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Selective activation of heterologously expressed G protein-gated K⁺ channels by M₂ muscarinic receptors in rat sympathetic neurones

Jose M. Fernandez-Fernandez, Nicolas Wanaverbecq, Pam Halley, Malcolm P. Caulfield and David A. Brown *

MS 8938 Received 6 November 1998; accepted after revision 22 January 1999.

	ABSTRACT

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1. G protein-regulated inward rectifier K⁺ (GIRK) channels were over-expressed in dissociated rat superior cervical sympathetic (SCG) neurones by co-transfecting green fluorescent protein (GFP)-, GIRK1- and GIRK2-expressing plasmids using the biolistic technique. Membrane currents were subsequently recorded with whole-cell patch electrodes.

2. Co-transfected cells had larger Ba²⁺-sensitive inwardly rectifying currents and 13 mV more negative resting potentials (in 3 mM [K⁺]_o) than non-transfected cells, or cells transfected with GIRK1 or GIRK2 alone.

3. Carbachol (CCh, 1-30 µM) increased the inwardly rectifying current in 70 % of GIRK1+ GIRK2-transfected cells by 261 ± 53 % (n = 6, CCh 30 µM) at -120 mV, but had no effect in non-transfected cells or in cells transfected with GIRK1 or GIRK2 alone. Pertussis toxin prevented the effect of carbachol but had no effect on basal currents.

4. The effect of CCh was antagonized by 6 nM tripitramine but not by 100 nM pirenzepine, consistent with activation of endogenous M₂ muscarinic acetylcholine receptors.

5. In contrast, inhibition of the voltage-activated Ca²⁺ current by CCh was antagonized by 100 nM pirenzepine but not by 6 nM tripitramine, indicating that it was mediated by M₄ muscarinic acetylcholine receptors.

6. We conclude that endogenous M₂ and M₄ muscarinic receptors selectively couple to GIRK currents and Ca²⁺ currents respectively, with negligible cross-talk.

	INTRODUCTION

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Molecular cloning studies have identified five distinct muscarinic acetylcholine receptor (mAChR) genes, termed m1-m5 (Hulme et al. 1990); the corresponding receptors are designated M₁-M₅ (Caulfield & Birdsall, 1998). Nearly all neurones in the rat superior cervical ganglion (SCG) express mRNAs for at least three of these (m1, m2 and m4: Brown et al. 1995). The principal effects of stimulating M₁ and M₄ receptors in rat SCG neurones have now been well defined: M₁ receptors inhibit the voltage-gated K⁺ current I_K(M) (Marrion et al. 1989; Bernheim et al. 1992), while both M₁ and M₄ receptors independently inhibit the N-type voltage-gated Ca²⁺ current I_Ca(N) through different mechanisms (Wanke et al. 1987; Bernheim et al. 1992; Hille, 1994). These effects contribute to the slow muscarinic excitatory action of synaptically released acetylcholine (Brown & Selyanko, 1985).

The role of the SCG M₂ receptor is less clear. While M₂ receptors strongly inhibit N-type Ca²⁺ currents in expression systems (Higashida et al. 1990) and in some other neurones (Allen & Brown, 1993), they do not seem to contribute to muscarinic inhibition of I_Ca(N) in rat SCG neurones (Bernheim et al. 1992). An obvious alternative effector current to which they might be linked is the G protein-regulated inward rectifier (GIRK/Kir3) K⁺ current (Doupnik et al. 1995). Prominent hyperpolarizing responses of several other neurones to muscarinic agonists, probably induced by M₂ receptor activation of GIRK channels, have been reported (summarized in Brown et al. 1997). However, although M₂-mediated hyperpolarizations of intact rat SCGs by muscarinic agonists have been seen using extracellular recording (Newberry & Priestley, 1987), these are very small, and no corresponding activation of GIRK currents in single SCG neurones by muscarine has been described (for example see Wang & McKinnon, 1996).

There are several possible reasons for such negative effects, perhaps the most obvious being that the density of GIRK channels in rat SCG neurones might be too low (see Wang & McKinnon, 1996). Accordingly, in the present experiments we have tested this by heterologous co-expression in rat SCG neurones of cDNAs for the two subunits of the neural GIRK channel, GIRK1 and GIRK2 (Kir3·1 and Kir3·2, respectively: Lesage et al. 1995; Velimirovic et al. 1996). Interestingly, we find that stimulation of endogenous M₂ receptors, but not endogenous M₄ receptors, activates the expressed currents.

	METHODS

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Cell culture

Sympathetic neurones were dissociated from superior cervical ganglia (SCG) of 15- to 21-day-old male Sprague-Dawley rats killed by CO₂ asphyxiation. The ganglia were incubated initially in collagenase (500 U ml^-1 for 15 min) and then in trypsin (1 mg ml^-1 for 30 min), followed by mechanical trituration with a fire-polished glass Pasteur pipette and subsequent centrifugation. Cells were plated on laminin-coated glass coverslips. Cultured neurones were kept at 37°C under an atmosphere of 5 % CO₂ in L-15 medium supplemented with 10 % fetal bovine serum, 2 mM glutamine, 24 mM NaHCO₃, 38 mM glucose, 50 U ml^-1 penicillin-streptomycin and 25 ng ml^-1 nerve growth factor. All culture reagents were obtained from Gibco except laminin, collagenase, trypsin (Sigma) and nerve growth factor (Tocris).

Particle-mediated gene transfer

Superior cervical ganglia neurones cultured for 1 day were transfected using a biolistic device (PDS-1000/He; Bio-Rad) and a standard protocol (Lo et al. 1994). Neurones were bombarded with 1·6 µm gold particles (Bio-Rad) pre-coated with three different plasmids encoding: (1) green fluorescent protein (GFP), (2) GIRK1 (Kir3·1) and (3) GIRK2 (Kir3·2). GIRK-expressing plasmids were generously donated by Dr F. Lesage (Institut de Pharmacologie Moleculaire et Cellulaire, Sophia Antipolis, Valbonne, France). For each preparation (3-6 transfections), 5 µg of each plasmid DNA, 50 µl of 2·5 M CaCl₂, and 20 µl of 100 mM spermidine free-base were added to 50 µl of gold particles (60 mg ml^-1 in glycerol 50 %) under continuous vortex. DNA was allowed to coat the gold particles for 15 min with vortexing, after which the gold particles were washed twice, and resuspended in 24-48 µl absolute ethanol. Then 8 µl of the gold particle suspension was applied to the centre of macrocarrier disks (Bio-Rad) in a desiccated atmosphere to reduce agglomeration of coated particles. After evaporation of ethanol, the macrocarrier disks were mounted in the biolistic device and the particles were accelerated by a 450 p.s.i. helium pressure transient (provided by a rupture disk, Bio-Rad) into target cultured neurones that were placed 6 cm away in a partial vacuum of 15 cmHg. Neurones were immediately returned to the incubator and maintained in culture for a further 1-3 days before recording. When choosing a neurone for study, the neurones were illuminated with 470-490 nm light to excite the GFP and viewed through a 515 nm filter. About 10 % of cells were GFP-positive at > 20 h after transfection. Those cells with a medium level of green fluorescence were selected for recording. For the pertussis toxin (PTX) experiments, transfected neurons were incubated in 500 ng ml^-1 PTX in culture medium at 37°C for 18-22 h before recording.

Potassium current (I_K) recording

K⁺ currents were measured at 32-35°C from SCG neurones after 1-4 days in culture, using the whole-cell patch-clamp technique. Borosilicate glass electrodes (2-4 M) were filled with a solution containing (mM): KCl, 60; potassium acetate, 60; MgCl₂, 2·5; Hepes, 30; BAPTA, 10; Na₂ATP, 2 and Na₃GTP, 0·1 (adjusted to pH 7·2 with KOH; 290 mosmol l^-1). Cells were initially superfused with Krebs solution containing (mM): NaCl, 110; NaHCO₃, 23; KCl, 3; MgCl₂, 1·2; CaCl₂, 2·5; Hepes, 5; glucose, 11; tetrodotoxin (TTX), 0·0005, and bubbled with a 95 % O₂-5 % CO₂ mixture (pH 7·4). After patch rupture, the resting potential was measured under current-clamp ('bridge') mode. Neurones were then voltage clamped at a holding potential of -60 mV and superfused with a solution consisting of (mM): NaCl, 101; NaHCO₃, 23; KCl, 12; MgCl₂, 5; Hepes, 5; glucose, 11; TTX, 0·0005 (bubbled with a 95 % O₂-5 % CO₂ mixture, pH 7·4). Membrane currents were recorded with an Axoclamp-2B amplifier (Axon Instruments). K⁺ currents were typically evoked by 500 ms voltage steps to between -30 to -160 mV in 10 mV increments, low-pass filtered at 1 kHz and sampled at 6·67 kHz. The amplitude of I_K was measured at the end of the test pulses. Activation of I_K was measured 2 min after perfusion with a 12 mM K⁺ solution containing the muscarinic agonist carbachol, using a gravity-fed perfusion system (10 ml min^-1). When 100 µM CCh was used, a transient inward current was observed, probably due to activation of nicotinic receptors. In such cases, this was allowed to subside before applying the voltage protocol to evoke I_K.

Muscarinic antagonists (tripitramine and pirenzepine), when used, were present 3 min before and during carbachol application. Tripitramine was a generous gift from Professor Carlo Melchiorre (Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Italy).

Calcium current (I_Ca) recording

Voltage-gated Ca²⁺ currents were recorded in whole-cell mode (32-35°C) from non-transfected neurones cultured for 1-4 days. The ionic composition of the solution filling the patch electrodes (2-4 M) was (mM): CsCl, 13; caesium acetate, 120; MgCl₂, 2·5; Hepes, 10; BAPTA, 10; Na₂ATP, 2 and Na₃GTP, 0·1 (adjusted to pH 7·2 with CsOH; 290 mosmol l^-1). Neurones were bathed in Krebs solution and voltage clamped at -80 mV using an Axopatch-1D amplifier (Axon Instruments). Ca²⁺ currents were evoked by a double-pulse voltage protocol consisting of a 10 ms test pulse to +10 mV applied before and after a 50 ms conditioning pulse to +100 mV. Currents were low-pass filtered at 1 kHz and sampled at 33·3 kHz. I_Ca amplitude was estimated by digitally subtracting the outward current remaining during the same voltage protocol in the presence of Krebs solution in which CaCl₂ had been replaced with equimolar CoCl₂. Inhibition of I_Ca was measured 1 min after the normal perfusion solution was changed to a solution containing carbachol. Antagonists, when used, were perfused (10 ml min^-1) in the bath for at least 3 min before testing with carbachol.

Chemicals and drugs

All chemicals and drugs were from Sigma unless otherwise stated above.

Statistics

All data are expressed as means ± S.E.M., except for resting potentials (means ± S.D.).

	RESULTS

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Representative membrane currents recorded in 12 mM [K⁺]_o in GIRK1 + GIRK2-transfected cells are illustrated in Fig. 1. In non-transfected cells, the mean current activated by a step-hyperpolarization from -60 to -120 mV was -400 ± 33 pA (n = 22). Currents in GIRK1- or GIRK2-transfected cells were within this range (-329 ± 36 pA, n = 5, and -306 ± 83 pA, n = 5, respectively) (data not shown), whereas currents recorded in (GIRK1 + GIRK2)-transfected cells were approximately 75 % greater (-701 ± 64 pA; n = 43). This additional current could be attributed to the additional expression of GIRK channels since a greater proportion of the current (55 ± 5 %, n = 4) was reduced by 0·1 mM Ba²⁺ (applied before any agonist) than that (30 ± 3 %, n = 20) in non-transfected cells. Thus, in these experiments, co-transfection of GIRK1 with GIRK2 nearly doubled the Ba²⁺-sensitive, inwardly rectifying component of membrane current. (The presence of a small component of endogenous inwardly rectifying current in control SCG neurones accords with previous reports: e.g. Wang & McKinnon, 1996.)

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Figure 1. Muscarinic activation of a Ba2+-sensitive inwardly rectifying current in rat SCG neurones transfected with GIRK1- and GIRK2-expressing plasmids Representative currents recorded from a non-transfected SCG neurone (A) and from a SCG neurone co-transfected with GFP-, GIRK1- and GIRK2-expressing plasmids (Ba), bathed in 12 mM [K+]o before (Control) and after application of 10 µM carbachol (CCh) and then with the subsequent addition of 100 µM BaCl2 in the presence of 10 µM CCh. Currents were generated by 500 ms voltage steps to between -30 and -160 mV in 10 mV increments from a holding potential of -60 mV (see Methods) (only traces in response to hyperpolarizing pulses are shown). Records in Bb show CCh-activated currents after subtracting the control currents from those in the presence of carbachol, and Ba2+-sensitive currents after subtracting currents in the presence of CCh but absence of Ba2+. Bc, current-voltage relation for the currents illustrated in Ba. Symbols: control (), CCh 10 µM (), 10 µM CCh + 100 µM BaCl2 (), and wash (). Dashed lines indicate the zero current level.

Effect of carbachol

Carbachol (10 µM) increased the inwardly rectifying component of membrane current in 31 of 44 neurones transfected with GIRK1 + GIRK2 cDNAs (Fig. 1B), by about 209 % at -120 mV (see Fig. 2A and B). In contrast, it had no discernible effect on membrane currents in 18 non-transfected cells (Fig. 1A), or in cells transfected with GIRK1 or GIRK2 alone (5 cells in each case) (data not shown). The additional current activated by carbachol, i.e. carbachol minus control (-1453 ± 145 pA, n = 22), was blocked by 0·1 mM Ba²⁺ (Fig. 1B).

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Figure 2. Muscarinic pharmacology of IGIRK activation versus ICa inhibition Muscarinic activation of GIRK currents (IGIRK) (A and B) and inhibition of calcium currents (ICa) (C and D) in SCG neurones. , mean activation of IGIRK (A and B) and the mean inhibition of ICa (C and D) in the absence of antagonists (n = 6-22 for each data point). Curves show least-squares Hill equation fits. The curve for log[CCh]-IGIRK activation has an extrapolated maximum of 278 ± 73 %, pEC50 (-logEC50) of 5·5 ± 0·3 (EC50 = 3·1 µM), and Hill coefficient of 1, and for log[CCh]-ICa inhibition has a maximum of 45 ± 6 %, pEC50 of 6·4 ± 0·4 (EC50 = 363 nM), and Hill coefficient of 0·5. (Given the number of concentrations tested these curves are empirical and were used solely for the purpose of estimating inhibitor constants: see text.) A, shift of logCCh concentration-response curve for IGIRK activation by tripitramine (Trp, 6 nM) (n = 6). B, pirenzepine (Pz, 100 nM) (n = 7) had no effect on muscarinic IGIRK activation. C, effect of Trp (6 nM) (n = 4) and Trp (100 nM) (n = 4) on the logCCh concentration-response curve for ICa inhibition. D, shift of logCCh concentration-response curve of ICa inhibition by Pz (100 nM) (n = 7).

Responses of GIRK1 + GIRK2-transfected cells to carbachol were abolished by 18-22 h pretreatment with 500 ng ml^-1 PTX (n = 7). However, resting potentials and membrane currents at -120 mV in PTX-treated cells were within the normal range for GIRK1 + GIRK2-transfected cells (resting potential, -72 ± 9 mV; membrane current, -961 ± 134 pA; proportion of current blocked by 0·1 mM Ba²⁺, 60 ± 2 %; n = 7).

Receptors

To define the receptor(s) responsible for the activation of the GIRK current (I_GIRK) by CCh in GIRK1 + GIRK2-transfected neurones, we used two muscarinic antagonists with inverse affinities (differing on average by at least 10-fold) for M₂ and M₄ receptors - tripitramine (-logK_B (pK_B): 9·4-9·6 for M₂, 7·8-8·2 for M₄) and pirenzepine (pK_B: 6·3-6·7 for M₂; 7·1-8·1 for M₄) (see Caulfield & Birdsall, 1998). To estimate K_B values we recorded the increase in expressed I_GIRK at -120 mV produced by incremental concentrations of carbachol in the absence or presence of 6 nM tripitramine or 100 nM pirenzepine (Fig. 2A and B). An approximate measure of K_B for each antagonist could then be obtained from the rightward shift of the concentration-response curve, using the equation r = 1 + ([B]/K_B) where r is the dose ratio (i.e. the ratio of the concentrations of carbachol producing equivalent currents in the absence and presence of antagonist) and [B] is the concentration of antagonist (in nM). As shown in Fig. 2A and B, 6 nM tripitramine produced a substantial (30-fold) increase in the requisite concentration of carbachol, corresponding to a K_B of 0·2 nM. In contrast, 100 nM pirenzepine had a negligible effect, implying a K_B well in excess of 100 nM. These values correspond reasonably well to those expected were the current generated by activating M₂ receptors (Table 1).

Table 1. Pharmacological dissection of M₂ and M₄ receptors

	Tripitramine	Pirenzepine
KB for M2 * (nM)	0·25-0·4	200-500
KB for M4 * (nM)	6·3-16	8-80
KB for IGIRK (nM)	0·2	> 100
KB for ICa (nM)	10	10

In parallel experiments we adopted the same procedure to identify the receptors responsible for inhibiting the Ca²⁺ current (I_Ca: see Methods). (Fig. 2C and D). As previously reported (Delmas et al. 1998; see also Beech et al. 1992) inhibition was partly voltage dependent (Fig. 3). At the concentration which strongly antagonized the activation of I_GIRK (6 nM), tripitramine had no effect on the inhibition of I_Ca (Fig. 2C). A stronger antagonism was observed at 100 nM, from which we deduced an apparent K_B of 10 nM. In contrast, 100 nM pirenzepine was much more effective in antagonizing I_Ca inhibition (Fig. 2D) than in opposing I_GIRK activation, yielding an approximate K_B against I_Ca inhibition of 10 nM. These values suggest that inhibition of I_Ca in non-transfected SCG neurones is mediated primarily by M₄ receptors under these (whole-cell) recording conditions (Table 1), which is in agreement with Bernheim et al. (1992).

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Figure 3. Voltage dependence of the carbachol-induced inhibition of ICa A, superimposed ICa traces before and after application of 10 µM carbachol (CCh) obtained from a non-transfected SCG neurone. Dashed line indicates the zero current level. Calcium currents were generated by a double-pulse voltage protocol consisting of a 10 ms test pulse to +10 mV applied before (left) and after (right) a 50 ms conditioning pulse to +100 mV (see Methods); the current response to the conditioning pulse is omitted for clarity. B, histogram showing the total inhibition of ICa induced by 10 µM carbachol (45 ± 6 %) and their voltage-dependent (31 ± 3 %) and voltage-independent (14 ± 3 %) components. Data are means ± S.E.M. for n cells (as indicated in parentheses). The voltage-independent component represents the inhibited current remaining after the conditioning pulse, while the voltage-dependent component was defined as the difference in inhibition between the postpulse and prepulse current.

	DISCUSSION

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The principal conclusion from the present experiments is that endogenous M₂ muscarinic receptors can indeed activate GIRK channels in rat SCG neurones when the latter are expressed heterologously by co-transfecting cDNAs for the constituent GIRK1 and GIRK2 subunits. This clearly establishes that M₂ receptors are present as fully functional receptors in these neurones. However, this in turn raises some interesting points concerning the selectivity of muscarinic receptor coupling in these (and perhaps other) native nerve cells.

First, M₄ receptors have also been reported to activate inward rectifier currents (Jones, 1992). M₄ receptors are present in these cells (Brown et al. 1995) and are functionally effective in inhibiting I_Ca (Bernheim et al. 1992; see also Fig. 2 and Table 1 above); yet the lack of any effect of pirenzepine (Fig. 2B) implies that they do not contribute at all to the activation of I_GIRK. Second, and conversely (but confirming Bernheim et al. 1992), the endogenous M₂ receptors do not appear to contribute at all to the inhibition of the Ca²⁺ current by muscarinic agonists, even though there is plenty of evidence from experiments on other cells that they are potentially capable of doing so (see Introduction). Thus, there is a remarkable degree of discrimination in the way in which these two similar receptors couple to different ion channels in these neurones.

One explanation might be that the two receptors preferentially activate different G proteins. There is strong evidence that M₄-induced Ca²⁺ current inhibition is normally mediated by G_o (Delmas et al. 1998), although this route may not be obligatory when G_o is deleted (Greif et al. 1998), whereas activation of I_GIRK might plausibly be mediated by one of the G_i family. However, this alone would not explain why M₄ receptors do not activate I_GIRK since GIRK channels are activated by -subunits, not -subunits, and available evidence suggests that cardiac GIRK channels, at least, show hardly any selectivity in their response to the different -subunits likely to be associated with _o or _i subunits (Wickman et al. 1994).

An alternative explanation is to suppose that there might be a selective compartmentation of receptors and channels. Thus, the selective effect of M₄ receptors on Ca²⁺ channels might result from a relatively tightly coupled receptor- G protein-channel complex (as suggested from the work of Stanley & Mirotznik, 1997), which in turn might 'tie-up' M₄ receptors with their associated G_o proteins and preclude access to GIRK channels. Conversely, the M₂ receptor may not have the requisite association with the Ca²⁺ channel-G protein complex, so precluding Ca²⁺ channel inhibition but allowing it to couple to expressed GIRK channels.

One other feature of interest concerns the degree of agonist-independent activation of the GIRK channels in transfected cells. Agonist-independent activation of GIRK channels has been described before in atrial myocytes (e.g. Ito et al. 1991; Okabe et al. 1991), and in Xenopus oocytes expressing GIRK channels (e.g. Chan et al. 1996). However, two further points arise from the present experiments. First (and in contrast to GIRK-overexpressed hippocampal cells: Ehrengruber et al. 1997), the transfected SCG neurones showed a 13 mV higher resting potential than non-transfected cells. This suggests that the heterologously expressed channels were partly open at resting potential and contributed a steady outward current. Second, this basal activity was clearly unaffected by pertussis toxin, since current amplitudes and membrane potential in PTX-treated cells were no less than those in untreated cells. This differs from previous observations in both atrial myocytes (Ito et al. 1991; Okabe et al. 1991) and in Xenopus oocytes (Chan et al. 1996). One explanation for this might be that the basal activity was generated by phosphatidylinositol phosphates such as PIP₂ (Huang et al. 1998), rather than by spontaneous activation of G proteins coupled to unoccupied receptors. Exposure of the neurones to nerve growth factor during culture may favour this route, by stimulating phophoinositide 3-kinase (Downes & Carter, 1991), and hence increasing levels of PIP₂ in the membrane.

Note added in proof

After the submission of this paper, Ruiz-Velasco & Ikeda (1998) reported the expression of GIRK currents in rat sympathetic neurones after injection of GIRK1 + GIRK2 cDNAs. They describe the activation of GIRK currents by noradrenaline, VIP, adenosine, somatostatin and prostaglandin E₂. They also noted that oxotremorine-M did not activate the current in cells pretreated with PTX (i.e. via M₁ muscarinic receptors) but did not report the effect of oxotremorine-M in cells not pretreated with PTX.

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Acknowledgements

We are grateful to Dr Florian Lesage (Institut de Pharmacologie Moleculaire et Cellulaire, Sophia Antipolis, Valbonne, France) who provided the GIRK1- and GIRK2-expressing plasmids and to Professor Carlo Melchiorre (Dipartimento di Scienze Farmaceutiche, Universita di Bologna, Italy) for the muscarinic antagonist tripitramine. We thank Dr Elena del Rio for help with the biolistic technique. This work was supported by The Wellcome Trust.

Corresponding author

J. M. Fernandez-Fernandez: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

Email: uckljmf{at}ucl.ac.uk

Author's present address

N. Wanaverbecq: Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

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