cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels

CARDIOVASCULAR SYSTEM

Cell membrane-derived lysophosphatidylcholineactivates cardiac ryanodine receptor channels

Yuki Nakamura & Midori Yasukochi & Sei Kobayashi &Kiyoko Uehara & Akira Honda & Ryuji Inoue &

Issei Imanaga & Akira Uehara

Received: 21 June 2006 /Accepted: 3 July 2006 / Published online: 7 October 2006# Springer-Verlag 2006

Abstract Lysophosphatidylcholine (LPC) is metabolizedfrom a membrane phospholipid and modulates a variety ofchannels in the plasma membrane (PM). We examined LPCmodulation of cardiac ryanodine receptor (RyR) channels inthe sarcoplasmic reticulum (SR) using the planar lipidbilayer method to measure the single-channel currents.Micromolar concentrations of LPC increased the openprobability of the reconstituted RyR channels irrespectiveof whether LPC was added to the cis or trans chamber. LPCalso increased the membrane capacitance of the bilayer. The

effects of LPC contrasted well with those of sphingosyl-phosphorylcholine (SPC). Taken together, these resultssuggest that amphipathic lipid LPC does not bind directlyto the RyR channel protein, but rather, is incorporated intothe bilayer membrane and activates the channel. Thus, weconsider cell membrane-derived LPC to be a putativeendogenous mediator that activates not only plasmamembrane channels but also RyR channels and inducesarrhythmogenic Ca2+ mobilization in cardiomyocytes.

Keywords Heart . Ca2+ release . Ryanodine receptor .

Lysophosphatidylcholine . Single channel . Ischemia

Introduction

Lysophosphatidylcholine (LPC) is metabolized by phos-pholipase A2 from phosphatidylcholine (PC), a majormembrane phospholipid [19, 22, 30]. Accumulation ofLPC leads to the development of cardiac arrhythmia duringmyocardial ischemia [4, 6, 17].

Exogenous LPC is known to alter the contractility, such ascontracture, of cardiacmyocytes and perfused hearts due to theelevation of intracellular Ca2+ concentration [3, 10, 20, 29,31]. Most of these studies ascribed the change in contractilityto increased entry of extracellular Ca2+ through plasmamembrane (PM) channels. However, Yu et al. [34] proposedthat the LPC-induced increase in intracellular Ca2+ concen-tration depends on both extracellular Ca2+ entry via PMNa+/Ca2+ exchangers and Ca2+ release from the sarcoplasmicreticulum (SR) store. Because the effects of LPC on SRryanodine receptor (RyR) channel proteins have not beendirectly examined to date, the hypothesis that SR Ca2+

release is involved in intracellular Ca2+ mobilization has notbeen confirmed. The present study was thus designed to

Pflugers Arch - Eur J Physiol (2007) 453:455–462DOI 10.1007/s00424-006-0141-y

Y. Nakamura :A. Honda : R. Inoue :A. Uehara (*)Department of Physiology, School of Medicine,Fukuoka University,45-1, 7-chome Nanakuma, Jonan-ku,Fukuoka 814-0180, Japane-mail: [email protected]

M. YasukochiLaboratory of Human Biology, School of Medicine,Fukuoka University,45-1, 7-chome Nanakuma, Jonan-ku,Fukuoka 814-0180, Japan

S. KobayashiFirst Department of Physiology, School of Medicine,Yamaguchi University,1144 Kogushi,Ube 755-0067, Japan

I. ImanagaGeneral Research Center for Medical Science,School of Medicine, Fukuoka University,45-1, 7-chome Nanakuma, Jonan-ku,Fukuoka 814-0180, Japan

K. UeharaDepartment of Cell Biology, School of Medicine,Fukuoka University,45-1, 7-chome Nanakuma, Jonan-ku,Fukuoka 814-0180, Japan

clarify whether LPC acts on cardiac RyR channels usingsingle-channel current measurements.

LPC is an amphipathic lipid and can, therefore, interactwith the RyR channels through direct binding to the RyRchannel protein or through incorporation into the mem-brane lipid bilayer. Here, we investigated which interac-tion mechanism accounts more plausibly for the possibleLPC modification of RyR channel function. Then, weattempted to clarify the actions of LPC by comparingwith those of SPC, metabolized from sphingomyelin(SM) of another membrane phospholipid, which iscapable of intermolecular binding to the cytoplasmicdomain of RyR channels [27, 33].

Our experimental results confirmed that RyR channelsare activated by LPC. This activation appears to be trig-gered by incorporation of LPC into the SR membrane,followed by membrane curvature. The present report is thefirst demonstration of LPC action on intracellular channelssuch as RyR; previous studies have been restricted to PMchannels.

Materials and methods

Chemicals

Lysophosphatidylcoline (LPC) was obtained from BiomolResearch Laboratory (Plymouth Meeting, PA) and fromSigma Chemical (St. Louis, MO). Phosphatidyl ethanol-amine (PE) and phosphatidylcholine (PC) were from AvantiPolar Lipids (Alabaster, AL). All other chemicals werefrom Sigma Chemical (St. Louis, MO).

Lipid bilayer experiments

Junctional SR membranes were prepared from rabbitventricular muscles according to the method of Hombergand Williams [9]. The SR membranes were solubilized withCHAPS, and then, purified RyR proteins were isolated witha continuous sucrose gradient according to the method ofUehara et al. [26]. Briefly, planar lipid bilayers were formedacross a 300-μm hole in a Derlin partition separating twoexperimental compartments (cis and trans) from 50 mg/mldecane–lipid mixture. The lipid mixture contained phos-phatidylethanolamine and phosphatidylcholine in a weightratio of 7:3. The capacitance of bilayer membranescontaining no RyR channels was measured by repetitiveramp pulses applied to bilayers. Bilayers had a capacitancethat ranged between 200 and 300 pF. The trans and cischambers were held at virtual ground and at variousmembrane potentials, respectively. RyR samples weredropped to the cis chamber and stirred until they wereincorporated into the bilayers. Single-channel current flow

through the bilayer was measured with a patch clampamplifier. Junction potentials were measured and thecorresponding corrections were made.

The sidedness of channels incorporated in the bilayerwas identified by ATP sensitivity of the cytoplasmic side.During single-channel recording, the chambers containedidentical solutions of 210 mM CsOH, 10 mM ethyleneglycol-bis (b-aminoethyl ether) N,N,N,N_-tetraacetic acid(EGTA), required CaCl2 to achieve desired pCa values, and140 mM piperazine-N,N_-bis (ethansulfonic acid) (PIPES)(pH 7.4). The pCa values were determined using themethod of Fabiato and Fabiato [5] corrected by Tsien andRink [25]. The RyR channel was activated not by caffeinebut by test pulses or holding potentials. Data were collectedfrom the bilayer membranes containing only a singletransition level between an open state and a closed statethroughout more than 10 min in the recording time. Afterthe experiments, the single channels were identified as RyRchannels by confirming the locked open state after the cisaddition of 10 μM ryanodine and channel activation by5 mM caffeine (see Uehara et al. [27]). All experimentswere performed at room temperature.

Data acquisition and analysis

Single-channel activity was measured with an Axopatch200B amplifier and was stored directly on a computer usingpCLAMP software (Axon Instruments, Foster City CA).Data were low-pass filtered at 1 kHz (fc), digitized at4 kHz, and analyzed by pCLAMP software. The minimumdetectable event duration (equal to 0.179�2/fc, i.e., thedead time of the system) was 358 μs. Gating parameterssuch as open probability, mean open and closed time wereobtained from idealized current traces. Distributions ofdwell time duration were fitted using the maximum loglikelihood method [2].

Statistical methods

Data are expressed as meansTSD Statistical differenceswere determined by Student_s t-test. Mean values wereconsidered significantly different when the P value was lessthan 0.05.

Results

LPC action on RyR single-channel currents

The stationary single-channel currents of control RyR werecharacterized with planar lipid bilayer methods (upperpanel of Fig. 1a,b). The chambers contained symmetricalsolutions of CsOH. The concentration of cis and trans Ca2+

456 Pflugers Arch - Eur J Physiol (2007) 453:455–462

was 1 μM. The holding potentials were +30 mV. Openevents are shown as upward deflections. The modifiedchannels at 1 min after the cis addition of 5 μM LPC of amembrane phospholipid appeared to open more frequentlythan the control channels (lower panel of Fig. 1a). Channelgating was unaltered by vehicle solution containing ethanolor albumin without LPC. The open time of the LPC-modified channels appears to be prolonged. The modifiedchannels exhibited no changes in current amplitude. LPCactivated RyR channels similarly when added to the transsolution (lower panel of Fig. 1b). LPC, thus, increased thesingle-channel activity of RyR, irrespective of whether itwas added to the cis or trans chamber.

Subsequently, the nonstationary single-channel currentsof RyR were compared before (upper panels) and after(lower panels) the addition of 5 μM LPC (Fig. 2a,b). Thetest pulses from the holding potential, 0 mV, for 3.5 s wereapplied to the bilayer. The test pulses to elicit therepresentative current traces shown in Fig. 2a,b were +40mV in amplitude. In this case, the Ca2+ concentration of thebath solution was 10 μM. The other experimental con-ditions in Fig. 2 were identical to those in Fig. 1. Thenonstationary currents at any voltages were consistentlyactivated by both cis (lower panel of Fig. 2a) and trans(lower panel of Fig. 2b) additions of LPC (Fig. 2a). LPCexerted more prominent effects on the RyR channels atmore negative potentials; the open probability of the LPC-modified channel, thus, became less voltage dependentthan that of the control channel (Fig. 2c). The slopeconductance, as calculated from I–V relation, was not

altered by LPC (not shown). The experimental data asshown in Fig. 2a were used for the following analyses ofgating parameters (Figs. 3, 4).

LPC-induced alteration of gating parameters

The fundamental gating parameters of mean open andclosed time were calculated from the nonstationary RyRcurrents (Fig. 3). The mean open time was 2.72T0.48 and11.35T2.91 ms in the absence and the presence of 5 μM cisLPC, respectively. The mean closed time was 0.91T0.06and 0.76T0.12 ms in the absence and the presence of cisLPC. The mean open time was significantly altered by LPCbut the mean closed time was not. In the case of SPC ofanother phospholipid metabolite, on the contrary, the meanopen time does not change, but the mean closed time issignificantly altered (see Uehara et al. [27], Yasukochi et al.[33]). Taken together, these findings suggest that LPCdecreases the rate of channel closing, while SPC decreasesthe rate of channel opening.

To characterize in more detail the changes in gatingkinetics, dwell time analysis before and after addition of5 μM LPC to the cis chamber was conducted, as shown inFig. 4. The time constants and the relative areas of dwellstate components were obtained by multiple exponentialfitting. Under the present condition, the histogram for thecontrol channel was described by four open state compo-nents with time constants of 0.20T0.16, 1.70T0.15,5.87T0.20, and 57.63T1.12 ms and by a closed statecomponent with a time constant of 0.58T0.67 ms (upper

Fig. 1 Effects of LPC on stationary RyR currents. a Representativecurrent traces obtained before (upper) and after (lower) cis addition ofLPC, respectively. -c Closed state. b Representative current traces

obtained before (upper) and after (lower) trans addition of LPC,respectively. -c Closed state

Pflugers Arch - Eur J Physiol (2007) 453:455–462 457

panels). After addition of LPC, the time constants of theopen state components were 0.21T0.13, 8.85T0.15,58.50T0.24, and 201.40T1.62 ms, and that of the closedstate was 0.54T0.50 ms (lower panels). The time constantsof the third and fourth open state components wereprolonged markedly by LPC. These results obtainedsuggest that the open state properties of the LPC-modifiedchannel are preferentially altered.

LPC-induced alteration of membrane capacitance

The effects of LPC on the capacitance of bilayer mem-branes, in which the RyR channel was not incorporated, areshown in comparison with those of SPC (Fig. 5). The LPCconcentration used here was 5 and 50 μM. The capacitance(% of control pF) was monitored by repetitive ramp pulses.The capacitance was significantly increasing with LPCconcentration but not altered by SPC. However, the bilayermembranes were so frequently ruptured by LPC doseshigher than the order of 10 μM that we failed to confirm a

dose-dependence of LPC action quantitatively. Theseresults suggest that LPC is incorporated into the bilayerwhile SPC is not.

Discussion

The present study demonstrated that intracellular channelssuch as cardiac RyR can be modified by LPC metabolizedfrom membrane phospholipid PC. LPC increased the single-channel open probability after addition to either the cis ortrans chamber. In addition, LPC increased membrane capac-itance. These results suggest that the LPC-induced activationof RyR channels is mediated not by direct intermolecularbinding between the lipid LPC and the RyR channel protein,but rather, by incorporation of LPC into the membrane itself.

Uehara et al. [27] and Yasukochi et al. [33] havereported the effects of SPC on RyR channels using thesame materials and methods as the present study. Therefore,the effects of LPC and SPC can be directly compared.

Fig. 2 Effects of LPC on nonstationary RyR currents. a Representa-tive current traces obtained before (upper) and after (lower) cis LPC,respectively. -c Closed state. b Representative current traces obtainedbefore (upper) and after (lower) trans LPC, respectively. -c Closed

state. c Open probability Po of the RyR currents plotted vs voltages.Solid and open circles denote the data points which were obtained bythe cis and trans addition of LPC, respectively. N=8


Fig. 4 Effects of LPC on dwell times of RyR channels. Open (left) and closed (right) time histograms before (upper) and after (lower) LPC areillustrated. N=10

Fig. 3 Effects of LPC on mean open time (left) and mean closed time (right) of RyR channels. Mean open time is significantly different (p<0.05),but mean closed time is not significantly different between control and LPC-modified channels. N=8


Figure 6a summarizes the metabolism of SPC and LPC andtheir hypothetical action mechanisms in heart myocytesunder physiological and pathophysiological conditions.During ischemia, PC is highly metabolized to LPC, whichfuses with the SR membrane and exerts a nonspecific actionon RyR channels in the SR membrane (Fig. 6b). On theother hand, during hyperlipidemia or apoptosis, SM ishighly metabolized to SPC, which binds directly to thecytoplasmic domain of RyR channel protein and exerts a

specific action on the RyR channels. Thus, LPC and SPCmetabolized from membrane phospholipids can modulateRyR channel activity via their respective interactionmachinery, which differs from each other greatly. Wepropose that the PM of T-tubule and the SR membraneare so closely adjacent in the diad junctions in the heartmuscle cells (see Fig. 6a) that PM-derived LPC and SPCcould diffuse to SR with a minimal dilution enough to exertsignificant effects on the RyR channels.

Fig. 6 Simultaneous illustration of LPC and SPC action machinery. aLipid metabolism and action in cardiomyocyte. Note the T-tubulestructure from which PM-derived LPC can diffuse near the SRchannel. b LPC incorporation into the bilayer membrane and SPC

binding on the RyR protein (modified after [8]). SM sphingomyelin,PC phosphatidylcholine, SR sarcoplasmic reticulum membrane, PLplasma membrane

Fig. 5 Effects of LPC and SPC on membrane capacitance. a Increase in capacitance by LPC. b No change in capacitance by SPC. Asterisk (*):p<0.05; double asterisks (**): p<0.01. N=5


LPC-induced activation has been reported in the nonse-lective cation channels of cardiac myocytes [13, 15] andsmooth muscle cells [11, 24]. Xu [32] suggested that thisLPC-induced activation in the nonselective cation channelsis triggered LPC and/or SPC receptors coupled withG-proteins and subsequent receptor-mediated signal trans-duction. Moreover, LPC-induced activation accompanied byincreased [Ca2+]i was reported in the TRPC Ca2+ channelsof smooth muscle cells [7, 21] and in the delayed rectifierHERG K+ channels of cardiac myocytes [30]. LPC-inducedactivation in HERG channels was inferred to be mediatedthrough direct interaction between the lipid and the channelprotein. In contrast, the inward rectifier K+ channels ofcardiac myocytes are known to be inhibited by LPC, thus,resulting in depolarization of membrane potential [1, 12].LPC augmented the sustained currents of the voltage-gatedNa+ channels, which was ascribed to the ischemic [Na+]irise and subsequent [Ca2+]i rise via Na+/Ca2+ exchangersleading to diastolic tension in the heart [23, 28].

Thus, previous studies have indicated that a variety ofPM channels are sensitive to LPC in cardiovascular cells. Ithas been demonstrated that LPC sensitivity is mediateddifferently by interaction between the ligand LPC and itsreceptor protein and by the more direct interaction of LPCbinding to the channel protein in nonselective cationchannels and HERG channels, respectively. The LPCsensitivity of RyR channels, which is suggested here to betriggered by incorporation of LPC into the bilayer mem-brane, is not identical to the sensitivities of nonselectivecation channels and HERG channels. Thus, the mechanismunderlying the action of LPC apparently varies fromchannel to channel.

LPC-induced channel activation mechanisms have beenpreviously examined in single-channel current experimentswith a planar lipid bilayer method. The mechanisms arewell understood in prokaryotic bacterial mechanosensitiveMscL channels [18]. Exogenously added LPC markedlyactivates single-channel currents in MscL. The manner ofthis channel activation, in which opening frequency andopen time are increased by LPC, appears to be similar tothat of RyR channels described by us [27, 33]. Perezo et al.[18] inferred that LPC induces membrane curvature byinsertion into the outer leaflet of the plasma membranebilayer and augments the activity of mechanosensitivechannels. Lundbaek and Andersen [14] similarly suggestedthat this membrane curvature alters the membrane distor-tion energy, and the mechanical stress of LPC insertion intothe bilayer membrane causes embedded channel activationin the primitive and small channels of prokaryotic bacteria.Maingret et al. [16] reported that this membrane curvaturemechanism also underlies the LPC-induced activation oftwo-pore domain K+ channels, such as eukaryotic TREK-1and TRAAK, which are also mechanosensitive. Thus, such

membrane curvature via lysophospholipid insertion that isrelevant to the mechanosensitivity is also very likely, as ageneral mechanism, to underlie the physiological andpathophysiological LPC-induced activation of cardiacRyR, the molecular weight of which is the highest amongprokaryotic and eukaryotic channels.

In our planar lipid bilayer experiments, isolated proteinsof RyR channels were used. These channels lacked receptorproteins specific for LPC and/or SPC [32] in the plasmamembrane. It is, thus, not likely that LPC receptor-mediatedmechanisms [32] are involved in the interaction betweenLPC and RyR. Moreover, the single channels of purifiedRyR in lipid bilayer are lacking in local control by most ofthe proteins naturally present in their microdomain (kinases,phospatases, FKBP12.6, mAKAP, junctin, calsequetrin,triadine). This local control should be taken into accountfor the LPC effects on the single RyR channel currents inthe cardiomyocytes.

In conclusion, the present results suggest that cardiacRyR channels are activated via incorporation of cellmembrane-derived LPC into the SR membrane. LPC couldphysiologically and pathologically act as an endogenousmediator that activates both PM channels and SR RyRchannels, thus, inducing arrhythmogenic Ca2+ mobilizationin cardiomyocytes. To confirm that LPC is such a mediator,further studies including knockout and knockdown experi-ments on RyR channels in intact cells are necessary.

Acknowledgements This work was supported in part by a Grant-inAid for Scientific Research from the Ministry of Education, Scienceand Culture of Japan.

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cell membrane-derived lysophosphatidylcholine activates cardiac ryanodine receptor channels

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