Cell Calcium 38 (2005) 557–567

Type-3 ryanodine receptor involved in Ca2+-induced Ca2+ release andtransmitter exocytosis at frog motor nerve terminals

Masakazu Kubotaa,1, Kazuhiko Naritad, Takashi Murayamac, Shinichi Suzukia,Satoko Sogaa, Jiro Usukurab, Yasuo Ogawac, Kenji Kubaa,e,∗

a Department of Physiology, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japanb Department of Anatomy, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan

c Department of Pharmacology, Juntendo University, School of Medicine, 2-2-1 Hongo, Bunkyoku, Tokyo 113-8421, Japand Department of Physiology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192, Japan

e Laboratory of Anatomy and Physiology, Faculty of Nutrition, Nagoya University of Arts and Sciences, 57 Takenoyama,Iwasaki-cho, Nissin, Aichi 470-0196, Japan

Received 9 July 2005; received in revised form 15 July 2005; accepted 22 July 2005Available online 12 September 2005

Abstract

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Ca2+-induced Ca2+ release (CICR) occurs in frog motor nerve terminals after ryanodine receptors (RyRs) are primed for activonditioning large Ca2+ entry. We studied which type of RyR exists, whether CICR occurs without conditioning Ca2+ entry and how RyRre primed. Immunohistochemistry revealed the existence of RyR3 in motor nerve terminals and axons and both RyR1 and RyRbers. A blocker of RyR, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) slightly decreased rises in inular Ca2+ ([Ca2+]i ) induced by a short tetanus (50 Hz, 1–2 s), but not after treatment with ryanodine. Repetitive tetani (50 Hz for 10 s) produced repetitive rises in [Ca2+]i , whose amplitude overall waxed and waned. TMB-8 blocked the waxing and waning compyanodine suppressed a slow increase in end-plate potentials (EPPs) induced by stimuli (33.3 Hz, 15 s) in a low Ca2+, high Mg2+ solu-

ion. KN-62, a blocker of Ca2+/calmoduline-activated protein kinase II (CaMKII), slightly reduced short tetanus-induced rises in [2+]i ,ut markedly the slow waxing and waning rises produced by repetitive tetani in both normal and low Ca2+, high Mg2+ solutions. Likeise, KN-62, but not KN-04, an inactive analog, suppressed slow increases in EPP amplitude and miniature EPP frequency d

etanus. Thus, CICR normally occurs weakly via RyR3 activation by single impulse-induced Ca2+ entry in frog motor nerve terminand greatly after the priming of RyR via CaMKII activation by conditioning Ca2+ entry, thus, facilitating transmitter exocytosis andlasticity.2005 Elsevier Ltd. All rights reserved.

eywords: Ca2+-induced Ca2+ release; Type-3 ryanodine receptor; Presynaptic terminal; Transmitter exocytosis; Plasticity; Frog neuromuscular jun

. Introduction

Much evidence has been accumulated for activation ofICR [1–4] via ryanodine receptors (RyRs) in neurons

4–11] (see reviews[12–14]). Its physiological roles in theegulation of the cell membrane excitability[15–20] and

∗ Corresponding author. Tel.: +81 561 75 2559; fax: +81 561 75 2559.E-mail address: kubak@nuas.ac.jp (K. Kuba).

1 Present address: Department of Sports Medicine, Graduate School ofedicine, Nagoya University, Nagoya 464-8601, Japan.

neuronal differentiation[21] have well been establisheEvidence for the role of CICR in synaptic transmisshowever, is limited to certain types of presynaptic termi[22–29].

Frog neuromuscular junction had been the preparatioprovided the basic concept of transmitter release mechaat presynaptic nerve terminals. Ca2+ entry through voltagegated Ca2+ channels is well known to activate a sequencevents for transmitter exocytosis[30]. Recent experimentsfrog motor nerve terminals, however, have shown strongdence for the role of CICR as an additional origin of Ca2+ in

143-4160/$ – see front matter © 2005 Elsevier Ltd. All rights reserved.oi:10.1016/j.ceca.2005.07.008

558 M. Kubota et al. / Cell Calcium 38 (2005) 557–567

the activation of transmitter exocytosis[22,23]. CICR in frogmotor nerve terminals has unique properties that the full acti-vation requires the conditioning large Ca2+ entry by repetitivenerve activity, which converts the state of ryanodine recep-tors from the ‘sleeping’ (incapable of being activated) to anactivable state[22]. Henceforce, we call this mechanism ofconversion the priming of RyR or CICR. A further studyshowed that this mode of activation of CICR contributes tothe generation of short-forms of synaptic plasticity, augmen-tation and potentiation[23].

Among many unsolved problems as to the mechanism ofCICR in frog motor nerve terminals, three specific questionswere asked in this study. First, what is the type of RyR in frogmotor nerve terminals? Two isoforms of RyR, type 1(RyR1)and type 3 (RyR3), which are also referred to as�-RyR and�-RyR, respectively, exist in almost equal amount in frogskeletal muscle[31,32]. mRNAs of both isoforms were alsodetected in frog brain[32]. However, it is not known whichtype of RyR exists in the frog motor nerve terminals. Second,does CICR occur in response to impulse-induced Ca2+ entryeven without the conditioning Ca2+ entry to prime RyRs?Third, how RyRs are primed by repetitive Ca2+ entries intothe terminals? The mechanism may involve a Ca2+-dependentsignaling process.

We studied these issues by applying histochemical, Ca2+

imaging and intracellular recording techniques to frog motorn andR kerso II)w Ther tiono lse-i n-t tent.T rgeC ofC

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RTKKKRRGDRYSVQ, which corresponds to 3478–3491of RyR1; for RyR3 antibody, KKRRRGQKVEKPE(4369–4381 of mammalian RyR3, which was the same asthat of frog RyR3 except for K at 4377 instead of V)[33]; andfor anti-pan RyR antibody, HPASKQRSEGEKVR (151–164of RyR1 and 152–165 of RyR3)[34]. These antibodieswere produced in rabbits and affinity-purified by proce-dures described previously[31]. Anti-RyR1 and anti-RyR3antibodies react specifically with RyR1 and RyR3, respec-tively, and there was no cross-reaction between them. Anti-pan RyR antibody reacts with all kinds of RyR isoforms,whose entire sequences were reported. Mouse monoclonalanti-chicken ryanodine receptor antibody (34C, Biomol, Ply-mouth, PA)[35] that reacts with both frog RyR1 and RyR3was also used. In some experiments, each of these pep-tides was added to the PBS containing the correspondingantibody.

The preparations were stained with Alexa 488-conjugatedrabbit or mouse IgG (Molecular Probes) at the concentrationof 15�g/ml in PBS containing 1% bovine serum albumin atroom temperature for 1 h. The muscles were mounted withan anti-fading agent (ProLong, Molecular Probes). Fluores-cence images were taken by a confocal scan unit (MRC-600, Nippon BIO-RAD) attached to an inverted microscope(Nikon TMD-300, Water immersion 40× objective, NA1.15). Stained muscles were scanned in aX–Y plane withb as urew ainedw he3 s of0 tis-s yR1,a f thec imum(

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hosed db witht er’ss ,5 TA-1 pre-v th ac atsuP tensi-fi VA)a bjec-t her n tot cos-mr n

erve terminals. Antibodies raised against frog RyR1yR3 or both were used to solve the first issue, while blocf RyR and Ca2+/calmoduline-activated kinase II (CaMKere applied to clarify the second and third issues.

esults indicate that CICR normally occurs via activaf RyR3 to some extent in response to single impu

nduced Ca2+ entry in frog motor nerve terminals, coributing to transmitter exocytosis to a considerable exhey further suggest that CaMKII activation by a laa2+ entry is involved in the priming of the mechanismICR.

. Materials and methods

.1. Immunohistochemistry

Frogs (Rana nigromaculata) were decapitated and cueous pectoris muscles were isolated with the innerverve. The muscles were fixed with 4% paraformaldehyodium phosphate-buffered solution (0.11 M, pH 7.4: Pt 4◦C for 2 h, washed with PBS, permeabilized with 0.riton X-100 in PBS containing 4% bovine serum alin (BSA) at room temperature for 2 h and rinsed wBS. The muscles were exposed to an antibody againsyR1, RyR3 or both in PBS with 1% bovine serum alin at room temperature at the IgG concentration of 1ilution.

Antibodies used were polyclonal antibodies raised agarticular sequences of peptides: for anti-RyR1 antib

lue laser (488 nm, 0.15–0.45 mW) atZ levels varied attep of 0.2 or 0.3�m. The diameter of the confocal apertas set to be 1.9–3.1 mm for the images of the tissues stith anti-RyR1, anti-RyR3 and anti-pan RyR antibody. T.1 mm diameter yielded the lateral and axial resolution.15�m and 1.25�m, respectively. For the images of theues stained with 34C antibody and those with anti-Rnti-RyR3 or anti-pan RyR antibody in the presence oorresponding epitope, the diameter was set to be max9.3 mm).

.2. Ca2+ imaging and intracellular recording

Experimental procedures are essentially similar to tescribed[23]. Frogs (R. nigromaculata) were anesthetizey cooling and cutaneous pectoris muscles were isolated

he innervating nerve. The composition of normal Ringolution (mM) was NaCl, 112; KCl, 2; CaCl2, 1.8; glucose.0; Hepes-Na, 5.0 (pH 7.4). K-salt of Oregon Green BAP(OGB-1) was loaded into the terminals as described

iously [22]. Fluorescence of OGB-1 was measured wiooled CCD-camera (Argus/HiSca, C6790-81, Hamamhotonics, Hamamatsu, Japan) through an image iner (Stardancer 2, Videoscope International, Sterling,ttached to an upright microscope (Zeiss Axioscope, o

ive 60× water/N.A. 0.95, Karl Zeiss Japan, Tokyo). Tatios of the images during and after nerve stimulatiohat before stimulation were analyzed (Argus or Aquaos, Hamamatsu Photonics, Hamamatsu, Japan[23]). The

atios were converted to [Ca2+]i values using the equatio

M. Kubota et al. / Cell Calcium 38 (2005) 557–567 559

with assumption of 70 nM for the resting level of [Ca2+]i and240 nM for the dissociation constant of OGB-1[22]. Twomodes of tetanus were applied to the nerve either to activateCICR or to monitor relative changes in Ca2+ entry by nerveimpulses in different conditions. First, a short tetanus of 15,50 or 100 pulses at 50 Hz was applied to record changes in[Ca2+]i mostly derived from Ca2+ entry by nerve impulses innormal Ringer containingd-tubocurarine (3–10�M). Sec-ond, long tetani of 50 Hz for 15 s with a 5 s pause betweeneach of tetani were repetitively applied for 4–6 min in nor-mal Ringer or a low Ca2+, high Mg2+ solution. This causedthe priming, subsequent activation and finally inactivation ofCICR [23].

For intracellular recording of end-plate potentials (EPPs)and miniature EPPs (MEPPs), frog (R. nigromaculata) cuta-neous pectoris or sartorius muscles with the innervating nervewere used. There was no difference in the results obtainedfrom these two types of muscle. The composition of normalRinger’s solution (mM) was NaCl, 112; KCl, 2.5; CaCl2,1.8; Hepes-Na, 5.0; glucose, 5.0 (pH 7.4). EPPs induced bya tetanus of 33.3 Hz for 20 or 30 s and stimuli at 1 Hz beforeand after the tetanus were recorded with an intracellular elec-trode (filled with 3 M KCl) in low Ca2+, high Mg2+ solutions(CaCl2, 0.2–0.5 mM and MgCl2, 10 mM) at 20–24◦C. Theywere stored on a PCM recorder (40 kHz, RD101T or RD125T,

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3. Results

3.1. Type of ryanodine receptor in frog motor nerveterminals

Antibody common to both RyR1 and RyR3, polyclonalanti-pan RyR antibody well stained the nerve terminals(Nt/Nip/Ni = 148/31/31), parent axons (Nip/Ni = 31/31)and muscle fibers (Nip/Ni = 31/31, Fig. 1A). The nerveterminal was identified by a number of criteria; a longprocess of homogenous diameter of 1.5–2�m extendingfrom the parent axon in the nerve bundle and running onthe surface of the muscle fiber in parallel with its longaxis. Stained muscle fibers showed clear striations at1.5–3�m interval. Some images (Nip/Ni = 3/31) showedstaining of cells like Schwann cells (not shown) havinga round cell body and two or three long processes[36].An epitope peptide for anti-pan RyR antibody blockedthe staining of all these structures (Nip/Ni = 5/5, Fig. 1B).Monoclonal antibody 34C, which recognizes the epitoperegion common to both RyR1 and RyR3[35] well stainedthe muscle in a striation pattern (Nip/Ni = 21/21), lightlythe nerve terminals (Nt/Np = 21/6) and axons (Nip/Ni = 6/21,Fig. 1C).

Anti-RyR3 antibody well stained the nerve terminals(N /N = 141/73), axons (N /N = 70/73) and muscle fibers( edc wannc ep ain-i dw nottP annc ra clefiT s inf aree ibed[

3c

ib-i talm s-m -i for3 s in[ tim-uP ft afterw n

TEAC, Tokyo, Japan). The quantal content (QC) of EPPcalculated by dividing the mean amplitude of EPP bymean amplitude of MEPPs recorded under the same ction.

2.3. Drugs

OGB-1 was obtained from Molecular Probes(Eugene, OR). TMB-8 (8-(N,N-diethylamino)octyl3,4,5trimethoxybenzoate hydrochloride) and ryanodine wereSigma Chemical Co. (St. Louis, MO) or Tokyo Kasei KogCo. (Tokyo, Japan). KN-62 and KN-04 were obtained fSigma or Seikagaku Kogyo Co. (Tokyo, Japan).

2.4. Statistics

The number of observations in immunohistochemwas expressed in two ways. The number of nerve termpositively stained with an antibody was shown asNt. Thenumber of the image plane that demonstrated positive sing of nerve terminals, axon or muscle fibers was showNip. The number of image plane examined was shown aNi .For the positive staining of the nerve axons, muscle fibecells like Schwann cells,Nip was shown withNi . The numbers of observations in Ca2+ imaging and electrophysiolog(n) were those of the terminal and end-plate, respectivelydent’st-test was applied, when necessary.χ2-test was appliefor fitting the distribution of the magnitude of the actionTMB-8 on short tetanus-induced rises in [Ca2+]i in the termi-nals to the normal distribution or the sum of the two nordistributions.

t i ip iNip/Ni = 93/93, Fig. 2A). Stained muscle fibers showlear striations. Positive staining was also seen in Schell-like cells (not shown:Nip/Ni = 31/73). An epitopeptide for anti-RyR3 antibody blocked all these st

ngs (Fig. 2B, Nip/Ni = 5/5). Anti-RyR1 antibody staineell the muscle fibers in a striation pattern, but

he nerve terminals and axons (Fig. 2C, Nip/Ni = 13/13).ositive staining was also seen in cells like Schwells (not shown:Nip/Ni = 3/13). An epitope peptide fonti-RyR1 antibody blocked the staining of the musbers and Schwann cell-like cells (Fig. 2D, Nip/Ni = 9/9).he results suggest that RyR3 but not RyR1, exist

rog motor nerve terminals, while the muscle fibersndowed with both RyR1 and RyR3 as already descr

31].

.2. Does CICR occur in response to Ca2+ entry withoutonditioning stimulation?

To answer this question, we used TMB-8, which inhted caffeine- and high K+-induced contractions of skele

uscle without effects on Ca2+ uptake into the sarcoplaic reticulum (SR)[37,38] and did not require the prim

ng of RyR for its action to take place. TMB-8 applied0–120 min reversibly decreased the amplitude of rise

Ca2+]i induced by a short tetanus (50 Hz, 15, 50 or 100 sli) to 93.7± 1.9% (n = 47: P < 0.01), 87.6± 5.0% (n = 20:< 0.05) and 91.1± 2.8% (n = 47:P < 0.01), respectively, o

he average of those before application and 30–60 minash (Fig. 3A). To confirm that the small blocking actio

560 M. Kubota et al. / Cell Calcium 38 (2005) 557–567

Fig. 1. Immunohistochemistry of ryanodine receptors in frog skeletal muscles with antibodies common to RyR1 and RyR3. All the images were taken byconfocal imaging. (A) Staining of RyRs in the nerve terminals, parent axons and muscle fibers with anti-pan RyR antibody. (B) Blockade by the epitope peptideof staining with anti anti-pan RyR antibody. The sensitivity of the imaging system was set to be 10 times higher than that for the image in A by increasingthesize of the confocal aperture and the photomultiplier amplifier gain. (C) Staining of ryanodine receptors in the nerve terminals, parent axons and muscle fiberswith 34C antibody. The sensitivity of the imaging system was set to be 21 times higher than that for the image in (A). Note the branching of the terminals fromthe parent axon and the striation of the muscle fibers in (A) and (C).

of TMB-8 results from the action on RyRs, the nerve waspre-stimulated by repetitive tetani in the presence of ryan-odine (10�M) applied for 60 min. Under this condition, thesmall blocking action of TMB-8 was not seen (98.6± 2.6% ofthe control (n = 23) for 50 stimuli (50 Hz) and 103.7± 5.0%(n = 35) for 100 stimuli,Fig. 3B).Fig. 3C shows the frequencydistributions of the effects of TMB-8 pooled from all the data.Although variability in each distribution is fairly large, it isevident that TMB-8 significantly suppresses short tetanus-induced rises in [Ca2+]i , but not after the blockade of RyRwith ryanodine. Thus, TMB-8 indeed blocks RyRs, albeitit is known to block nicotinic acetylcholine receptors[39],affect cell membrane excitability[40] and facilitate Ca2+

mobilizations[41] in other types of cell. Consequently, itis clear that CICR is normally activated by impulse-inducedCa2+ entry in a fraction of terminals. Furthermore, the dis-tribution in the absence of ryanodine is nicely fitted bythe normal distribution (Fig. 3C), indicating no dichotomyin the population of terminals as to the activation ofCICR.

3.3. CICR is normally involved in transmitter exocytosis

The foregoing results raise a possibility that activationof CICR is normally involved in impulse-evoked transmitterexocytosis. We examined this possibility by using ryanodine,instead of TMB-8, because TMB-8 had a weak blockingaction on nicotinic acetylcholine receptors[39,42]. EPPsevoked by a short tetanus (33.3 Hz for 20 or 30 s) in a lowCa2+, high Mg2+ solution were decreased to 31.3± 21.6%(for the initial 33 EPPs) and 33.8± 4.7% (the last 33 EPPs:n = 5) by ryanodine (20�M) applied for 60 min (Fig. 3D(b)and D(c)). Thus, ryanodine blocked the RyRs in the activablestate at the beginning of the tetanus and decreased the rateof their priming. The inhibition of EPP by ryanodine wasmuch stronger than that of tetanus-induced rises in [Ca2+]iby TMB-8. This was presumably due to the cooperativeCa2+-dependence of exocytotic machinery (see Section4).Consequently, the activation of CICR by impulse-inducedCa2+ entry normally enhances transmitter exocytosis to a con-siderable extent without a conditioning nerve activity.

M. Kubota et al. / Cell Calcium 38 (2005) 557–567 561

Fig. 2. Immunohistochemistry of ryanodine receptors in frog skeletal muscles with antibodies specific to frog RyR1 or RyR3. All the images were taken byconfocal imaging. (A) Staining of RyRs in the nerve terminals, parent axons and muscle fibers with anti-RyR3 antibody. Note the branching of the terminalsfrom the parent axon and the striation of the muscle fibers. (B) Blockade by the epitope peptide of staining with anti-RyR3 antibody. The sensitivity oftheimaging system was set to be 13 times higher than that for the image in (A). (C) Staining of RyRs in muscle fibers with anti-RyR1 antibody. Note the absenceof fluorescence in the regions of the nerve axon bundles with branching, the ‘shadow’ of which are seen as a reversed Y-shaped dark region in the right side ofthe image and note also the striation of the muscle fibers. (D) Blockade by the epitope peptide of staining of the muscle fibers with anti-RyR1 antibody. Thesensitivity of the imaging system was set to be nine times higher than that for the image in (C).

3.4. Possible involvement of CAMKII in the priming ofCICR

The slow priming, activation and subsequent inactiva-tion of CICR were demonstrated by application of repetitivetetani in the previous study[23]. Repetitive tetani (50 Hz,15 s) in combination with a pause (5 s) between each tetanusproduced complex changes in [Ca2+]i in normal Ringer(Fig. 4A). Aside from the time course of changes in [Ca2+]iinduced by each of tetani (see[23], for their detailed descrip-tion and interpretation), notable changes are the slow waxingand waning of the peak amplitude of each tetanus-inducedrise in [Ca2+]i (shown by a gray dotted curve). This waxingand waning component was reduced to 52.9± 4.0% (n = 33)with an increase in time to peak by TMB-8 (10�M: appliedfor 60–120 min,Fig. 4A). Similar effects of ryanodine, ablocker or activator of RyR, and thapsigargin, a blocker ofCa2+ pump at Ca2+ stores, were observed (Kubota et al.,unpublished observations). Thus, the slow waxing and wan-

ing component of repetitive tetani-induced rises in [Ca2+]ireflects the time course of priming, activation and inactiva-tion of CICR as suggested previously[23]. Furthermore, thedependence of the priming process on large Ca2+ entry sug-gests that the mechanism of priming of CICR is likely toinvolve Ca2+-dpendent signaling processes.

We first examined a possibility that CAMKII is activatedto some extent even under the resting condition and nor-mally sets RyRs in an activable state. A blocker of CAMKII, KN-62 (15�M) slightly reduced the amplitude of risesin [Ca2+]i by a short tetanus (50 or 100 pulses) at 50 Hz innormal Ringer solution (Fig. 4B). The rises evoked by 50stimuli decreased to 95.1± 4.4% (n = 39) and 81.1± 7.5%(n = 14: p < 0.05) of the control at 30 and 60 min, respec-tively, after application, while they recovered to 97.6± 4.1%at 60 min. Likewise, reductions of 100 pulse tetanus-inducedrises at 30 and 60 min were 91.5± 4.3% (39) and 82.7± 9.0%(n = 14), respectively, while the recovery at 60 min were to92.5± 4.2%.

562 M. Kubota et al. / Cell Calcium 38 (2005) 557–567

Fig. 3. Effects of TMB-8 on short tetanus- and repetitive tetani-induced rises in [Ca2+]i in the nerve terminals. (A) Effects of TMB-8 on tetanus-induced risesin [Ca2+]i in the normal nerve terminals in normal Ringer containingd-tubocurarine (10�M). A tetanus of 50 or 100 stimuli at 50 Hz was applied to thenerve in normal Ringer. Changes in the fluorescence intensity of OGB-1 loaded in the nerve terminals were measured at 2.5 Hz with a cooled CCD-camera.The averaged intensity in a rectangular window enclosing the terminal was measured, converted to [Ca2+]i values and plotted against time. Effects of TMB-8(10�M) was observed at 60 min after the application. The gray trace is the control record, while the black trace is that after the application of TMB-8. (B)Effects of TMB-8 on tetanus-induced rises in [Ca2+]i after the use-dependent blockade of RyR with ryanodine. The nerve was first pre-stimulated without Ca2+

imaging by repetitive tetani (50 Hz, 15 s with a 5 s pause, repeated 12 times) in the presence of ryanodine applied for 60 min. (The control responses werenottaken to keep the terminals in the more intact condition by omitting photodynamic actions of irradiation before applying TMB-8.) Then, Ca2+ imaging wasmade to observe the effects of TMB-8 as in A. The gray trace is the control record, while the black trace is that after the application of TMB-8. (C) The frequencydistributions of changes in the magnitude of short tetanus-induced rises in [Ca2+]i by the action of TMB-8 in the terminals. All the data on the magnitude ofchanges in [Ca2+]i in each terminal obtained at different stimulation frequencies were treated as independent ones and pooled into one population. The changesin [Ca2+]i expressed in relative values to the control were classified by the bin width of a 10% net change relative to the control and plotted in a frequencyhistogram. Gray columns indicate changes in [Ca2+]i by TMB-8 (10�M) in the normal muscles, while black columns are those in the muscles treated withryanodine (10�M) and given repetitive tetani. The black and gray curves are the normal distributions fitted to the distributions of the experimental data in thepresence and absence of ryanodine, respectively (the probabilities for the null hypothesis were 0.81 and 0.95, respectively). When the data withouttreatmentwith ryanodine were fitted to the sum of the two normal distributions, the fraction that had the mean of 100% and the same variance with that in the presence ofryanodine was negligibly small. (D) Effects of ryanodine on the time course of changes in the amplitude of short tetanus-induced EPPs. A tetanus of 33.3 Hzfor 30 s was applied in a low Ca2+, high Mg2+ solution. Before and after the tetanus, stimuli at 1 Hz were applied to show the basal level of EPP amplitude. Thegraphs show the time courses of changes in the amplitude of EPPs before, during and after the tetanus in the absence (a) and presence (b) of ryanodine (20�M)applied for 68 min. The expanded time courses of changes in the mean amplitudes of EPPs (averaged over one sec period) during a short tetanus (33.3 Hz) aresuperimposed in (c).

We then tested effects of KN-62 on the full activa-tion of CICR in normal Ringer solution. KN-62 (15�M)decreased the waxing and waning component of rises in[Ca2+]i induced by repetitive tetani to 55.1± 3.6% of thecontrol (n = 49: p < 0.001) with no or small increase in time

to peak (124.7± 14.1%,n = 27) and no change in half decaytime (97.2± 9.3%, Fig. 4C). Thus, the rate of priming ofCICR estimated from the ratio of the amplitude to the time topeak decreased to 38.4± 4.8% (n = 18: p < 0.001). We nextexamined effects of KN-62 in a low Ca2+, high Mg2+ solu-

M. Kubota et al. / Cell Calcium 38 (2005) 557–567 563

Fig. 4. Effects of TMB-8 on repetitive tetani-induced rises in [Ca2+]i in the nerve terminals and KN-62 on short tetanus- and repetitive tetani-induced rises.(A) Effects of TMB-8 on repetitive tetani-induced rises in [Ca2+]i in the nerve terminals. Repetitive tetani (50 Hz, 15 s with a 5 s pause, 10 times) were appliedto the nerve in normal Ringer containingd-tubocurarine (10�M). Ca2+ imaging was made as inFig. 3A. Effects of TMB-8 were observed at 60 min after theapplication of TMB-8. Black and gray traces are the time courses of changes in [Ca2+]i in the presence and absence of TMB-8. Black and gray dotted curveswere fitted by eyes to show the slow waxing and waning phases of rises in [Ca2+]i induced by repetitive tetani. The waxing phase predominantly reflects therate of the priming of CICR, while the waning phase, particularly here in normal Ringer, would represent mostly the rate of the inactivation of CICR, depletionof Ca2+ stores and to some extent the rate of the Ca2+-dependent inactivation of Ca2+ channels. The Ca2+ channel inactivation as well as the delay in the time topeak could be the reasons for the greater magnitudes of the fifth and sixth rises in the presence of TMB-8 than the corresponding controls. (B) Effects ofKN-62on short tetanus-induced rises in [Ca2+]i in normal Ringer containingd-tubocurarine. A tetanus of 50 or 100 stimuli at 50 Hz was applied to the nerve. Grayand black traces are the time courses of changes in [Ca2+]i before and after the applicarion of TMB-8, while a dotted trace is that after wash. Recording in thepresence of KN-62 was made 60 min after the application. (C) Effects of KN-62 on repetitive tetani-induced rises in [Ca2+]i in the nerve terminals in normalRinger containingd-tubocurarine (10�M). Repetitive tetani (50 Hz, 15 s with a 5 s pause) were applied to the nerve. The effects were observed at 60 min afterthe application of KN-62 (10�M). Black and gray traces are the time courses of changes in [Ca2+]i in the presence and absence of KN-62. Black and graydotted curves were fitted by eyes to show the slow waxing and waning phases of rises in [Ca2+]i induced by repetitive tetani. (D) Effects of KN-62 on repetitivetetani-induced rises in [Ca2+]i in the nerve terminals in a low Ca2+, high Mg2+ solution. Repetitive tetani (50 Hz, 15 s with a 5 s pause) were applied to thenerve. The effects were observed at 60 min after the application of KN-62 (10�M). Other explanations are the same as those in (C).

tion, because CICR is also primed, activated and inactivatedby repetitive tetani in such a condition[22,23] (see below).KN-62 (10–15�M) indeed suppressed the waxing and wan-ing component of repetitive tetani-induced rises in [Ca2+]iin a low Ca2+, high Mg2+ solution (Fig. 4D). The maximumamplitude of the slow waxing and waning component wasreduced to 51.9± 4.2% of the control (n = 21:p < 0.001), withno change in time to the peak (90.8± 10.1%,n = 21) and anincreased half decay time (234.9± 57.6%:p < 0.05).

If activation of CAMKII is involved in the priming ofCICR, KN-62 should also block the enhancement of transmit-ter release produced by continuous tetanus or repetitive tetanithat prime the mechanism of CICR. This was the case. KN-62(10�M) decreased the maximum rises in MEPP frequencycaused by continuous tetanus (MEPP hump: to 54.7± 4.5%of the control,n = 8: p < 0.001), increased the time to peak(142.1± 15.1%: p < 0.05, Fig. 5A), and thus, reduced therate of rise (to 40.4± 4.1%: p < 0.001) without a changein the half decay time (91.1± 12.2%). Likewise, KN-62

(10�M) reduced the maximum increase in QC of EPP pro-duced by repetitive tetani (to 44.0± 15.4%,n = 8: p < 0.01),increased the time to peak (164.2± 8.5%,n = 8: p < 0.001),and decreased the rate of increase (to 27.8± 3.9%:p < 0.001)with no change in the half decay time (113.0± 17.0%,Fig. 5C). An inactive analogue of KN-62, KN-04, however,did not affect much the rate and amplitude of rises in MEPPfrequency induced by a continuous tetanus (Fig. 5B) andthose of slow increases in QC of EPPs produced by repetitivetetani (Fig. 5D). These results suggest that the activation ofCAMK II is involved in the priming process of the mecha-nism of CICR.

4. Discussion

There are three major findings in the present study. First,immunohistochemistry using antibodies to two types of frogRyR demonstrates that RyR3 exists in the motor nerve

564 M. Kubota et al. / Cell Calcium 38 (2005) 557–567

Fig. 5. Effects of KN-62 and KN-04 on the amplitude of EPPs induced by repetitive tetani and miniature EPP frequency during a continuous tetanus. (A)and (B) Effects of KN-62 and KN-04 on miniature EPP frequency during a continuous tetanus. Continuous tetanus of 50 Hz was applied with a pause of 30 safter the subsidence of a transient rise in miniature EPP frequency. Effects of KN-62 (10�M: A) and KN-04 (10�M: B) were observed at 62 and 73 min afterapplication of each drug. (C) and (D) Effects of KN-62 and KN-04 on the amplitude of EPPs induced by repetitive tetani. Combination of tetanus of 33.3 Hz(30 s) and low rate stimulation (1 Hz) for 30 s was repeated in a low Ca2+, high Mg2+ solution. Effects of KN-62 (10�M: C) and KN-04 (10�M: D) wereobserved at 66 and 56 min after application of each drug. Black and gray traces are the time courses of changes in QC of EPPs in the presence and absence ofeach drug. Black and gray dotted curves were fitted by eyes to show the slow waxing and waning phases of rises in QC of EPPs.

terminals and parent axon of the frog, while both RyR1and RyR3 are in the muscle fibers. Second, RyRs in frogmotor nerve terminals, whose activation in a full extentrequires high frequency tetanic nerve activity, are normallyactivated to some extent by impulse-induced Ca2+ entry,even when the nerve activity is low. The resultant CICRis found to have a significant facilitatory effect on impulse-induced transmitter exocytosis. Third, activation of CAMKII appears to be needed to maintain RyRs at, and primeRyRs into an activable state. These findings are quite sur-prising and important for the understanding of the mecha-nism of transmitter release at frog motor nerve terminals,which has been thought to depend fully on Ca2+ entry [30].The significance and mechanism of these modes of acti-vation of CICR in frog motor nerve terminals is discussedbelow.

4.1. How do the properties of RyR3 explain thecharacteristics of CICR in the motor nerve terminals?

The RyR1 in the sarcoplasmic reticulum of frog skeletalmuscle is predominantly activated by the depolarization ofthe T-tubule membrane[43]. By contrast, the RyR3 of theSR opens the gate of the Ca2+ release channel by a CICRmechanism[44,45]. CICR activity in frog skeletal muscles

is regulated by the bindings of Ca2+ and Mg2+ to RyR.CICR occurs, when the high affinity activating Ca2+ sites (A-sites) are occupied by Ca2+ (the dissociation constant (Kd),3�M) and the low affinity inactivating Ca2+ sites (I-sites) arefree of Ca2+ and Mg2+ (Kd, 0.3–0.4 mM:[44]). Mg2+ com-petes with Ca2+ at the A-sites (Kd, around 75�M), while itacts as an agonist at the I-sites. Thus, Mg2+ decreases theapparent Ca2+ dependence of CICR activity with a reducedpeak activity. Since the resting cytoplasmic Mg2+ concen-tration was estimated to be 0.5–1.0 mM, the CICR activityin the frog skeletal muscle fibers, predominantly via theactivation of RyR3 would normally be much suppressed[44,46].

These properties and functional roles of the RyR3 in frogskeletal muscles nicely fit the characteristics of CICR in thefrog motor nerve terminals except for inactivation. CICR inthe frog motor nerve terminals is slightly activated by theCa2+ entered through voltage-gated Ca2+ channels, althoughit considerably contributes to transmitter exocytosis. Thislimited activation of CICR would obviously be ascribed, atleast in part, to the strong depressant actions of Mg2+ onRyR3, as shown above. Furthermore, the distance betweenthe orifice of a Ca2+ channel and Ca2+-activation site of RyRwas estimated to be less than 100 nm[23]. The increase in[Ca2+]i in such a region by Ca2+ entry through a Ca2+ channel

M. Kubota et al. / Cell Calcium 38 (2005) 557–567 565

may not be high enough for the full activation of the RyR3in the terminals. Thus, it seems that CICR is only slightlyactivated by impulse-induced Ca2+ entry. Then, further acti-vation of CICR would require the priming of RyR by theconditional repetitive Ca2+ entries[22,23].

4.2. Subtle activation of CICR significantly contributesto transmitter exocytosis

The second major finding is that the small activation ofCICR, 10–20% of that caused by Ca2+ entry, produced a sig-nificant contribution to transmitter exocytosis to 1.5–2-foldof that produced only by Ca2+ entry. There are two possibleexplanations for this difference in the extents of contributionof CICR. First, the cooperative Ca2+-dependence of exocy-totic machinery would amplify the additional rise in [Ca2+]iproduced by CICR. Reduction of rises in [Ca2+]i to 80–90%would cause the decrease of transmitter exocytosis to 41(0.84 × 100) to 66% (0.94 × 100). Second, The local rise in[Ca2+]i produced by CICR may be much greater than the risein [Ca2+]i in the bulk phase which was actually seen by flu-orescence changes. A possibility that a small component ofCICR in short tetanus-induced rises in [Ca2+]i assessed bythe action of TMB-8 might result from the priming of CICRthat occurs even during such a short tetanus can be ruled outfor the reasons. First, the duration of the tetanus (1 or 2 s) wasm theb en at

T bil-i lsov hisd CRc ofa m-b ctedf o-p ac

4p

thes es yo oust uts tiono IIb ti-v ase.A ev-e t ofC

First, phosphorylation of RyRs or associated proteins byCAMKII may alter the conformation of RyR molecule to anactivable state. This is consistent with the high activity ofCAMKII in the presynaptic terminals (see a review[47]) andthe existence of potential phosphorylation sites for CAMKIIin frog muscle RyRs[32]. In addition, phosphorylation ofRyRs by CAMKII was reported to remove Mg2+-inducedblockade of RyR in rabbit skeletal or canine cardiac mus-cles[43,48,49]. Secondly, RyRs could be recruited to a siteclose enough for activation by Ca2+ entry. Such recruitmentof RyRs may be mediated by mobilization of Ca2+ stores,in which they reside. Although endoplasmic reticulum iswell known as Ca2+ stores (see[14]), synaptic vesicles couldalso be a possible candidate for Ca2+ stores in presynapticterminals. This idea nicely fits the mobilization of synap-tic vesicles to exocytotic sites via activation of CAMKII[50–53]. If RyRs reside on synaptic vesicles, their mobi-lization would result in the location of RyR close enoughfor activation by Ca2+ entry at the cell membrane. Further-more, electron spectroscopic imaging and electron energyloss spectroscopy analysis revealed the existence of Ca2+

in synaptic vesicles in frog motor nerve terminals[54]. Thethird possible mechanism for priming of RyR could be theproduction of a modulator, say cyclic ADP-ribose (cADP-ribose)[55,56] via the activation of CAMK II. In supportof this, cADP-ribose applied in liposomes enhanced evoked

cti-

sted

cedig-fre-

thistosis

rmi-to atheus-

ajoritionervecy of

t the

uch shorter than that for full priming of CICR. Second,locking action of ryanodine on EPPs was observed ev

he initial phase of nerve stimuli.The blockade of short tetanus-induced rises in [Ca2+]i by

MB-8 was variable depending on terminals. This variaty must be due to not only experimental variability, but aariable extent of activation of CICR in each terminal. Tifference in the magnitude of normal activation of CIould arise from differences in the availability or locationctivable RyRs relative to Ca2+ channels at the terminal meranes. The variability of RyR location may also be expe

rom variable arrangement of Ca2+ stores, presumably endlasmic reticulum and/or synaptic vesicles, relative to C2+

hannels in each terminal.

.3. Possible modes of involvement of CAMKII in theriming of CICR

The present study showed that KN-62 slightly blockedhort tetanus-induced rises in [Ca2+]i and considerably thlow waxing and waning rises in [Ca2+]i , MEPP frequencr QC of EPPs induced by repetitive tetani or continu

etanus. Thus, it is highly likely that CAMKII is normally, blightly, active under the resting condition and sets a fracf RyRs in an activable state. The full activation of CAMKy a large Ca2+ entry would cause the priming and full acation of RyR to CICR and so enhance transmitter relelthough the evidence is not fully enough for this idea, sral possible mechanisms for the mode of involvemenAMKII in priming RyRs are discussed below.

ttransmitter release in frog neuromuscular junctions[57]. Thisfinding is consistent with this facilitatory action on the avation of RyR3 in the rat diaphragm muscles[58]. The roleof cADP-ribose in transmitter exocytosis was also suggein Aplysia synapses[24].

4.4. Physiological significance of the unique mechanismof CICR in the nerve terminals

CICR normally occurs in response to impulse-induCa2+ entry in a fraction of frog motor terminals, but snificantly contributes to transmitter exocytosis. Highquency nerve activity causes a large Ca2+ entry into theterminals, which fully primes and activates CICR andmarkedly enhances impulse-induced transmitter exocyand its plasticity. Thus, RyRs in the frog motor nerve tenals are in a critical state for activation and conversiongreatly activable state. This mode of activation of CICR inmotor nerve terminals may operate during physiological mcle activity, say, during tetanic contraction that plays mroles in maintaining the tone of a muscle and the posof the related joint. Synaptic depression during such ntetanic activity may be overcome by the increased efficaexocytosis by the activation of CICR.

Acknowledgements

We thank Dr. Wang Jing for her technical assistance ainitial stage of immunohistochemical experiments.

566 M. Kubota et al. / Cell Calcium 38 (2005) 557–567

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