicing mechanisms have been shown to be expressed in GH₃ cells (de la Peña et al. 1992). However, no significant differences in binding or signalling properties of these receptors seem to exist (de la Peña et al. 1992; Lee et al. 1995; Gershengorn & Osman, 1996). Furthermore, the presence of two different molecular species of TRH-R could not explain the dominant-negative specificity found in HEK-H36/T1 cells in which only an isoform of the receptor is expressed. Clearly, further work will be necessary to understand the reason(s) for the observed specificity of the dominant-negative effects.

Regardless of the exact mechanism by which the negative dominance takes place, the demonstration of the functionality and specificity of the G-QL/DN variants allowed us to explore the TRH-R coupling to defined G proteins for transducing the inhibitory signal to ERG channels. Our results suggest that G_s plays a crucial role transducing the TRH signal to native r-ERG channels in GH₃ cells, since dominant-negative G_s causes the greatest reduction of the hormonal effect as well as the smallest data dispersion in individual cells. These data are consistent with previous results showing that the TRH-induced r-ERG inhibition is enhanced by short-term treatment with cholera toxin (an agent able to specifically modify G_s functionality) and nearly abolished when the treatment with the toxin is prolonged (Barros et al. 1993, 1994; Bauer et al. 1994). They will be also coherent with the reported inhibition of TRH-induced PLC stimulation in Xenopus oocytes using nucleotides antisense to G_s (de la Peña et al. 1995; but see Gershengorn & Osman, 1996). The reasons why coupling of TRH-R to G_s in GH₃ cells only modestly stimulates adenylyl cyclase (Paulssen et al. 1992; Gershengorn & Osman, 1996) and why prolonged stimulation with TRH down-regulates G_q/11 but has no effect on the G_s protein levels (Kim et al. 1994) remain to be established. As suggested previously (Barros et al. 1993, 1994; Bauer et al. 1994), it is possible that a G_s-like protein that is not G_s itself, but sensitive to cholera toxin and blocked by G_s-QL/DN expression, is involved in GH₃ cell r-ERG inhibition by TRH.

Using the dominant-negative approach we also studied the possible implication of G₁₃ and RhoA, two entities recently proposed as mediators of r-ERG modulation by TRH (Storey et al. 2002). In this case, expression of dominant-negative G₁₃-QL/DN significantly reduced the TRH-induced inhibition, although a slightly smaller effect was observed than that with G_s-QL/DN and only in around 50% of the recorded cells was the hormonal effect clearly antagonized. It is unlikely that this situation is caused by an unspecific inhibition of G_s by dominant-negative G₁₃ because smaller TRH-induced inhibitions and wider dispersion of the data were also observed with dominant-negative RhoA, a component located downstream of G₁₃ in many cellular systems and (supposedly) not directly related to G_s. On the other hand, it seems difficult to conceive a transduction mechanism involving G_s, G₁₃ and RhoA (but not G_q) either simultaneously or indistinctly. Our data following the overexpression of G_t, an agent known to sequester free G-protein ß dimers (Crespo et al. 1994; Faure et al. 1994; Palomero et al. 1998), offer a possible explanation of this apparent paradox. Thus, prominent reductions of the TRH-induced r-ERG inhibition equivalent to those obtained with dominant-negative G_s are observed in G_t