Dual Modulation of an Inwardly Rectifying Potassium Conductance

Neurophannacology, Vol.36, No. ~ pp.209-215, 1997 Copyright @1997ElsevierScienceLtd.Allrightsreserved PrintedinGreatBritain

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Dual Modulation of an Inwardly Rectifying Potassium Conductance S. V. P. JONES* Molecular Neuropharmacology Section, Departments of Psychiatry, Pharmacology and Molecular Physiology and Biophysics, Universi~ of Vermont College of Medicine, Burlington, VT 05405, U.S.A. (Accepted 29 August 1996)

Summary-The modulationofa constitutively activeIRK1-likeinwardlyrectifyingpotassiumchannel,thatis endogenouslyexpressedin the RBL-2H3cell, was studiedwith the whole-cellpatch-clamptechnique. Activationof G-proteinsby intracellularapplicationof GTPySrevealeda dual modulationof the inward rectifier.An initialincreasein inwardcurrentamplitudewas inducedby GTPyS, followed by a profound inhibition of the current. The stimulation of the inward rectifier by GTPySwas abolishedby pretreatmentwith pertussis toxin. The inhibitory phase of the GTPyS-induced response was pertussis toxin-insensitive. Stimulation of the ml-muscarinic receptor expressed in the RBL cell after stable transection, induced an inhibition of the inwardly rectifying currents. Application of protein kinase C activators such as phorbol 12myristate 13-acetateand phorbol 12,13-dibutyrate,resultedin a stronginhibitionof the currents.Applicationof the cAMP-dependentprotein kinase activator 8-bromocAMP also inducedan inhibitionof the inwardrectifier. It is concluded that the inward rectifier of the RBL-2H3 cell may be inhibited both by activation of protein khtase C and by cAMP-dependentprotein kinase. As this type of inward rectifier is widely expressed in the nervoussystem, these data imply that the channel can be inhibitedby receptorsthat stimulate phospholipaseC and/or stimulate adenylyl cyclase, and can be activated by receptors that inhibit adenylyl cyclase activity. @ 1997 Elsevier Science Ltd. All rights reserved.

Keywords-Potassiumchannel,inwardrectifier,muscarinicreceptor,patch-clamp,proteinkinaseC, cAMPdependentproteinkinase.

Inwardly rectifying potassium channels are widely expressed throughout the nervous system and play an important role in maintaining the membrane potential. Modulation of these types of channel have profound effects on the excitability of neurons, therefore, understanding the mechanisms by which they are regulated may prove to be useful in identifying novel targets for therapeutic agents (Goldstein and Colatsky, 1996). Over the past few years, numeroustypes of inwardlyrectifying potassium channel have been cloned, greatly expanding the knowledge of their structure and function. The channels can be divided into six classes designated as KIR 1-6 (Doupnik et al., 1995). These include the socalled G-protein coupled, or GIRK-type potassium channels (KIR 3.1–3.5) and the IRK-type potassium channels (HR 2.1–2.3) that are normally active under control conditions. The GIRK-type potassium channels (GIRK1-5) (Dascal et al., 1993; Kubo et al., 1993b; Ashford et al., 1994; Lesage et al., 1994; Bond et al., *Towhom correspondenceshould be addressed.

1995;Krapivinsky et al., 1995;Tsaur et al., 1995;Hedin 1996) have been the focus of a large number of studiesand havebeen shownto be enhancedby G-protein /?y subunits (Reuveny et al., 1994; Takao et al., 1994; Huang et al., 1995; Kofuji et al., 1995; Kunkel and Peralta, 1995; Lim et al., 1995) and inhibited by the ail G-protein subunit (Schreibmayer et al., 1996). IRK-type inwardly rectifying potassium channels (Kubo et al., 1993a; Koyama et al., 1994; Mrtkhina et al., 1994; Morishige et al., 1993, 1994; Perier et al., 1994; Takahashi et al., 1994; Tang and Yang, 1994), differ from the GIRK-type, in that they are normally constitutively active under control conditions, display strong rectificationand have not been shown to be modulatedby ~y or by G-proteina-subunitsto date. However,IRK1 has been shownto be inhibitedby the ml muscarinicreceptor (Jones, 1996) and by protein kinase C (PKC) (Fakler et al., 1994; Jones, 1996). The rat basophillic leukemia cell line (RBL-2H3) has been shown to express an inwardly rectifying potassium channelwith propertiessimilarto those of the IRK family (Lindau and Fernandez, 1986; McCloskey and Cahalan, et al.,

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1990; Lewis et al., 1991; Mukai et al., 1992; Piguet and North, 1992; Jones, 1993) with greatest homology to IRK1 (Wischmeyer et al., 1995). This channel has been shown to be inhibited by GTPyS (McCIoskey and Cahalan, 1990; Wischmeyer et al., 1995), indicating the modulation of the inward rectifier by G-proteins. This study set out to investigate the mechanism by which GTPyS inhibits the inward rectifierof theRBL-2H3 cell. However, it was noted that GTPyS also induced a transient enhancement of the inwardly rectifying potassium conductance. Some of these results have been presented in abstract form (Jones, 1991).

contained (in mM): 150 K–gluconate or 150 KC1, 2 MgC12,1.1 EGTA, 0.1 CaC12,5 HEPES, 5 Mg–ATP, 0.1 Li-GTP. The pH was adjusted to 7.2 and the osmolarity to 315–320mmol/kg. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), acetylcholine (ACh) and 8bromoadenosine3’:5’-cyclicmonophosphate(8brcAMP) were obtained from Sigma (St. Louis, MO, U.S.A.). Pertussistoxin (PTX) was obtainedfrom List Biological (Campbell, CA, U.S.A.). Guanosine-5’-O-(3-thiotriphosphate) (GTPyS) was obtained from Boehringer Mannheim (Indianapolis,IN, U.S.A.).

MATERIALSAND METHODS

RBL-2H3 cells RBL-2H3 cellswere grown in 35 mm dishesat 37°C in 5% C02 and in Dulbecco’smodifiedEagles mediumwith 15% fetal bovine serum. The cells were transected by viral infectionwith the expressionvector pDs containing both the human ml muscarinicreceptor subtypegene and the neomycin resistancegene.(Gutkind et al., 1991).The cells were selected with G418 for 2 weeks and subsequently subcloned, as described previously (Jones et al., 1991a). Culture and transection

of

Electrophysiology

The whole-cell patch-clamp recording technique was performed at room temperature (21–23”C)using a LIST EPC-7 patch clamp amplifier.Cellswere visualizedusing an inverted phase contrast microscope (AXIOVERT 35) at a magnificationof 350X. Patch electrodeswere pulled from thin wall borosilicate glass capillary tubing on a three stage horizontalpuller(MecanexBB.CH.PC,Basel, Switzerland)and had resistancesof about5 MQ. Currents were filteredat 1 KHz, digitizedthrougha LabmasterTL1-125 DMA interface (12 bit +-10 V range, Axon Instruments Inc., Foster City, CA, U.S.A.) and sampled at 2 KHz by a Compaq 386 computer using pClamp software (Axon Instruments). Cells were voltage-clamped at a holding potential of –60 mV and the currents evoked by a series of depolarizing steps were recorded. Current–voltage (I-V) relationships were constructed from the currents measured at the end of 200 msec stepsfrom —120 mV to 50 mV in 10 mV increments.Acetylcholinewas applied by a low pressure ejection system from a micropipette placed close to the cell. Drugswere applieddirectlyto the bath and were washed out by perfusion of the bath with extracellular solution (2 ml/min). Results are expressed as mean ~ standard error. Solutions and reagents

The extracellular recording solution consisted of (in mM): 150 NaCl, 5 KC1,2 CaC12,1 MgC12,5 HEPES, 20 glucose and the pH was corrected to 7.4 with 1 M NaOH. The osmolarity of the solution was adjusted to 325– 330 mmol/kg. The intracellular patch pipette solution

RESULTS The inwardly rectifying potassium conductance was elicited in the RBL-2H3 cell, by voltage steps from a holding potential of –60 mV to a variety of potentials (Fig. 1A).A large inward current was noted that reversed at around —60mV and a small outward current was displayedwhich peaked at about –50 mV. The inwardly rectifying potassium currents were both cesium- and barium-sensitive.Applicationof 500PM cesium resulted in a reduction and 1 mM abolished both the inward and outward currents (n = 5) (Fig. 1A). Application of 1 mM barium to the recording chamber fully inhibited the inward rectifier (n = 3) (Fig. IB). Control current measurements showed minimal rundown of the inwardly rectifying current measured at –120 mV over a 30-60 min period (n= 9) (Fig. 2B). These measurementswere made with 0.1 mM GTP and 5 mM ATP included in the patch pipette. Omission of GTP from the patch pipetteresultedin a gradual rundown of the inwardly directed current amplitude (n = 3). In cells in which the GTP was replacedwith 200 ~M GTPyS (n= 8), current amplitudes were greatly increased in comparison to control values, on first breaking through into whole cell recording mode (Fig. 2A) and continued to increase in amplitudeover the first 5 min of recording (Fig. 2B). The current increase was followed by a sharp inhibition of the current amplitude over the following 5 min period. This rather surprising result was not observed in previous studies of the inward rectifier in RBL cells, with administration of GTPyS (McCloskey and Cahalan, 1990; Wischmeyer et al., 1995) and might be indicative of a dual modulationof the current by two distinct G-proteins. Therefore, the initial increase in current amplitude may be mediated via one G-protein, which was subsequently overcome by a profound activation of another G-protein with opposing activity. To test this hypothesis,the cells were pre-incubatedin 100 rig/mlpertussistoxin (PTX) for approximately16 hr. Inclusionof GTPySin the patch pipettenow only induced an inhibition of the current amplitude (Fig. 2C) and current amplitudeswere similar to those observed under ccmtrolconditions on initial..lweakthroughinto the w“hole cell mode (n = 4) (Fig. 2A). This result‘indicatesthat the

Dual modulation of an inward rectifer

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Fig. 1. RBL-2H3cells express an inwardlyrectifyingpotassiumchannel. Cells were held at a holdingpotential of –60 mV and stepped to a variety of potentials in 10 mV increments.(A) Under control conditions(.) an inward current was displayed at potentials more negative than –60 mV and a small outward current was noted at –50 mV. Both the outwardand inwardcurrentswere reducedby 500 PM cesium(~) and fully inhibitedby 1 mM cesium (A). (B) Controlinwardlyrectifyingpotassiumcurrents(0) werefullyinhibitedby 1 mMbarium(~).

stimulation of inwardly rectifying potassiumcurrents by GTPyS is mediated by a PTX-sensitive G-protein. The mechanisms involved in the modulation of the inward rectifier were further studied by stable transection of the RBL-2H3 cells with the ml muscarinic receptor. This receptor has been shown to couple with PTX-insensitiveG-proteins to activate PLC, resulting in release of calcium from intracellularstores and formation of the PKC activator, diacylglycerol(DAG) (for review see: Jones et al., 1992; Brann et al., 1993a,b; Caulfield 1993;Jones, 1993).Both calcium and stimulationof PKC have been implicated in modulation of inwardly rectifying potassium conductance (Kramer and Levitan, 1988; Mukai et al., 1992; Uchimura and North, 1990;Fakler et al., 1994; Takano et al., 1995;Jones, 1996).Application of 10 PM ACh from a pressure ejection pipette, resulted in a decrease in the inwardlyrectifyingconductance(Fig. 3). Both inward and outward currentswere reduced (Fig. 3C). Current amplitudesmeasured at the end of a 200 ms step to —120 mV were reduced from —301 f 39 to –214 +29 nA (n = 16) in the presence of 10PM ACh. The 31 ~ 5% (n = 16)reductionin current amplitudewas reversed on washout of the ACh. No change in the kinetics of the currents were observed (Fig. 3B). Under current-clamp conditions, application of 50PM ACh resulted in a depolarization of the cell membrane potential (Fig. 3D). The muscarinic receptor-induced inhibition of the inward rectifier remained robust after pretreatmentof the cells with 100 rig/mlof PTX for about 16 hr. Fifty micromolarACh produced a 50 + 5% (n = 3) inhibitionof the inward current amplitudein the presence of Pm. As the ml muscarinic receptor has been shown to inhibit an inwardly rectifying potassiumconductancevia activationof PKC (Jones, 1996),it is highly likely that in the RBL cell, the ml muscarinic receptor activates PKC via formation of DAG. Therefore, a further dissectionof the mechanism of action of the inhibition of the inward rectifier was carried out by application of the phorbol

.

esters PMA or PDBu to the cells (n = 9) to activate PKC (Fig. 4A). One hundred nanomolar PMA mimicked the effects of ACh application, showing a 33 ~ 9% (n =5) reduction in current amplitude. Pre-incubation of the RBL cells with 200 nM PMA reduced the inhibition of the inward rectifierby 50PM ACh to 5 +4% (n= 6). An increase in inwardly rectifying potassium current was noted, with activation of PKA in studies expressing IRK1 inXenopusooctyes(Fakler et al., 1994).Therefore, it is possible that the increase in current amplitude induced by GTPyS in the RBL cell may be due to stimulationof increasedcAMP levels,althoughthis is not usuallyvia a PTX-sensitiveG-protein.This was tested by applicationof the membrane permeant analog of cAMP, 8-bromo cAMP (Fig. 4B). Application of 500PM 8bromo cAMP resulted in a small but consistent decrease of 17 + 270 (n = 5) in inward current amplitude.Application of 10 UMforskolin,an activatorof adenylylcyclase, induced a 25 + 1370 reduction in inwardly rectifying potassiumcurrent (n= 2). Application of 50 PM ACh to the RBL cells pre-incubated with 500 PM 8-bromo cAMP, resulted in a 17 ~ 6?Z0(H= 3) inhibition of inwardlyrectifyingpotassiumcurrent amplitude,indicating that the muscarinic receptor-induced response was unlikely to be mediated entirely by increases in cAMP levels. DISCUSSION

The inward rectifierof the RBL-2H3 cell was found to be dually modulated by G-proteins.A PTX-sensitiveGprotein induced an initial increase in current amplitude followed by a PTX-insensitive G-protein-mediated reduction in potassium current amplitude. This differs from the studies of Wischmeyer et al. (1995) and McCloskey and Cahalan (1990), where only a decrease in inwardly rectifying potassium current amplitude was noted, with application of GTPyS. This may be due to differencesbetween the cell lines or due to the later time

S. V. F’.Jones

been observed in locus coeruleus neurons (Velimirovic et

1995). An increase in IRK1 current amplitude was noted on applicationof a PKA activator(Fakler et al., 1994)when IRK1 was expressed in Xenopus oocytes. However, this

al.,

:OIW’ROLGTPYS NOGTP

was not observed with IRK1 when expressed in a mammalian cell line (Jones, 1996). The activation of PKA, by increased cAMP levels is usually mediated via a PTX-insensitive G-protein such as Gs and, thus, is unlikely to induce an increase in current amplitude in the RBL-2H3 cell, which was PTX-sensitive. This is supported by the observation that stimulation of the ml muscarinic receptor did not result in an initial enhancement of the KIR amplitudein the RBL cell, although the

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Fig. 2. GTPyS induces an initial activation followed by inhibition of the inward rectifier. (A) Current amplitudes measured on breakthroughinto the whole cell mode at the end of 200 msec steps to – 110 mV were enhanced with 200 PM GTPyS(GTPYS)includedin the patch pipette in comparisonto control conditions (CONTROL).Omission of GTP (NO GTP) from the patch pipette, had no effect on current amplitudes at breakthrough. Preincubation of cells with 100rig/ml PTX abolished the effects of 200 PM GTPyS (PTX). (B) Time course of current amplitude normalized to the amplitudeof the current on breakthroughinto the whole cell mode, measured at the end of a 200 msec step to – 110 mV in control (0), without GTP (+) and with 200 KMGTPySincludedin the patch pipette (H). (C) Comparison of effects of 200PM GTPYSin control conditions (.) and after incubation with 100rig/ml PTX (H).

points, chosen to obtain measurementsin these other two studies. A similar dual modulationof an inward rectifier by PTX-sensitive and PTX-insensitive G-proteins has

ml muscarinic receptor has been shown to raise intracellular cAMP levels in a variety of cells (Jones et al., 1991b;Dittman et al., 1994;Gutkind et al., 1991).In addition, stimulationof PKA by application of 8-bromo cAMP to the RBL cell, resultedin a reductionin inwardly rectifyingpotassiumcurrent.Therefore, it is possiblethat the GTPyS-inducedenhancement of the inward rectifier in the RBL cell, may be mediated by a reduction in PKA activity,via inhibitionof adenylylcyclase, a mechanism known to involve PTX-sensitiveG-proteins. Alternatively, GTPyS may activate a G-protein that directly interacts with the channel. This has not been demonstrated with IRK1 to date, but is a major mechanism of activation of the GIRK-type channels, via the /?y G-protein subunits (Reuveny et al., 1994; Takao et al., 1994;Huang et al., 1995;Kofujiet al., 1995; Kunkel and Peralta, 1995; Lim et al., 1995). The identity of the inwardly rectifying potassium channel in RBL-1 cells was exhaustively studied by Wischmeyer et al. (1995). They used degenerate oligonucleotidescomplementaryto regions of homology acrossall inward rectifierssequencedto date, to clone the inwardly rectifying potassium channel endogenously expressed in the RBL cells. They only found evidence for a channel with very high homology to IRK1. Therefore, it is conceivable that the RBL-2H3 cell of this study also expresses IRK1, however, the possibility that other inwardlyrectifyingpotassiumchannelsare also expressed cannot be fully ruled out. The inhibitionof the inward rectifier in the RBL-2H3 cell appears to correlatewell with other studies on IRK1. Activation of the ml-muscarinic receptor, inclusion of GTPySin the patch pipette and phorbol esters all induced a reduction in current amplitude in the RBL cell. Inhibition of constitutively active inwardly rectifying potassium channels by stimulation of muscarinic (Uchimura and North, 1990) and substance P receptors (Takano et al., 1995) have been shown to be mediated via activationof PKC in variouscentral neurons.The mlmuscarinicreceptorhas been shown to reduce IRK1 via a mechanism involving PKC (Jones, 1996) and PKC activators,such as PDBu and SC1O,reduce IRK1 current amplitude(Jones, 1996;Fakler et al., 1994).Therefore, it is likely that the same pathway is involved in the RBL-

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Fig.3. Stimulationof ml muscarinic receptor inhibits inward rectifier.(A) Superimposedcurrent traces recorded from an RBL-2H3 cell during steps from a holding potential of –60 mV to a variety of potentials ranging from – 120 to –40 mV under control conditions. (B) The same steps superimposedmeasured during application of 10 pM ACh. (C) Currentvoltage curves measuredfrom a differentRBL cell showingthe inwardcurrent activation in control (0), during application of 10 ,uM ACh (~) and after washout of the ACh (+). (D) Voltage trace measured during current clamp mode shows membrane depolarization on application of 50 ~M ACh.

2H3 cell. This is supportedby the lack of effect of ACh on the inward rectifier of the RBL cell, when the cells were pre-incubatedwith phorbol ester. Increasing the concentration of cAMP by application of 8-bromo cAMP or forskolin also induced a reduction in KIR. Thk indicated that the reduction in current amplitude induced by GTPyS was also probably mediated, in part, by stimulation of adenylyl cyclase, althoughthe overallreductionin KIRinducedby 8-bromo cAMP was smaller than that observed with the phorbol esters. Increased levels of cAMP either via applicationof forskolin, membrane perrneant analogs of cAMP or via, ~-adrenergic receptor activation, have been shown to inhibit constitutively active inwardly rectifying potassium conductance in glial cells (Roy and Sontheimer, 1995)and in the ventricle(Koumi et al., 1995),indicating that this type of activity maybe widespread.No effect of increased cAMP levels was noted with IRK1 when expressed in a mammalian cell line (Jones, 1996).

Conversely, as stated earlier, IRK1 was enhanced by increased cAMP when expressed in Xenopus ooctyes (Fakler et al., 1994). As the recording conditions were virtually identical in this study and in that of Jones (1996), it is unlikely that this is the cause of the differences in the results with raised cAMP levels. However, it is possiblethat the small differencesnoted in the sequence of the IRK channel cloned from the RBL cell (Wischmeyer et al., 1995), when compared with IRK1 (Kubo et al., 1993a), may be responsible for the inhibitory activity of cAMP noted in the present study. The effect of cAMP could be mediatedvia protein kinase A and, thus, involve a potential phosphorylation site, althoughthe predicted sites are not differentbetween the clones.However, it is also possiblethat cAMP itself may modulate the channel. From this study it appears that a constitutivelyactive IRK1-like inwardly rectifying potassium conductance, showing similarity to inward rectifiers observed in a

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Fig. 4. Stimulationof PKC and PKA results in an inhibition of the inward rectifier.(A) Applicationof 500 nM PDBu (~) greatly reducesthe inwardcurrentamplitudepresent undercontrolconditions(.). (B) Five hundredmicromolar8-bromo-cAMP(B) also reduces the inward rectifier in comparisonto control conditions (.). In both cases cells were held at —60mV and stepped to a variety of potentials for 200 msec. Current amplitudes were measured at the end of the steps.

mechanism for coupling of m4 muscarinic acetylcholine variety of central and other neurons, can be dually receptors to calmodulin-sensitiveadenylyl cyclases: Crossmodulatedby second messengerssuch as PKC and PKA. over from G protein-coupled inhibition to stimulation. These mechanisms may well underlie many of the Biochemistry 33: 943–951. observed modulationsof inward rectifiersby various GDoupnik C. A., Davidson N. and Lester H. A. (1995) The protein coupled receptors in the CNS. inward rectifier potassium channel family. Curr. Opin.

Acknowledgements—The author would like to thank Drs M. R.

Brann and J. S. Gutkind for the kind gift of the ml muscarinic receptor viral construct and Dr C. Heilman for assistance with the muscarinic receptor transections. This work was fundedby National Institute of Neurological Diseases and Stroke Grant NS 29634. REFERENCES

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