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Heterologous expression and coupling of G protein-gated inwardly rectifying K⁺ channels in adult rat sympathetic neurons

Victor Ruiz-Velasco and Stephen R. Ikeda

Received 8 July 1998; accepted after revision 14 September 1998.

	ABSTRACT

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1. G protein-gated inwardly rectifying K⁺ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole-cell patch-clamp technique.

2. Following coinjection of either GIRK1-2 or GIRK1-4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K⁺ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA-activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed.

3. NA-mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the G_i/G_o subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin-sensitive pathway suggesting coupling through G_s. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP-mediated GIRK channel currents when compared with untreated cells.

4. Application of adenosine, prostaglandin E₂ and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine M to PTX-treated neurons) failed to elicit overt GIRK channel currents.

5. GIRK channel overexpression decreased basal Ca²⁺ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA-mediated inhibition of Ca²⁺ channels was significantly attenuated.

6. In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor-G protein-effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.

	INTRODUCTION

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It is well established that G protein-mediated signal transduction pathways play a major role in neuronal excitability and cardiac rate (for a review see Clapham & Neer, 1997). Neurotransmitters acting via G protein-coupled receptors cause the dissociation of the heterotrimeric G proteins into the G-subunit and the G-complex, both of which are capable of regulating a number of effectors. For instance, the G-dimer mediates the voltage-dependent inhibition of N- and P/Q-type Ca²⁺ channels, as well as activation of G protein-gated inwardly rectifying K⁺ (GIRK) channels (Logothetis et al. 1987; Kofuji et al. 1995; Ikeda, 1996; Herlitze et al. 1996). Although the G-dimer appears to be the active moiety underlying voltage-dependent inhibition of N-type Ca²⁺ channels, the exact molecular events associated with the modulation of these channels remain unknown. A particularly interesting question we wanted to address is that of coupling specificity between neurotransmitter receptors and two G-modulated effectors, N-type Ca²⁺ and GIRK channels.

Consequently, the purpose of the present study was to test whether GIRK channels (GIRK1 (Kir 3.1), GIRK2 (Kir 3.2) and GIRK4 (Kir 3.4)) could be heterologously expressed in acutely isolated rat superior cervical ganglion (SCG) neurons and whether the expressed channels couple to native signalling pathways. Introduction of another G-modulated effector, not normally expressed in this cell type, would thus aid in delineating factors involved in receptor-G protein specificity and N-type Ca²⁺ channel modulation. The hypothesis to be tested was that heterologously expressed GIRK channels would couple to signal transduction elements normally associated with N-type Ca²⁺ channel modulation. Moreover, we reasoned that introduction of an additional G-binding effector might influence N-type Ca²⁺ channel modulation, possibly through competition for signalling elements. Our results show that coexpression of either GIRK1 and GIRK2 (GIRK1-2) or GIRK1 and GIRK4 (GIRK1-4) leads to greater channel activation with the ₂-adrenergic agonist noradrenaline (NA) than expression of any of the GIRK channel subunits alone. The data also indicate that neurons coexpressing GIRK channels exhibit neurotransmitter- receptor coupling via members of the G_i/G_o and G_s subfamilies. In this expression model, basal Ca²⁺ current facilitation in GIRK1-2 and GIRK1-4-expressing neurons was decreased significantly when compared with uninjected neurons. Finally, the NA-mediated inhibition of Ca²⁺ currents in GIRK-expressing neurons was significantly attenuated. Our results suggest that competition for G-subunits by different ion channels (N-type Ca²⁺ and GIRK channels) can influence modulation and apparent receptor- effector coupling specificity. Portions of this work have been published previously in abstract form (Ruiz-Velasco & Ikeda, 1998).

	METHODS

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Neuron isolation

Single neurons from adult rat SCG were prepared using the method described previously (Ikeda, 1997). The experiments carried out were approved by the Institutional Animal Care and Use Committee (IACUC). Briefly, male Wistar rats (175-225 g) were killed by decapitation using a laboratory guillotine without prior anaesthesia and the SCG dissected in cold Hanks' balanced salt solution. The ganglia were desheathed and multiple parallel slits (1 mm apart) were made perpendicular to the long axis prior to incubation with 0·7 mg ml^-1 collagenase Type D, 0·3 mg ml^-1 trypsin (both from Boehringer Mannheim) and 0·1 mg ml^-1 DNase Type I (Sigma) for 1 h in a shaking water bath at 35°C. Following the incubation period, the cells were dissociated in a culture flask by vigorous shaking. The dispersed neurons were centrifuged twice for 6 min at 50 g and then resuspended in minimal essential medium (MEM; Mediatech, Inc., Herndon, VA, USA) supplemented with 10 % fetal calf serum (Atlanta Biologicals, Atlanta, GA, USA), 1 % glutamine and 1 % penicillin- streptomycin solution (both from Mediatech, Inc.). The neurons were then plated into 35 mm tissue culture plates coated with poly-L-lysine and stored in a humidified incubator containing 5 % CO₂ in air at 37°C.

cDNA microinjection

Microinjection of cDNA plasmids was performed with an Eppendorf 5246 microinjector and 5171 micromanipulator (Madison, WI, USA) 3-5 h after plating, as described previously (Ikeda, 1997). Plasmids coding for human GIRK1 and GIRK4 (subcloned in pcDNA3; Invitrogen Corp., Carlsbad, CA, USA) and human GIRK2 (subcloned in pcDNA1; Invitrogen Corp.) were stored in TE buffer (10 mM Tris, 1 mM EDTA, pH 8·0) and diluted to a final concentration of 0·01-0·1 µg µl^-1 per K⁺ channel subunit in TE buffer. A plasmid (pEGFP-N1; Clontech Laboratories, Palo Alto, CA, USA; final concentration, 5 ng µl^-1) coding for an 'enhanced' jellyfish green fluorescent protein (EGFP) was mixed with the GIRK plasmids and served as a coinjection 'marker' for identification of neurons receiving a successful nuclear injection. Neurons expressing EGFP were identified 12-24 h later using an inverted microscope (Diaphot 300; Nikon, Tokyo, Japan) equipped with an epifluorescence unit (XF100 filter cube; Chroma Technologies, Brattleboro, VT, USA).

Electrophysiology and data analysis

GIRK and Ca²⁺ channel currents were recorded using the whole-cell variant of the patch-clamp technique (Hamill et al. 1981). Patch pipettes were pulled from borosilicate glass capillaries (Corning 7052; Garner Glass Claremont, CA, USA) on a P-97 Flaming-Brown micropipette puller (Sutter Instrument Co., San Rafael, CA, USA), coated with Sylgard (Dow Corning, Midland, MI, USA) and fire polished on a microforge. Whole-cell currents were acquired with a patch-clamp amplifier (Axopatch 200A, Axon Instruments, Foster City, CA, USA), analog filtered at 1-2 kHz (-3 dB; 4-pole Bessel) and digitized using custom-designed software (S3) on a Macintosh Quadra 700 computer (Apple Computer, Cupertino, CA, USA) equipped with a 12-bit analog-to-digital converter board (MacADIOS II; G.W. Instruments, Bedford, MA, USA). Cell membrane capacitance and series resistance (80-85 %) were electronically compensated. All experiments were performed at room temperature (21-24°C). Data and statistical analysis was performed with Igor (Lake Oswego, OR, USA) and GB-Stat PPC (Silver Spring, MD, USA) software packages, respectively, using one-way analysis of variance (ANOVA) with P < 0·05 considered statistically significant. Graphs and current traces were produced with Igor and Canvas (Deneba Software, Miami, FL, USA) software packages.

The time course plots indicate the maximal inward current amplitude obtained during the voltage ramp protocol. Maximal inward current typically occurred between -135 and -125 mV. Except where noted, the current traces were obtained by digitally subtracting currents obtained before from those obtained after application of agonists. Some GIRK1-4-injected cells exhibited basal activation prior to agonist application. In order to avoid an underestimate of peak current, digital subtraction was not performed under these conditions. Data are presented as means ± S.E.M.

Solutions and drugs

For recording GIRK channel currents, the pipette solution contained (mM): 135 KCl, 11 EGTA, 1 CaCl₂, 2 MgCl₂, 10 Hepes, 4 Mg-ATP, and 0·3 Na₂ATP; pH 7·2 and 299-302 mosmol kg^-1. The external solution consisted of (mM): 130 NaCl, 5·4 KCl, 10 Hepes, 10 CaCl₂, 0·8 MgCl₂, 15 glucose, 15 sucrose, and 0·0003 tetrodotoxin (TTX); pH 7·4 and 317-319 mosmol kg^-1. In experiments in which reversal potentials (Vrev) were examined, external K⁺ was replaced by an equal amount of Na⁺. For recording Ca²⁺ currents, the pipette solution contained (mM): 120 N-methyl-D-glucamine, 20 tetraethylammonium hydroxide (TEA-OH), 11 EGTA, 10 Hepes, 10 sucrose, 1 CaCl₂, 4 Mg-ATP, 0·3 Na₂ATP, and 14 Tris creatine phosphate; pH 7·2 with methanesulphonic acid and HCl and 299-302 mosmol kg^-1. The external solution consisted of (mM): 140 methanesulphonic acid, 145 TEA-OH, 10 Hepes, 15 glucose, 10 CaCl₂, and 0·0003 TTX; pH 7·4 and 312-319 mosmol kg^-1.

Stock solutions of noradrenaline bitartrate salt, adenosine (Ado), cholera toxin (CTX) (Sigma), vasoactive intestinal polypeptide (VIP), somatostatin (SS; Bachem, Torrance, CA, USA), pertussis toxin (PTX; List Biological Laboratories, Inc., Campbell, CA, USA) and oxotremorine M (oxotremorine methiodide, Oxo-M; Research Biochemicals Int., Natick, MA, USA) were prepared in H₂O. Prostaglandin E₂ (PGE₂) (Cayman Chemical Co., Ann Arbor, MI, USA) was prepared in ethanol at a stock concentration of 10 mM. All drugs were diluted in the external solution from stock solutions to their final concentrations just prior to use. The external solution containing SS was prepared in 0·05 mg ml^-1 cytochrome c (Sigma) to minimize the binding of SS to the capillary columns used for drug delivery (Boland et al. 1994). Neurons pretreated with PTX or CTX were incubated overnight (12-14 h) in tissue culture medium containing 500 ng ml^-1 of the toxin.

Application of drugs to the neuron under study was performed by positioning a custom-designed gravity-fed perfusion system 100 µm from the cell. The end of this perfusion system consisted of a single fused silica gas chromatography column (i.d., 210 µm) that was attached in series to seven parallel columns of the same diameter. The flow of the seven columns was controlled with a two-way stopcock and had a switching time of approximately 200 ms. To wash off drugs as well as to avoid flow-induced artifacts, the capillary column containing normal external solution was kept open continuously until the time at which the desired solution was employed.

	RESULTS

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Heterologous expression of GIRK1, 2, 4, 1-2 and 1-4 in rat SCG neurons

Although GIRK1 and GIRK2 are expressed in neuronal tissue (Lesage et al. 1994; Karschin et al. 1996), the rat SCG neurons used in this study do not express overt functional GIRK-type channels. In addition, both GIRK1 and GIRK4 (a homologue of GIRK2) subunits form the heteromultimeric muscarinic-gated atrial channel (Krapivinsky et al. 1995). In neurons previously injected with only the EGFP cDNA, voltage ramps from -140 to -40 mV (200 ms) elicited from a holding potential of -60 mV evoked little discernible inward current. Furthermore, exposure of these neurons to NA (10 µM) did not induce additional inward current (Fig. 1A). Application of NA to neurons previously injected with GIRK1 cDNA resulted in inward current in only 4 of 16 cells tested. Maximal inward currents during the voltage ramp ranged from 80 to 340 pA for those neurons responding to NA (Fig. 1B). In GIRK2-injected cells, application of NA activated strongly rectifying inward current (80-500 pA) in 5 of 7 cells. Exposure of NA to neurons injected with GIRK4 cDNA activated current in 4 of 5 cells tested, with inward currents ranging from 200 to 740 pA (Fig. 1D). In contrast, application of NA to neurons previously injected with both GIRK1 and GIRK2 or GIRK1 and GIRK4 cDNA (hereafter denoted GIRK1-2 and GIRK1-4) produced much greater inward current (Fig. 1E and F) when compared with neurons injected with cDNA encoding any of the GIRK subunits alone (cf. Fig. 1B -F). Furthermore, NA-induced currents were detected in every GIRK1-2- and GIRK1-4-injected neuron tested. At very hyperpolarized voltages (ca -140 to -120 mV), the ramp current-voltage (I-V) relationship exhibited a region of negative slope conductance. Following this region, the I-V curve was sigmoidal demonstrating a profound decrease in slope conductance at voltages positive to Vrev (ca -80 mV). Figure 1G summarizes the means (± S.E.M.) NA-induced inward current for neurons injected with cDNA encoding EGFP or EGFP plus the indicated GIRK subunit(s). NA-induced current amplitude was calculated by digitally subtracting current traces obtained before from those obtained after application of NA (10 µM) and then measuring the maximal inward current of the subtracted current trace. These results show that SCG neurons have minimal basal or NA-activated inward currents over the voltage range -80 to -140 mV under the recording conditions utilized and thus provide a suitable null-background for the expression of GIRK channels. Moreover, injection of GIRK1-2 and GIRK1-4 cDNAs results in reliable and robust expression of GIRK channels which readily couple to natively expressed ₂-adrenergic receptors and signal transduction elements (e.g. heterotrimeric G proteins).

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Figure 1. Heterologous expression of GIRK1, 2, 4, 1-2 and 1-4 in SCG neurons Whole-cell inwardly rectifying K+ currents evoked by 200 ms voltage ramps from -140 to -40 mV from a holding potential of -60 mV. Cells were microinjected with cDNA encoding the 'marker' EGFP alone (A), GIRK1 (B), GIRK2 (C), GIRK4 (D), GIRK1-2 (E), and GIRK1-4 (F), together with EGFP. Traces shown are superimposed currents from the same neuron before (Control) and after application of 10 µM NA. Dashed lines indicate the zero current level; scale bar applies to all current traces. G, summary graph showing the mean (± S.E.M.) peak normalized current activated by NA for EGFP alone, GIRK1, GIRK2, GIRK4, GIRK1-2 and GIRK1-4. Numbers in parentheses indicate the number of experiments.

Characterization of GIRK channels expressed in SCG neurons

The biophysical properties of channels arising from injection of GIRK1-2 cDNA are shown in Fig. 2. GIRK channels are highly selective for K⁺ ions, thus varying the external [K⁺] should result in a shift of V_rev consistent with that predicted by the Nernst equation. Figure 2A (left panel) depicts the I-V relationships for NA-induced currents in a GIRK1-2-injected neuron recorded in 5·4, 20 and 60 mM external [K⁺] (traces shown are from the same neuron). Increasing the external [K⁺] resulted in a shift of V_rev and the region of negative slope conductance to more depolarized potentials and produced an increase in maximal slope conductance. It should be noted that the increase in slope conductance at high external [K⁺] was probably underestimated due to desensitization during the NA application (see below). A summary plot of mean V_rev values versus the logarithm of external [K⁺] is shown in Fig. 2A (right panel). The slope of the data (continuous line), as calculated from linear regression, was 55 mV per 10-fold change in external [K⁺], a value close to that predicted by the Nernst equation for a K⁺-selective ion channel (58 mV at 20°C).

Heterologously expressed GIRK channels were tested for their susceptibility to block by external Ba²⁺ (Fig. 2B). Although specific blockers of GIRK channels have yet to be discovered, external Ba²⁺ has been shown to efficiently block the flow of K⁺ through GIRK channels (Dascal et al. 1993). Figure 2B shows current traces obtained from a GIRK1-2-injected neuron under control conditions (i.e. in the absence of NA) and in the presence of 10 µM NA prior to and following the application of external solution containing 1 mM Ba²⁺. The NA-induced current was abolished by the application of external Ba²⁺. Moreover, the inward current amplitude in the presence of NA and Ba²⁺ was less than that obtained under control conditions, possibly indicating a small basal activation of the expressed GIRK channels in the absence of agonist. Similar responses were observed in a total of seven neurons.

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Figure 2. K+ selectivity and Ba2+ sensitivity of GIRK1-2 channel currents A, left panel: inwardly rectifying K+ currents evoked by 200 ms voltage ramps from -140 to -40 mV from a holding potential of -60 mV for 5·4 mM external K+ (K); from -130 to -30 mV from a holding potential of -50 mV for 20 mM K; and from -110 to 0 mV from a holding potential of -20 mV for 60 mM K. Right panel shows reversal potential (Vrev) values as a function of external K+ concentration, with Na+ replacing K+. Each data point represents the mean (± S.E.M.) and points were fitted with a straight line. B, superimposed current traces from the same neuron of NA-activated GIRK1-2 currents before and after application of 1 mM Ba2+ to the external solution. Dashed lines indicate the zero current level.

Figure 3A and B illustrates step current traces recorded from a GIRK1-2-expressing neuron in the absence and presence of 10 µM NA, respectively. Neurons were maintained at a holding potential of -60 mV and step currents evoked every 3 s by 50 ms square pulses to test potentials of -160 to -40 mV. In the absence of NA, the inward currents were small and exhibited no apparent time- or voltage dependence (Fig. 3A). In contrast, currents evoked in the presence of NA were larger, demonstrated time-dependent activation over the voltage range -160 to -40 mV, and produced outward tail currents upon repolarization to -60 mV (Fig. 3B). I-V plots of the current traces depicted in Fig. 3A and B are shown in Fig. 3C. The plots show that the I-V relationship was relatively linear in the absence of NA (open circles), but displayed marked inward rectification following exposure to 10 µM NA (closed circles). These results are typical of those obtained from twelve different neurons. The time course of the NA-induced inward current is shown in Fig. 3D. Currents were evoked every 5 s by a 200 ms voltage ramp from -140 to -40 mV from a holding potential of -60 mV. The maximal current obtained during the ramp is plotted versus time. Upon application of NA (filled horizontal bars), the inward current activated rapidly, usually reaching a maximal amplitude within 5-10 s. During the continued presence of the agonist, the current amplitude declined over several minutes, often reaching a plateau. Recovery from desensitization during repeated applications of agonist was variable depending on the interval between agonist application and duration of the preceding agonist application (Fig. 3D; 2nd and 3rd filled bars). This phenomenon was not further investigated; however, in the present study, NA was typically not applied for long periods to avoid the desensitization process.

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Figure 3. Current-voltage (I-V) and desensitization of NA-activated GIRK1-2 channel currents A and B, whole-cell GIRK1-2 channel currents from the same neuron before (A) and after (B) application of 10 µM NA, evoked by 50 ms pulses from a holding potential of -60 mV to potentials between -160 and -40 mV, in 10 mV increments applied every 3 s. Dashed lines indicate the zero current level. C, I-V relationships of the neuron in A and B. D, time course of the absolute peak GIRK channel current amplitudes evoked every 5 s by a 200 ms voltage ramp from -140 to -40 mV from a holding potential of -60 mV. The filled bars indicate application of 10 µM NA.

Figure 4 depicts the concentration-response relationship for NA activation of GIRK1-2 channel currents. Currents were evoked as described for Fig. 3D. A single NA concentration (0·1-30 µM) was applied for about 10 s after which the current was allowed to return to baseline before the next higher concentration of NA was applied (Fig. 4A). The concentration-response curve shown in Fig. 4B was constructed by averaging the normalized current amplitude evoked by NA from several neurons. Current amplitude was normalized to the amplitude obtained on application of 30 µM NA. Significant GIRK channel current was evoked at NA concentrations greater than 0·3 µM and reached a plateau around 10 µM. The estimated EC₅₀ of NA for GIRK1-2-expressing neurons was 1·9 µM.

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Figure 4. NA concentration-response relationship of GIRK1-2-expressing neurons A, time course of NA-activated GIRK1-2 channel currents in a representative neuron. Currents were evoked by 200 ms voltage ramps from -140 to -40 mV from a holding potential of -60 mV applied every 5 s. B, concentration-dependent GIRK1-2 channel current activation by NA. Currents were determined as in A. Peak current amplitudes were normalized to the peak current evoked by 30 µM NA. Each data point represents the mean (± S.E.M., n = 4), except for 0·1 µM NA where n = 2.

Coupling of heterologously expressed GIRK channels to natively expressed neurotransmitter receptors and G proteins

To characterize the coupling of heterologously expressed GIRK channels to natively expressed neurotransmitter receptors and signal transduction elements, GIRK1-2- and GIRK1-4-injected neurons were exposed to agents previously shown to inhibit N-type Ca²⁺ channels in these cells (Hille, 1994). Figure 5A shows the peak inward current amplitude plotted as a function of time for a GIRK1-2-expressing neuron exposed sequentially to 10 µM NA and 10 µM VIP. The values of the peak currents plotted in Fig. 5A-C were determined from non-subtracted current traces. When compared with the typical responses observed for NA, exposure of GIRK1-2-expressing neurons to VIP resulted in smaller maximal inward currents and a slower time-to-peak activation (ca 15-30 s). Following overnight treatment with PTX (500 ng ml^-1), the response to NA was nearly abolished (Fig. 5B and D). However, the effect of VIP in PTX-treated neurons was maintained and in fact was significantly greater than that seen in non-PTX-treated cells (Fig. 5B and D). In contrast, pretreatment of GIRK1-2-injected neurons with CTX (500 ng ml^-1) nearly abolished the VIP-mediated GIRK channel current activation while leaving the NA-induced current activation intact (Fig. 5C). A summary of the peak current (in pA pF^-1) activated by NA or VIP in GIRK1-2-expressing neurons which were untreated, pretreated with PTX or pretreated with CTX is shown in Fig. 5D. These results demonstrate that heterologously expressed channels readily couple to neurotransmitter receptors natively expressed in rat SCG neurons via discrete signal transduction pathways. NA activates GIRK channels via a PTX-sensitive pathway whereas VIP utilizes a CTX-sensitive pathway.

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Figure 5. Comparison of NA and VIP coupling to GIRK1-2 channels A, time course of both 10 µM NA- and 10 µM VIP-activated GIRK1-2 channel currents. Currents were evoked by 200 ms voltage ramps from -140 to -40 mV from a holding potential of -60 mV applied every 5 s. B, time course of PTX-treated (500 ng ml-1; for 12-14 h) neurons exposed to 10 µM VIP and 10 µM NA. Currents were evoked as in A. C, time course of CTX-treated (500 ng ml-1; for 12-14 h) neurons exposed to 10 µM NA and 10 µM VIP. Currents were evoked as in A. Filled bars in A-C indicate application of drugs. D, summary graph showing the mean (± S.E.M.) peak current activated by NA, NA + PTX, NA + CTX, VIP, VIP + CTX and VIP + PTX. Numbers in parentheses indicate the number of experiments. * P < 0·05, P < 0·05 vs. NA and VIP, respectively; ** P < 0·05, P < 0·05 vs. NA + CTX and VIP + CTX, respectively.

We next compared the ability of Ado, SS and PGE₂ to activate currents in GIRK-expressing neurons. These agonists were chosen as previous studies from our laboratory and others have demonstrated that receptors for these agonists are present in SCG neurons and couple to N-type Ca²⁺ channels via a PTX-sensitive G protein (Ikeda & Schofield, 1989; Ikeda, 1992; Zhu & Ikeda, 1993). Figure 6A-C shows representative time courses of GIRK1-2-expressing cells when exposed to 10 µM Ado (A), 0·1 µM SS (B) or 10 µM PGE₂ (C). Voltage ramps (-140 to -40 mV) were applied every 5 s from a holding potential of -60 mV. In each case, NA (10 µM) was applied to the neuron following application of the test agonist. The insets in Fig. 6A-C depict digitally subtracted current traces representing the current induced by application of the agonist. Although all three agonists activated GIRK channel currents, their respective magnitude was less than that produced by NA (cf. Figs 5D and 6D). Similar results were obtained in neurons injected with GIRK1-4 subunits (data not shown).

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Figure 6. Effect of other neurotransmitters on GIRK1-2 channel currents A-C, comparison of time courses of GIRK1-2 channel currents activated by 10 µM NA, with 10 µM Ado (A), 0·1 µM SS (B) and 10 µM PGE2 (C). Currents were evoked by 200 ms voltage ramps from -140 to -40 mV from a holding potential of -60 mV applied every 5 s. Insets show current traces obtained by digitally subtracting the currents before from those after application of Ado, SS and PGE2, respectively. D, summary graph showing the mean (± S.E.M.) peak current activated by Ado, PGE2, SS and 10 µM Oxo-M. Oxo-M was applied to PTX-treated (500 ng ml-1; for 12-14 h) neurons. Numbers in parentheses indicate the number of experiments.

Finally, the muscarinic acetylcholine receptor (mAChR) agonist Oxo-M was employed to determine whether receptors that couple via members of the G_q/G₁₁ subfamily activate heterologously expressed GIRK channels. Rat SCG neurons contain mAChRs which couple to ion channels (e.g. N-type Ca²⁺ channels and M-type K⁺ channels) through both PTX-sensitive and -insensitive pathways (Bernheim et al. 1992). In order to isolate the PTX-insensitive pathway, GIRK1-2-injected neurons were treated overnight with PTX (500 ng ml^-1). Under these conditions, application of Oxo-M (10 µM) failed to activate significant GIRK channel currents (Fig. 6D).

Reduced modulatory effect of G-dimers on N-type Ca²⁺ channels in GIRK-expressing neurons

After establishing the expression of functional GIRK channels, we wanted to determine whether overexpression of this G-effector would result in an alteration of the native coupling of neurotransmitter receptors to N-type Ca²⁺ channels. To this end, 0·1 µg µl^-1 of GIRK cDNA per subunit was injected. In rat SCG neurons, binding of NA to ₂-adrenergic receptors results in modulation of N-type Ca²⁺ channels via a signal transduction pathway that is: (1) membrane-delimited, (2) utilizes a PTX-sensitive G protein (G_i/G_o subfamily), and (3) results in voltage-dependent inhibition of the channels. Several neurotransmitters are known to cause this alteration of N-type Ca²⁺ currents, which results in 'kinetic slowing' of the rising Ca²⁺ currents during a depolarizing test pulse (Hille, 1994). Kinetic slowing is believed to be a result of a voltage-dependent relief of block during the test pulse. The voltage-dependent relief of Ca²⁺ channel inhibition also leads to 'facilitation'. Facilitation refers to the ratio of Ca²⁺ current amplitude determined from the test pulse (+10 mV) occurring after (postpulse) and before (prepulse) the conditioning pulse to +80 mV ('double pulse' voltage protocol) as illustrated in the bottom panel of Fig. 7A.

Figure 7A (top) shows superimposed Ca²⁺ current (I_Ca) traces evoked with the 'double pulse' voltage protocol illustrated, in the absence (lower trace) and presence (upper trace) of 10 µM NA in an uninjected neuron (Control). Following receptor-mediated G protein activation with NA, I_Ca displayed the typical voltage-dependent block as well as kinetic slowing as evidenced by the biphasic rising phase (upper trace). The middle panels of Fig. 7A demonstrate superimposed I_Ca traces for GIRK1-2- and GIRK1-4-expressing neurons. Figure 7B and C summarizes the mean basal and NA-mediated facilitation and inhibition, respectively, of all three groups. Mean basal facilitation in both GIRK1-2- and GIRK1-4-expressing neurons was significantly decreased compared with control cells. Upon addition of 10 µM NA to the bath, facilitation was enhanced in all three groups, though it was more prominent in control neurons. In addition, the inhibitory effect of NA was significantly decreased in both GIRK1-2- and GIRK1-4-expressing neurons (Fig. 7C). Thus, the overexpression of functional GIRK channels results in a significant attenuation of NA-mediated inhibition of N-type Ca²⁺ channels, presumably as a consequence of GIRK channels acting as a G 'sink' and preventing the majority of N-type Ca²⁺ channels from coupling to the free G-dimers following ₂-adrenergic receptor activation.

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Figure 7. Facilitation and NA-mediated inhibition of Ca2+ currents (ICa) in GIRK-expressing neurons A, superimposed ICa traces evoked with the 'double pulse' voltage protocol (shown at bottom) in the absence (lower traces) and presence (upper traces) of 10 µM NA for control, GIRK1-2- and GIRK1-4-expressing neurons. B, summary graph of mean (± S.E.M.) facilitation for control, GIRK1-2- and GIRK1-4-expressing neurons in the absence () or presence () of 10 µM NA. Facilitation was calculated as the ratio of ICa amplitude determined from the test pulse (+10 mV) occurring after (postpulse) and before (prepulse) the +80 mV conditioning pulse. ICa was measured isochronally 10 ms after initiation of the test pulse. C, summary graph of mean (± S.E.M.) ICa amplitude inhibition produced by application of 10 µM NA to control, GIRK1-2- and GIRK1-4-expressing neurons. Inhibition was determined from ICa amplitude measured isochronally at 10 ms into the test pulse (+10 mV) in the absence or presence of 10 µM NA. * P < 0·05 vs. control. Numbers in parentheses indicate the number of experiments.

	DISCUSSION

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N-type Ca²⁺ and GIRK channels share a common G protein signalling mechanism, i.e. both channels appear to be modulated by a direct interaction with the G-dimer (Logothetis et al. 1987; Ikeda, 1996; Herlitze et al. 1996). Following interaction with G, GIRK channels are activated whereas N-type Ca²⁺ channels are inhibited in a voltage-dependent manner. In some neurons, GIRK and N-type Ca²⁺ channels are natively co-expressed, modulated by the same neurotransmitters, and influence neuronal excitability. Thus it is of interest to understand whether the modulation of ion channels which 'share' a common signalling molecule exhibit an interdependence as a result of this interaction. To address this question, we have taken advantage of a neuronal preparation, i.e. SCG neurons, which has been extensively studied in regard to N-type Ca²⁺ channel modulation and in which proteins can be heterologously expressed (Ikeda, 1997). Accordingly, the goals of the study were to determine: (1) whether GIRK channels expressed in SCG neurons exhibit properties similar to those of natively expressed channels; (2) whether the heterologously expressed GIRK channels are functionally coupled to the same endogenous signalling pathways involved in N-type Ca²⁺ channel modulation; and (3) whether overexpression of GIRK channels alters N-type Ca²⁺ channel modulation.

Expression of GIRK channels in SCG neurons

Although GIRK channels are widely distributed in nervous tissue (Lesage et al. 1995; Slesinger et al. 1996), native expression of GIRK channels in rat SCG neurons was not apparent since application of 10 µM NA to cells injected with EGFP cDNA alone failed to activate overt inward current (Fig. 1A). When GIRK1, 2 and 4 subunits were expressed alone, NA-induced GIRK channel currents were inconsistent and small in amplitude. However, co-expression of GIRK1-2 or GIRK1-4 resulted in robust and reliable inward currents following NA application. These results are consistent with previous reports demonstrating that coexpression of GIRK1-2 in Xenopus oocytes resulted in greater activation of agonist-induced inward currents than either of the isoforms alone (Kofuji et al. 1995; Duprat et al. 1995). In similar fashion, coinjection in Xenopus oocytes or Chinese hamster ovary cells of both GIRK1 and GIRK4 subunits - the functional and structural GIRK1 and GIRK2 analogues found in atrial tissue - has been shown to be essential for GIRK channel activity (Krapivinsky et al. 1995; Hedin et al. 1996). Our results provide additional support for the concept that coexpression of GIRK isoforms is required to form fully functional GIRK channels.

In native tissues, GIRK channels are highly K⁺ selective, inwardly rectifying and blocked by external Ba²⁺. When expressed in SCG neurons, heteromultimeric GIRK channels possess similar properties. Thus, GIRK channels expressed in SCG neurons gave rise to currents which displayed strong inward rectification and reversed polarity near the potential predicted by the Nernst equation for a K⁺-selective electrode (Fig. 2A), i.e. V_rev shifted approximately 55 mV per 10-fold change in external [K⁺]. Application of 1 mM Ba²⁺ to the external bath solution completely blocked the NA-activated GIRK channel currents (Fig. 2B). Rapid desensitization of current following agonist application is another characteristic of GIRK channels seen in atrial cells and GIRK-injected Xenopus oocytes. In this study, prolonged and repeated stimulation with 10 µM NA likewise led to desensitization of GIRK channel currents (Fig. 3D) indicating that machinery required for GIRK channel desensitization is present in SCG neurons. Although the exact mechanism underlying desensitization is unknown, it has been suggested that the process is mediated by a cytosolic protein acting in a G protein-independent manner (Hong et al. 1996).

Modulation of GIRK channels in SCG neurons

The modulation of N-type Ca²⁺ channels in SCG neurons occurs through several convergent parallel signal transduction pathways (Hille, 1994). The activation of GIRK channel currents via receptors coupled to G proteins of the G_i/G_o subfamily (i.e. NA) was inhibited with PTX pretreatment. Pretreatment of GIRK-expressing neurons with CTX inhibited VIP-induced GIRK channel currents, which couple to G proteins of the G_s subfamily. Similar findings have been reported by Lim et al. (1995) who found that Xenopus oocytes expressing GIRK1 channels and the ₂-adrenergic receptor (G_s linked) activated GIRK channel currents in the presence of isoprenaline. They also found that the magnitude of the induced GIRK1 channel current was comparable to that of the muscarinic-activated current, which couples to the G_i/G_o subfamily. Together, these findings support the idea that the activation of GIRK channels is capable of occurring via two pathways that are to some extent both separate (i.e. G protein subfamilies) and convergent (i.e. free G-dimer). These results can be extended to those observed with N-type Ca²⁺ channel modulation by several neurotransmitters. That is, both G_i/G_o- and G_s-linked receptors inhibit Ca²⁺ channels in a membrane-delimited pathway, though the inhibition is voltage dependent.

Furthermore, pretreatment of SCG neurons with PTX caused a significant increase in VIP-activated GIRK channel current magnitude when compared with non-PTX-treated cells. Although the mechanism(s) underlying this effect is at present unknown, similar enhancements of signalling via PTX-insensitive G proteins following PTX treatment have been reported. For example, VIP-mediated inhibition of N-type Ca²⁺ channels in SCG neurons (Zhu & Ikeda, 1994) and metabotropic glutamate type 1 receptor-mediated increase in phosphoinositide turnover (Carruthers et al. 1997) are enhanced following PTX treatment. One possibility is that the VIP receptors couple to both inhibitory and stimulatory G protein pathways. Selective uncoupling of the inhibitory pathways would then result in an enhanced response. In this regard, it has been reported that activated G_i2 inhibits GIRK channels activated by G (Schreibmayer et al. 1996). Another possibility is that uncoupling of PTX-sensitive G proteins from receptors allows greater numbers of PTX-insensitive G proteins to couple with receptors. Finally, PTX pretreatment may alter the synthesis and/or degradation of proteins involved in the signalling pathway (Watkins et al. 1989). At present, experimental evidence supporting any of these mechanisms in SCG neurons is lacking.

Additional experiments were undertaken to determine whether other neurotransmitters (Ado, SS and PGE₂), which couple to G_i/G_o proteins, would activate GIRK-expressing neurons. Although the three agents activated GIRK channel currents, current magnitude was greater when NA was employed as the agonist (cf. Figs 5D and 6D). The activation of GIRK channels by Ado was probably mediated via A₁ receptors, which are believed to be the primary Ado receptor type in SCG neurons (Zhu & Ikeda, 1993). This receptor subtype has also been recently found to activate GIRK1-2 channel currents in cultured rat hippocampal neurons (Ehrengruber et al. 1997). SS has been reported to activate GIRK channel currents in cultured locus coerulus neurons from newborn rats and in AtT-20 cells, a mouse pituitary cell line (Takano et al. 1997). To our knowledge, this is the first report showing the activation of GIRK channel currents by PGE₂.

Another signalling pathway in SCG neurons which causes N-type Ca²⁺ and M-type K⁺ current inhibition but is voltage independent and PTX insensitive (Bernheim et al. 1992) was studied in order to see if it coupled to GIRK channels. Oxo-M, a muscarinic agonist, utilizes this pathway via the G_q/G₁₁ subfamily and a diffusible messenger produces the inhibition. Exposure of GIRK-expressing neurons (pretreated with PTX to eliminate G_i/G_o pathway) to Oxo-M did not activate significant GIRK channel currents. Under the experimental conditions used, it does not appear that stimulation of this pathway results in GIRK channel activation. When Sharon et al. (1997) coexpressed metabotropic glutamate receptors, mGluR1a and mGluR5, and GIRK1-2 in Xenopus oocytes, the channel activity was inhibited when exposed to glutamate. Both types of receptor activate phospholipase C, presumably via G_q proteins. They reported that this pathway was protein kinase C mediated and PTX insensitive. In the present study we did not observe this biphasic behaviour, although the expression systems and recording conditions employed could explain this discrepancy.

Unlike modulation of N-type Ca²⁺ channels by neurotransmitters in SCG neurons, our results suggest that there is a degree of specificity concerning GIRK channel activation. That is, most known and putative neurotransmitters exert an inhibitory effect that is either voltage dependent or -independent. However, activation of GIRK channel currents in our model was obtained with neurotransmitters that couple to either G_i/G_o or G_s subfamilies but not to the G_q/G₁₁ subfamily.

GIRK channels alter modulation of N-type Ca²⁺ channels

We envisioned two scenarios in regard to overexpression of GIRK channels in SCG neurons and N-type channel modulation. In the first case, GIRK and N-type channels would both have relatively equal 'access' to G following receptor stimulation. Thus, if GIRK channels were overexpressed, mass action would predict that the majority of G would bind to GIRK thereby depriving N-type channels of G and decreasing modulation. In the second case, the Ca²⁺ channels, G proteins and receptors would be 'compartmentalized' into functional microdomains perhaps through a scaffolding protein (Neubig, 1994). Under these circumstances, it might be anticipated that overexpression of GIRK would have minimal impact on G-mediated modulation. Prior to activation of GIRK channel currents with the agonist NA, both GIRK1-2- and GIRK1-4-expressing neurons showed a decreased basal facilitation, suggesting that the G protein-Ca²⁺ channel stoichiometry had been altered. This was more apparent following agonist application, since the NA-mediated inhibition of Ca²⁺ channels was significantly attenuated (Fig. 7B and C). These results would suggest that following receptor activation and heterotrimeric G protein dissociation, the free G-dimer is capable of acting on either the N-type Ca²⁺ (Ikeda, 1996; Herlitze et al. 1996) or GIRK channels (Logothetis et al. 1987). Alternatively, overexpression of GIRK channels may affect heterotrimeric G protein levels that are capable of coupling Ca²⁺ channels to ₂-adrenergic receptors. It has been reported that an N-terminal GIRK1 fusion protein binds heterotrimeric G proteins (Huang et al. 1995). Thus, overexpression of GIRK channels may sequester sufficient heterotrimeric G protein to functionally uncouple the ₂-adrenergic receptor from N-type Ca²⁺ channels. In addition, the spatial localization potentially afforded by GIRK-heterotrimeric G protein interaction could restrict 'released' G-dimers to GIRK channels. At present we cannot determine the exact mechanism by which GIRK overexpression disrupted N-type Ca²⁺ channel modulation.

Although under our experimental conditions we have no method of determining the number of N-type Ca²⁺ channels expressed per neuron, we can, however, estimate the number of GIRK channels expressed. The reported values of GIRK single channel conductance under symmetrical [K⁺] conditions range from 35 to 39 pS (Dascal et al. 1993; Duprat et al. 1995; Kofuji et al. 1995). The mean peak current for GIRK1-2- and GIRK1-4-expressing neurons was 1290 and 1840 pA, respectively, at -130 mV. By applying Ohm's law, the whole-cell conductance ranges from 26 000 to 37 000 pS. Thus, the approximate number of expressed GIRK channels ranges from 700 to 1000 per neuron. (We believe this to be an underestimate, since the value of the whole-cell conductance we calculated was recorded under asymmetrical conditions: 5·4 mM K/135 mM K⁺_i.) The robust expression of such a high number of GIRK channels per neuron allows them to behave as a G sink following receptor activation.

With this heterologous expression system, we have 'switched' the specificity of G protein-coupled receptors from one effector (N-type Ca²⁺ channel) to another (GIRK channel). Although the conditions we have imposed on this cell system are probably not likely to be encountered under physiological conditions, it can serve as a model of other similar native systems. For example, Li & Bayliss (1998) found that activation of the ₂-adrenergic receptors in caudal raphe neurons caused an inhibition of the Ca²⁺ channel currents, yet GIRK channels were not activated. On the other hand, exposure of the same neurons to 5-hydroxytryptamine (which acts via PTX-sensitive G proteins) both activated GIRK channels and produced inhibition of Ca²⁺ currents. These differential effects of neurotransmitters indicate the possibility of spatial location of G protein-coupled receptors and effectors. That is, depending on the location of the receptor-effector unit, the same neurotransmitter will exert a different form of cellular response inhibition of Ca²⁺ entry or membrane hyperpolarization. Within this context, it is possible that the relative abundance of two G effectors may influence apparent receptor specificity.

In summary, our results show the successful heterologous expression of functional GIRK channels in rat SCG neurons. Upon agonist application, the induced currents displayed electrophysiological properties of inward rectifier K⁺ channels. Like N-type Ca²⁺ channels, GIRK channels effectively coupled to native G protein signalling pathways. The approach described here provides an additional and novel tool that offers an opportunity to better understand the basis of receptor-G protein specificity and Ca²⁺ channel (effector) modulation. Consequently, we may be able to further our understanding concerning the molecular events associated with neurotransmitter regulation of synaptic transmission.

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Acknowledgements

We thank Marina King for excellent technical assistance, Drs S. W. Jeong and P. J. Kammermeier for providing critical comments to the manuscript and the following for providing cDNA clones: Diomedes E. Logothetis for GIRK1 and GIRK4, and Michel Lazdunski for GIRK2. This work was supported by a grant from the National Institutes of Health (grant no. GM 56180; to S. R. I.) with a research supplement for V. R.-V.

Corresponding author

S. R. Ikeda: Laboratory of Molecular Physiology, Guthrie Research Institute, One Guthrie Square, Sayre, PA 18840, USA.

Email: sikeda{at}inet.guthrie.org

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