Introduction

Top Summary Introduction Materials and Methods Results Discussion References

G protein-coupled inwardly rectifying K⁺ (GIRK) channels are important in regulating cell excitability in both the heart and the central nervous system (CNS; Dascal, 1997). In the CNS, GIRK channels are coupled to many types of inhibitory neurotransmitter receptors via pertussis toxin (PTX)-sensitive G proteins, e.g., -aminobutyric acid type B (GABA_B), 5-hydroxytryptamine type 1A (serotonin),  and µ opioid, somatostatin, and adenosine A₁ receptors (Andrade et al. 1986; Trussell and Jackson, 1987; Penington et al., 1993; Velimirovic et al., 1995). In agreement with this, Lüscher et al. (1997) recently showed that GIRK channels perform an important role in inhibition of hippocampal neurons using Kir3.2 knockout mice. Furthermore, immunocytochemical staining has revealed the presence of Kir3.1 on presynaptic nerve terminals in many areas of the brain, suggesting a role for GIRK channels in presynaptic inhibition (Morishige et al., 1996; Ponce et al. 1996).

The mechanism by which neurotransmitters interact to activate the GIRK channel is now well established. In the presence of agonist, G_i/G_o-coupled receptors catalyze the turnover of trimeric G proteins releasing subunits, which directly bind to the GIRK channel complex, stabilizing phosphatidyl inositol 4,5-bis-phosphate binding, with consequent channel activation (Huang et al., 1997, 1998).

In addition to their role in neuronal inhibition, there is increasing evidence to suggest that GIRK channels have an important function in neuronal excitation induced by G_q-coupled neurotransmitter receptors (Velimirovic et al., 1995; Farkas et al., 1997). However, in contrast to the wealth of knowledge concerning G_i/G_o-mediated activation of this channel complex, little is known regarding how G_q-coupled receptors inhibit these channels. In view of this, in the present study we have used the Xenopus oocyte expression system to examine the mechanism by which G_q-coupled receptors of the bombesin family interact with and inhibit GIRK channel activity. Some of these findings have recently been reported to The Physiological Society (Stevens et al., 1998).

	Materials and Methods

Top Summary Introduction Materials and Methods Results Discussion References

Molecular Biology. Rat Kir3.1 and Kir3.2 cDNAs engineered into pBG7.2 were linearized using NdeI, whereas rat bombesin receptor (BB₁ and BB₂) cDNAs engineered into pBluescript SK (Invitrogen, Netherlands) were linearized using BamHI and Spe1, respectively. Capped cRNAs were transcribed in vitro from linearized cDNAs using T7 polymerases (Promega, Southampton, UK).

Isolation of Xenopus Oocytes. Xenopus laevis were anesthetized by immersion in 0.3% (w/v) 3-amino benzoic acid (Sigma, Poole, U.K.) and ovarian lobes were removed. Oocytes were dissociated using 0.3% (w/v) collagenase (Sigma) in Ca²⁺-free solution (82.5 mM NaCl, 2.5 mM KCl, 1 mM MgCl₂, and 5 mM HEPES, pH 7.6). Prepared oocytes were microinjected with 50 nl of cRNAs dissolved in water. Oocytes were incubated at 18°C in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES).

Two-Electrode Voltage Clamp Recording. Two-electrode voltage clamp recordings were performed 3 to 6 days after microinjection of cRNAs using a GeneClamp 500 amplifier (Axon Instruments, Burlingame, CA) interfaced to a Digidata 1200 A/D board with Clampex software (version 6, Axon Instruments) and recorded on DAT (Sony, Toyoko, Japan). Records were replayed on a Gould TA240 chart recorder. Oocytes were continually perfused with standard recording solution (90 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 1 mM HEPES, pH 7.4). Microelectrodes filled with 3 M KCl had resistances between 0.5 and 2 M. Ten-millivolt hyperpolarizing steps were applied every second from a holding potential of 80 mV. Currents were filtered at 1 kHz. Data were analyzed using Clampfit (version 6; Axon Instruments) and Prism (version 2; GraphPad Software, San Diego, CA). Oocytes were preincubated with chelerythrine, staurosporine, U-73122 ({1-[6-((17-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione}), U73343 ({1-[6-((17-3-Metho-xyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione} or BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester) for 2 h. Statistical analyses were performed using a Student's unpaired t test. Data were assumed to be normally distributed and a significant difference was accepted when the two-tailed P value was less than 0.01. Averaged data are presented as means ± S.E.M. Dose-response data were fitted with the equation:

(1)

where EC₅₀ is the half-maximally effective concentration of neuromedin B (NMB), [NMB] is the test concentration of NMB, n is the Hill coefficient, and y is the amount of inhibition of current relative to the control level.

Single-Channel Recording. Single-channel recordings were performed using an Axopatch 200B patch clamp amplifier (Axon Instruments). Currents were recorded at 10 kHz and filtered at 2 kHz. The electrode solution contained: 140 mM KCl, 2 mM MgCl₂, 1.8 mM CaCl₂, and 0.1 mM GdCl₃, pH 7.2, whereas the bathing solution contained: 140 mM KCl, 3 mM MgCl₂, 5 mM EGTA, and 5 mM HEPES, pH 7.2. Patch electrodes were coated with dental wax and had resistances between 1 to 2 M. Data was acquired using Fetchex (versin 6; Axon Instruments) and analyzed using TAC and TACfit (version 5; Bruxton Corp., Seattle, WA). All experiments were performed at 22 to 24°C.

(2)

	Results

Top Summary Introduction Materials and Methods Results Discussion References

Inhibition of Kir3 by Bombesin Receptors. Oocytes coinjected with GIRK channel and bombesin receptor cRNA were recorded using two-electrode voltage clamp. Oocytes expressing heteromeric Kir3.1-Kir3.2 channels and the BB₁ receptor gave rise to Ba²⁺-sensitive currents at 80 mV in standard recording solution. A 2-min application of the preferred BB₁ agonist, NMB, inhibited the GIRK current in a concentration-dependent manner that was slowly reversible on washout (Fig. 1, a, b, and d). Voltage ramps applied from 120 mV to +40 mV before and after NMB application, suggested that NMB reduced current amplitude, but not rectification properties (Fig. 1b). In initial experiments, the time course of GIRK channel inhibition by NMB was obscured due to the activation of an endogenous Ca²⁺-dependent Cl current (Barish, 1983; Fig. 1a) at NMB concentrations above 50 pM (Fig. 2a-c). However, preincubation of oocytes with the membrane-permeant Ca²⁺ chelator BAPTA-AM abolished this Cl conductance at all tested NMB concentrations while having no effect on the inhibition of the GIRK current (Fig. 1c and Fig. 3). Under these conditions, inhibition of the GIRK current was preceded by a small activation, which could be due to either residual Ca²⁺-activated C currents or promiscuous coupling of the bombesin receptor to G proteins of the G_i/G_o family. Using this approach, it was possible to construct a concentration-inhibition curve for NMB. Fitting these data to eq. 1 gave a Hill coefficient of 1.09 ± 0.25 and an IC₅₀ of 5.4 ± 0.04 pM (Fig. 2a).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Inhibition of GIRK channels by coupling to bombesin receptors. a, inward GIRK currents were recorded from oocytes injected with cRNAs for Kir3.1, Kir3.2, and BB1 receptor. Application of 10 nM NMB both evoked an endogenous inward Ca2+-activated Cl current and inhibited the GIRK current, which was also selectively blocked by 300 µM Ba2+. b, current-voltage relationships were obtained using voltage ramps between 120 and +40 mV. Voltage ramps were applied before and after application of NMB and in the presence of Ba2+. Current responses to ramps in the presence of Ba2+ were subtracted from those before (i) and after (ii) NMB application. c, GIRK currents recorded from oocytes incubated in BAPTA-AM. Endogenous Ca2+-activated Cl current is completely abolished by the Ca2+ chelator. GIRK current was reversibly inhibited by a 2-min application of NMB. d, histogram of inhibitory effects of bombesin-like peptides on different bombesin receptor-coupled Kir3 subunit combinations (n = 3-6). BB1 receptors were activated with 10 nM NMB, whereas BB2 receptors were activated with 10 nM GRP. Relative inhibition is current amplitude after drug application relative to current amplitude before drug application. All Ba2+-insensitive currents were subtracted.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Dose-dependent inhibition of Kir3.1-Kir3.2 currents by BB1 receptors in response to NMB. Large transient inward current in c is due to activation of endogenous Ca2+-activated Cl current. d, dose-response relationship for NMB. Inhibited currents were normalized relative to current amplitude before application of NMB. Each data point represents n = 5. Line is best least-squares fit to eq. 1, where EC50 = 5.4 pM.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Effect of PKC inhibitors and BAPTA-AM on Kir3.1-Kir3.2 currents. Histogram shows effect of preincubation of oocytes with the PLC inhibitor U73122 (10 µM) and its inactive analog U73343 (10 µM), PKC inhibitors chelerythrine (10 µM) and staurosporine (10 µM), and Ca2+ chelator BAPTA-AM (200 µM) on amount of inhibition of GIRK currents in response to 10 nM NMB (n = 4-7). Chelerythrine, staurosporine, and U73122 significantly reduced amount of current inhibition in response to NMB, whereas BAPTA-AM and U73343 had no effect relative to untreated oocytes (*p < .05, **p < .01).

Inhibition Is Mediated by Protein Kinase C (PKC). Bombesin receptors have previously been shown to couple to phospholipase C (PLC) via G_q in Xenopus oocytes, which activates both the inositol 1,4,5-triphosphate (IP₃) and PKC pathways (Shapira et al., 1994). To determine whether PLC performed a role in inhibition of GIRK currents via interaction with the BB₁ receptor, oocytes were preincubated in the PLC inhibitor U73122. GIRK current inhibition was significantly reduced by U73122, whereas the inactive analog, U73343 had no effect (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of PKC and PKA activators on Kir3.1-Kir3.2 currents. a, inhibitory effect of 1 µM PMA on GIRK currents. b, inhibitory effect of 1 µM PBDu on GIRK currents. c, lack of inhibition of GIRK currents by 4-phorbol. d, histogram showing effects of phorbol esters PMA, PBDu, and 4-phorbol, and PKA activators 8-bromo cAMP and forskolin. Each bar represents n = 5.

Single-channel Properties in Presence of PMA. Single-channel recordings were performed in the cell-attached recording configuration. Inhibition of the GIRK current by the BB₁ agonist NMB was of insufficient duration (<10 min) for complete single-channel analysis due to desensitization of the receptor (Corjay et al., 1991). Therefore, PKC-mediated inhibition of the GIRK current with PMA was used to examine the effect of PKC activation on GIRK channel activity. The single-channel properties of the Kir3.1-Kir3.2 complex were compared before and after treatment with PMA. Under control conditions, GIRK currents were found to exhibit a unitary amplitude of 2.71 ± 0.08 pA at 80 mV (n = 3). As previously reported in both native and heterologous expression systems, GIRK channel activity was characterized by bursting behavior (Grigg et al., 1996; Luchian et al., 1997), i.e., clusters of openings separated by brief closures (Fig. 5, a and b).

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Change in single-channel properties of Kir3.1-Kir3.2 in response to PMA. Typical single channel records before (a) and after (b) application of 100 nM PMA showing a reduction in open probability. Single-channel amplitude histograms were fitted with a Gaussian function. Single-channel amplitudes before (c) and after (d) application of PMA were identical (2.7 pA). Open-time distributions were fitted with two exponential components. Before application of PMA (e), open times were described with time constants of 0.3 and 1.5 ms, whereas after application of PMA (f), open times were described by time constants of 0.3 and 1.1 ms. Closed-time distributions were fitted with two exponential components. Before application of PMA (g), closed times were described with time constants of 0.4 and 35 ms, whereas after application of PMA (h), closed times were described by time constants of 0.4 and 1480 ms.

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	Discussion

Top Summary Introduction Materials and Methods Results Discussion References

This study demonstrates that activation of G_q-coupled bombesin receptors using the bombesin-related peptides NMB or GRP reversibly inhibits GIRK currents in Xenopus oocytes. The BB₁ receptor has previously been shown to activate phospholipase C via a G_q-mediated pathway in oocytes (Shapira et al., 1994; Woodruff et al., 1996). This results in production of diacylglycerol and IP₃. IP₃ releases Ca²⁺ from intracellular organelles, whereas diacylglycerol activates PKC (Sternweis et al., 1992). The involvement of PLC in coupling between BB₁ receptor and GIRK currents was demonstrated using the specific PLC inhibitor U73122, which decreased the degree of inhibition of GIRK currents in response to NMB. The increase in intracellular Ca²⁺ after binding of NMB to BB₁ receptor is clearly seen in the present study by the activation of the endogenous oocyte Ca²⁺-dependent Cl channel (Barish, 1983). Treatment of oocytes with the Ca²⁺ chelator, BAPTA-AM, abolishes this Cl current, but has no effect on inhibition of GIRK current, suggesting that an increase in intracellular Ca²⁺ concentration is not involved in modulation of GIRK channel activity. In contrast, we demonstrate that PKC performs an important role in the modulation of GIRK channel activity using both PKC inhibitors and activators. The PKC inhibitors staurosporine and chelerythrine reduce the amount of GIRK current inhibition in response to NMB, whereas the PKC activators, the phorbol esters PMA and PDBu, induce inhibition of GIRK channel activity in the absence of receptor activation. The specificity of PDBu and PMA for PKC has been demonstrated using the inactive phorbol ester 4-phorbol, which does not inhibit the GIRK current.

Previous studies on interactions between GIRK channels and the G_q/G₁₁-coupled metabotropic glutamate receptors have given conflicting results. Saugstad et al. (1996) reported that type 1a metabotropic glutamate receptors (mGluR1a) activate Kir3.1-Kir3.4 heteromeric channels by coupling to PTX-sensitive G proteins, whereas Sharon et al. (1997) reported that mGluR1a receptors inhibit Kir3.1-Kir3.2 heteromeric channels by coupling to PTX-insensitive G proteins. Sharon et al. (1997) also showed that Kir3.1-Kir3.2 channels display an initial weak activation before the strong inhibition due to promiscuous coupling of the mGluR1a receptor to PTX-sensitive G proteins. In agreement with the present study, Sharon et al. (1997) showed that inhibition of the GIRK channel complex occurs through a PKC-dependent mechanism.

In the present study, single-channel recordings in the presence of PMA provide an insight into the mechanism of channel inhibition. Single-channel conductance, open-time distributions, and burst lengths remain unaffected, whereas the interburst interval is altered. Ivanova-Nikolova and Breitweiser (1997) reported that binding changes the proportion of channels within three bursting modes, but does not change the burst duration. Termination of bursting occurs independently of binding and has been shown to be caused by a C-terminal inactivation particle blocking the pore (Luchian et al., 1997). Because intraburst kinetics were unaffected by PKC activation in the present study, inhibition is unlikely to occur through a change in binding. However, an increased interburst duration suggests that PKC activation stabilizes the inactivated state.

Although there is no direct evidence that phosphorylation by PKC acts directly on the GIRK channel rather than an accessory protein, Kir3.1, Kir3.2, and Kir3.4 contain consensus PKC sites on both the N and C termini of the channel subunits. Another inward rectifier, Kir2.3, has been shown to couple to the G_q-linked muscarinic M₁ receptor and is inhibited by the phorbol esters PMA and PDBu (Henry et al., 1996), suggesting the involvement of PKC. Because Kir3 subunits and Kir2.3 possess a consensus PKC site at identical positions in the N terminus, it is possible to speculate that phosphorylation of this site may be involved in the channel inhibition process.

In order for GIRK channels to be a target for inhibition by coupling to G_q-linked receptors, a proportion of the channels must be activated. GIRK channels have been shown to display tonic activity in hippocampal neurons. Using patch clamp recording from brain slices, the membrane potentials of Kir3.2 knockout mouse CA1 neurons is 8 mV more depolarized than wild-type mouse CA1 neurons (Lüscher et al., 1997). Sodickson and Bean (1996) showed that dissociated rat hippocampal CA3 neurons give rise to GABA_B-activated GIRK currents (EC₅₀ = 1.6 µM) at extracellular GABA concentrations present basally in the brain (0.2-0.8 µM), suggesting that tonic activity of GIRK channels could arise from tonic activation of receptors. However, because GABA_B and A₁ antagonists do not affect resting potentials of CA1 neurons in slices, Lüscher et al. (1997) suggest that tonic activity of GIRK channels is independent of receptor activation. Tonic activity of GIRK channels in the absence of agonist has been shown to operate through constitutive turnover of G proteins, releasing , in Xenopus oocytes (Stevens et al., 1997) and through a Na⁺-gating mechanism in both myocytes and oocytes (Lesage et al. 1995; Sui et al. 1996).

Negative coupling of GIRK channels to G_q-linked receptors has been demonstrated in several neuronal preparations. For example, in cultured rat locus coereleus neurons a GIRK channel activated by somatostatin and [Met]enkephalin has been identified by its single-channel conductance (30 picosiemen) and membrane-delimited activation (Grigg et al., 1996). The same GIRK channel is also inhibited by Substance P via a PTX-insensitive G protein (Velimirovic et al., 1995). Similarly, in GTPS-loaded locus ceruleus neurons application of somatostatin induced a persistent increase in K⁺ conductance that was reversed by application of Substance P (Velimirovic et al., 1995). In cultured rat dopaminergic neurons of the midbrain ventral tegmental area a putative GIRK current is activated by dopamine (D₂ receptors) via PTX-sensitive G proteins and inhibited by neurotensin via PTX insensitive G proteins (Farkas et al., 1997)

The distribution of GIRK channels shares a high degree of overlap with BB₁ and BB₂ receptors throughout the CNS (Wada et al. 1991; Ladenheim et al., 1992; Murer et al., 1997; Karschin et al. 1996). Furthermore, inhibition of a resting K⁺ conductance by bombesin and related peptides has been reported in neurons of the rat dorsal raphe and suprachiasmatic nucleus (Pinnock and Woodruff, 1991; Reynolds and Pinnock, 1997). Because GIRK channels have been reported to be coupled to 5-hydroxytryptamine type 1A receptors in dorsal raphe neurons (Penington et al., 1993), it is possible that GIRK channels are the target for inhibition induced by bombesin.

In summary, in the present study we demonstrated unequivocally that G_q-coupled neuropeptides are able to inhibit GIRK channels in a nonmembrane-delimited manner involving PKC. Furthermore, we showed at the single-channel level this inhibition by PKC is mediated by a change in interburst interval of the channel complex. In future studies it will be important to examine the interaction between GIRK channels and G_q-coupled receptors in native neurons to test whether a similar mechanism is involved in postsynaptic excitation.