The K9K10 nicotinic acetylcholine receptor is permeable to and ismodulated by divalent cations

Noelia Weisstaub a, Douglas E. Vetter b, Ana Bele¤n Elgoyhen a, Eleonora Katz a;c;�

a Instituto de Investigaciones en Ingenier|¤a Gene¤tica y Biolog|¤a Molecular (CONICET-UBA), Vuelta de Obligado 2490, 1428 Buenos Aires, Argentinab Tufts University School of Medicine, Boston, MA, USA

c Departamento de Biolog|¤a, FCEyN, UBA, Buenos Aires, Argentina

Received 10 December 2001; accepted 12 March 2002

Abstract

The native cholinergic receptor that mediates synaptic transmission between olivocochlear fibers and outer hair cells of thecochlea is permeable to Ca2þ and is thought to be composed of both the K9 and the K10 cholinergic nicotinic subunits. The aim ofthe present work was to study the permeability of the recombinant K9K10 nicotinic acetylcholine receptor to Ca2þ, Ba2þ and Mg2þ

and its modulation by these divalent cations. Experiments were performed, by the two-electrode voltage-clamp technique, inXenopus laevis oocytes injected with K9 and K10 cRNA. The relative divalent to monovalent cation permeability was high (V10)for Ca2þ, Ba2þ and Mg2þ. Currents evoked by acetylcholine (ACh) were potentiated by either Ca2þ or Ba2þ up to 500 WM butwere blocked by higher concentrations of these cations. Potentiation by Ca2þ was voltage-independent, whereas blockage wasstronger at hyperpolarized than at depolarized potentials. Mg2þ did not potentiate but it blocked ACh-evoked currents (IC50 =0.38 mM). In the absence of Ca2þ, the EC50 for ACh was higher (48 WM) than that obtained with 1.8 mM Ca2þ (14.3 WM),suggesting that potentiation by Ca2þ involves changes in the apparent affinity of the K9K10 receptor for ACh. 9 2002 ElsevierScience B.V. All rights reserved.

Key words: Nicotinic receptor; Outer hair cell ; Olivocochlear e¡erent synapse; Ca2þ permeability; Voltage-dependent blockage;Neurotransmitter receptor channel; Ligand-gated channel

1. Introduction

In the vertebrate inner ear, hair cells of the cochleahave the ability to detect sound stimuli and transducethem into electrical signals. In mammals there are twotypes of hair cells involved in this process, inner haircells (IHCs) and outer hair cells (OHCs). IHCs are theprimary acoustic transducers and receive most of thea¡erent innervation while OHCs are thought to be in-

volved in sound ampli¢cation and receive a prominente¡erent innervation from the olivary complex in thebrainstem (for reviews see Fuchs, 1996; Hudspeth,1989; Dallos, 1996). It is hypothesized that OHCs, byvirtue of their voltage-driven length changes, feed backmechanical force to the cochlear partition, enhancing itssensitivity and frequency selectivity (see Holley, 1996).At the synapse between the e¡erent ¢bers and OHCs,

a non-classical ionotropic cholinergic receptor allowsCa2þ entry to the cell, activating a Ca2þ-sensitive Kþ

current (IKCa) that hyperpolarizes the plasma mem-brane (Blanchet et al., 1996; Doi and Ohmori, 1993;Dulon et al., 1998; Erostegui et al., 1994a; Evans,1996; Housley and Ashmore, 1991; Nenov et al.,1996; Oliver et al., 2000). The physiological conse-quence of this e¡ect to the mammalian inner ear is areduction in sensitivity due to suppression of basilarmembrane motion, thereby altering the dynamic rangeof hearing (for review see Guinan, 1996).

0378-5955 / 02 / $ ^ see front matter 9 2002 Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 5 9 5 5 ( 0 2 ) 0 0 3 8 0 - 5

* Corresponding author. Fax: +54 (11) 4786-8578.E-mail address: ekatz@dna.uba.ar (E. Katz).

Abbreviations: OHC, outer hair cells ; IHCs, inner hair cells ; ACh,acetylcholine; nAChR, nicotinic acetylcholine receptor; NFS, normalfrog saline; NMG, N-methyl glucamine; Erev, reversal potential; Pdiv/Pmono, relative divalent to monovalent permeability; GHK,Goldman^Hodgkin^Katz; IKCa, Ca2þ-sensitive potassium current;IClCa, Ca2þ-sensitive chloride current; IBa, barium current; ICa,calcium current

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The K9 nicotinic acetylcholine subunit is a main com-ponent of the cholinergic receptor that mediates synap-tic transmission between e¡erent olivocochlear ¢bersand OHCs (Elgoyhen et al., 1994; Glowatzki et al.,1995; Morley et al., 1998; Park et al., 1997; Vetter etal., 1999). Expression of K9 cRNA in Xenopus laevisoocytes results in the formation of a homomeric recep-tor^channel complex with pharmacological propertiesthat greatly di¡er from other cloned nicotinic acetylcho-line receptors (nAChR) (Elgoyhen et al., 1994; Rothlinet al., 1999; Verbitsky et al., 2000), but which are al-most identical to those of the cholinergic receptorpresent at the base of the OHCs (Chen et al., 1996;Erostegui et al., 1994a). Until the cloning of theK10 subunit (Elgoyhen et al., 2001), our working hy-pothesis was that the native OHC receptor was a ho-mopentamer composed of K9 subunits. However, therewere some biophysical characteristics of the recombi-nant homomeric K9 receptor that di¡ered from thosereported for the native OHC receptor, namely, its Ca2þ

sensitivity, its current^voltage relationship and its de-sensitization kinetics (Blanchet et al., 1996; Dulonand Lenoir, 1996; Evans, 1996; Katz et al., 2000;McNiven et al., 1996). The cloned K10 subunit is notable to form a homomeric functional channel, however,the expression of K9 together with K10 results in theformation of a heteromeric receptor with pharmacolog-ical and functional properties very closely resemblingthose described for the vertebrate native cholinergic re-ceptor (Elgoyhen et al., 2001). The stoichiometry of thisheteromeric receptor is so far unknown and its estima-tion would imply performing site-directed mutagenesison regions of both the K9 and K10 subunits that couldsigni¢cantly alter channel properties (see Cooper et al.,1991).The K9 and K10 subunits form part of the nAChR

family of ligand-gated ion channels, which includes re-ceptors that are distributed throughout the nervous sys-tem and at the neuromuscular junction. Muscle andneuronal nAChRs are permeable to monovalent cationslike Naþ and Kþ and also to Ca2þ and other divalentcations (Bertrand et al., 1993; Decker and Dani, 1990;Mulle et al., 1992a,b; Se¤gue¤la et al., 1993; Vernino etal., 1992, 1994). Neuronal nAChRs have a high Ca2þ

permeability and agonist-evoked currents through thesereceptors are potentiated by Ca2þ (Galzi et al., 1996;Mulle et al., 1992a,b; Vernino et al., 1992). The re-combinant homomeric K9 receptor is, like the neuronalK7 receptor (Se¤gue¤la et al., 1993), highly permeable toCa2þ (Katz et al., 2000). However, acetylcholine (ACh)-evoked currents through the K9 receptor are not poten-tiated but strongly blocked by this cation (IC50 100 WM)in a voltage-dependent manner (Katz et al., 2000). Pre-liminary studies have suggested that the recombinantK9K10 receptor is permeable to Ca2þ and that it is

tightly modulated by divalent cations (Elgoyhen et al.,2001).Since there is strong evidence supporting the hypoth-

esis that the functional native cholinergic receptorpresent at the base of OHCs is composed of both K9and K10 subunits and given the key role Ca2þ plays atthat sensory inhibitory synapse, the aim of the presentstudy was to perform an extensive characterization ofthe permeability to and the modulation by Ca2þ of therecombinant K9K10 nAChR. This work provides evi-dence that the K9K10 receptor is highly permeable toCa2þ and that it is also potentiated and blocked byphysiological external Ca2þ concentrations through dif-ferent mechanisms.

2. Methods

2.1. Expression of the K9K10 receptor in X. laevisoocytes and electrophysiological procedures

Full length rat K9 and K10 cDNAs constructed in amodi¢ed pGEMHE vector suitable for X. laevis oocyteexpression studies were used as described previously(Elgoyhen et al., 2001). cRNA was synthesized usingthe mMessage mMachine T7 transcription kit (Ambion,Austin, TX, USA) with plasmid linearized with NheI.The isolation and maintenance of oocytes was carriedout as described previously (Elgoyhen et al., 1994).Brie£y, oocytes were surgically removed from the ova-ries of X. laevis frogs anesthetized with 3-aminobenzoicacid ethylester (V1 g/ml). Oocytes were incubated with1 mg/ml of Worthington (Freehold, NJ, USA) type I ortype II collagenase for 60^90 min in Ca2þ-free salinesolution (in mM): 96 NaCl, 2 KCl, 5 MgCl2, 5 HEPESpH 7.6, with slow agitation to remove the follicular celllayer. Oocytes were then washed extensively in the samesolution and maintained at 17‡C in a Barth’s solutioncomprised of (in mM): 88 NaCl, 1 KCl, 0.91 CaCl2, 24NaHCO3, 0.33 Ca(NO3)2, 0.82 MgSO4, 10 HEPES, pH7.6 supplemented with 100 Wg/ml gentamicin sulfate.Oocytes (stages V and VI) were injected with 50 nl ofRNase-free H2O containing 0.01^1 ng of both K9 andK10 cRNAs (at 1:1 molar ratio) and maintained inBarth’s solution at 17‡C.Electrophysiological recordings were performed 3^7

days after injection, under two-electrode voltage-clampwith a Geneclamp 500 ampli¢er (Axon Instruments,Foster City, CA, USA). Voltage and current electrodeswere ¢lled with 3 M KCl and had resistances of 0.9^2M6. The preparation was grounded by means of eitherthe VG-2a U100 bath probe of the ampli¢er or an Ag/AgCl wire in a 3 M KCl solution connected to the baththrough a 3 M KCl agar bridge. Current^voltage (I^V)relationships were obtained by applying 2 s voltage

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ramps from 3120 to +50 mV, 10 s after the peak re-sponse to 10 WM ACh from a holding potential (Vhold)of 370 mV. Leakage correction was performed by dig-ital subtraction of the I^V curve obtained by the samevoltage ramp protocol prior to the application of ACh.Generation of voltage protocols and data acquisitionwere performed using a Digidata 1200 and the pClamp6.1 or 7.0 software (Axon Instruments). Data were an-alyzed using Clamp ¢t from the pClamp 6.1 software.During electrophysiological recordings, oocytes were

continuously superfused (£ow rate V10 ml/min) with anormal frog saline (NFS) comprised of (in mM): 115NaCl, 2.5 KCl, 1.8 CaCl2, 10 HEPES, bu¡ered to pH7.2 with NaOH. ACh was applied along with the per-fusion solution of the oocyte chamber. In the experi-ments in which the Ca2þ concentration was varied from0 to 3 mM or in which Ca2þ was replaced by di¡erentconcentrations of Ba2þ or Mg2þ, the other componentsof the frog saline remained constant. The solution usedfor the divalent cation permeability studies was 10 mMHEPES, 0.2^5 mM Ca2þ or 0.2^10 mM Ba2þ and 100^120 mM N-methyl glucamine (NMG) in order to com-pensate for changes in osmolarity, pH adjusted to 7.2with HCl. In all the experiments, oocytes were super-fused for 1 min with the test solution before the appli-cation of ACh and were transferred back to NFS (1.8mM Ca2þ) for at least 3 min before changing to adi¡erent test solution.In order to minimize the activation of the native oo-

cyte’s Ca2þ-sensitive chloride current, IClCa (Barish,1983; Boton et al., 1989), by Ca2þ entering throughthe K9K10 receptor (Elgoyhen et al., 2001), all experi-ments, unless otherwise indicated, were carried out inoocytes incubated with the membrane permeant Ca2þ

chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NP,NP-tet-raacetic acid-acetoxymethyl ester (BAPTA-AM, 100WM) for 3 h prior to electrophysiological recordings.This treatment has been shown previously to e¡ectivelychelate intracellular Ca2þ ions and, therefore, to impairthe activation of the IClCa (Gerzanich et al., 1994).As most of the experiments in this work involved

changes in the extracellular concentration of divalentcations, in order to minimize the activation of the oo-cyte’s non-selective inward current through a hemigapjunction channel in response to the reduction of theexternal divalent cation concentration, all experimentswere carried out in oocytes injected with 7.5 ng of anoligonucleotide (5P-GCTTTAGTAATTCCCATCCTG-CCATGTTTC-3P) antisense to connexin C38 mRNA(Arellano et al., 1995; Ebihara, 1996). This treatmentdid not alter the expression of the K9K10 receptor, asjudged by the fact that the temporal course and theamplitude range of the currents elicited by ACh in theseoocytes were similar to those obtained in oocytes in-

jected with cRNAs of both K9 and K10 without theantisense oligonucleotide.

2.2. Evaluation of divalent cation permeability

The relative divalent to monovalent permeability(Pdiv/Pmono) was evaluated as described in our previouspaper (Katz et al., 2000). Brie£y, it was calculated bythe Goldman^Hodgkin^Katz (GHK) constant ¢eld vol-tage equation assuming no anion permeability and ex-tended to include divalent cations (Lewis, 1979). Theinternal concentrations of Naþ and Kþ used in thecalculations were 20 and 150 mM, respectively. Perme-ability ratios were calculated for each oocyte and thenaveraged. Under our experimental conditions and as-sumptions, PCa/PNa (or PBa/PNa) was obtained from:

Erev ¼ RT=F ln ð4P0Ca½Ca�o=ð½Na�i þ PK=PNa½K�iÞÞ

where PPCa= (PCa/PNa)/(1+exp(F/RTErev)) ; R is themolar gas constant, F is the Faraday constant, T isthe absolute temperature (RT/F is 25.3 mV at 20‡C),[Ca]o is the external concentration of Ca2þ (or Ba2þ),[Na]i and [K]i are the internal concentrations of Naþ

and Kþ, respectively.

2.3. Evaluation of the modulation of the K9K10 receptorby divalent cations

The e¡ects of extracellular Ca2þ, Ba2þ and Mg2þ onthe ionic currents through the K9K10 receptor werestudied by measuring the amplitudes of the responsesto 10 WM ACh upon varying the concentration of thesecations from nominally 0 to 5 or 10 mM. Amplitudevalues obtained at each concentration were normalizedto that obtained in the same oocyte at a 1.8 mM con-centration of the divalent cation being studied. Valuesfrom di¡erent oocytes were averaged and expressed asthe meanSS.E.M.

2.4. Evaluation of inhibition by Mg2+

The IC50 values for the Mg2þ inhibition were eval-uated in the presence of either 0.5 mM or 1.3 mM Ca2þ

and calculated by:

I=Imax ¼ 1=ð1þ 10ðlog IC503X ÞnHÞ

where I is the current obtained at the di¡erent concen-trations of extracellular Mg2þ, Imax is the current ob-tained in the absence of Mg2þ, X is the logarithm of theMg2þ concentration and nH is the Hill coe⁄cient. IC50values were obtained for each oocyte and then aver-aged. Data are presented as meanSS.E.M.

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2.5. Evaluation of the e¡ect of divalent cations on thea⁄nity of the K9K10 receptor for ACh

To evaluate changes in a⁄nity and maximal responseto ACh, concentration^response curves to ACh werecarried out in the same oocytes at 0 and 1.8 mMCa2þ. Current amplitudes were normalized to the valueobtained at 1 mM ACh and 1.8 mM Ca2þ in eachoocyte in order to compare maximal amplitudes atboth Ca2þ concentrations. EC50 values were calculatedby:

I=Imax ¼ 1=ð1þ 10ðlog EC503X ÞnHÞ

Statistical signi¢cance was evaluated by the Student’st-test (two-tailed, unpaired samples) or by a one-wayANOVA followed by the Tukey test. P values 6 0.05were considered signi¢cant.

2.6. Materials

ACh chloride was purchased from Research Bio-chemicals (Natick, MA, USA). It was dissolved in dis-tilled water as a 100 mM stock and stored in aliquots at320‡C. BAPTA-AM (Molecular Probes, Eugene, OR,USA) was stored at 320‡C as aliquots of a 100 mMsolution in dimethyl sulfoxide, thawed and diluted

1000-fold into saline solution shortly before incubationof the oocytes.

3. Results

3.1. Permeability of the recombinant K9K10 nAChR todivalent cations

Ionic currents through the K9K10 receptor werestudied in X. laevis oocytes that had been injectedwith a mixture of K9 and K10 cRNAs (molar ratio1:1). The ability of ACh to activate the native IClCain oocytes expressing recombinant nAChRs can beused to evaluate whether the expressed receptor is per-meable to Ca2þ. As we have previously shown (Elgoy-hen et al., 2001), currents elicited by bath application of100 WM ACh to K9K10-expressing oocytes superfusedwith NFS (1.8 mM Ca2þ) were reduced by V85% after3 h of incubation with the fast calcium chelator BAP-TA-AM (Fig. 1A). This result is similar to what hasbeen reported for the K7 and K9 nAChRs (Katz etal., 2000; Se¤gue¤la et al., 1993) and indicates that theK9K10 receptor is permeable to Ca2þ. The Ca2þ perme-ability of the K9K10 receptor is also demonstrated bythe fact that ACh was able to elicit inward currentswhen all cations in the extracellular NFS were replaced

Fig. 1. The K9K10 nAChR is permeable to Ca2þ, Ba2þ and Mg2þ. (A) Representative records of responses evoked by ACh (100 WM) in anK9K10-expressing oocyte superfused with a NFS solution containing 1.8 mM Ca2þ before (left) and after (right) a 3 h incubation with the fastcalcium chelator BAPTA-AM (Vhold =370 mV). (B) Representative records of responses elicited by ACh (100 WM) in an K9K10-expressing oo-cyte (n=3 oocytes) superfused with saline solutions containing either Ca2þ, Ba2þ or Mg2þ as the only extracellular permeant ions (Vhold =390mV).

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by an iso-osmotically equivalent amount of Ca2þ

(Fig. 1B). Besides, ACh was able to elicit inward cur-rents when this experiment was done with either Ba2þ

or Mg2þ as the sole charge carriers through this channel(Fig. 1B), indicating that the K9K10 receptor is alsopermeable to these divalent cations.Due to the fact that it is not possible to control the

internal ionic composition of the oocyte, this is not theoptimal system with which to evaluate relative ion per-meability. However, one must consider that so far, as isalso the case for the K9 homomeric receptor (see Katzet al., 2000), it has not been possible to express the

recombinant heteromeric K9K10 receptor in any otherheterologous expression system. Given that in OHCsthe activation of the native receptor by ACh activatesan IKCa due to Ca2þ entry from the extracellular me-dium (see Section 1), it seems important to evaluate theCa2þ permeability of the recombinant receptor, despitethe inherent disadvantages of the oocyte system in ob-taining an exact value for this parameter, and compareit with values obtained in the same system for otherrecombinant nicotinic receptors.In order to make an estimate of Pdiv/Pmono, I^V

curves were elicited by a 2 s voltage ramp protocol

Fig. 2. Relative divalent to monovalent permeability of the K9K10 nAChR. Representative I^V curves obtained upon application of voltageramps (3120 to +50 mV, 2 s) 10 s after the peak response to 10 WM ACh in oocytes voltage-clamped at 370 mV and superfused with NMG-based solutions containing di¡erent concentrations of CaCl2 (A) or BaCl2 (B). Insets are an ampli¢cation of the I^V curves around the Erev,showing the shifts in this parameter upon variation of the concentration of either Ca2þ or Ba2þ. (C) The same voltage ramp protocol as in Aand B, applied to an oocyte superfused with an NMG-based saline solution containing either 5 mM MgCl2 or 5 mM BaCl2. Note that Erev isthe same in both solutions (inset). (D) Plots of Erev as a function of the concentration of Ca2þ (n=5 oocytes), Ba2þ (n=6 oocytes) or Mg2þ

(n=5 oocytes, only Erev obtained in 5 mM MgCl2 is plotted).

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(3120 to +50 mV), 10 s after the peak response to AChfrom a Vhold of 370 mV in the presence of di¡erentCa2þ concentrations. As I^V curves through theK9K10 receptor show recti¢cation around the reversalpotential (Erev) (see Elgoyhen et al., 2001), all monova-lent cations in the extracellular medium were replacedby the impermeant monovalent cation NMG in orderto enhance the shift in the Erev upon variation of Ca2þ.Fig. 2A shows that the Erev becomes more depolarizedas a function of the Ca2þ concentration. The same de-polarizing shift in Erev was observed when the experi-ment was carried out in the presence of NMG withBa2þ as the sole permeant cation in the external solu-tion (Fig. 2B). Inward currents elicited by ACh in the

presence of Mg2þ as the only permeant ion in the ex-ternal solution were very small for 0.2 and 2 mM Mg2þ,therefore evaluation of the Erev was di⁄cult under theseconditions. However, as illustrated in Fig. 2C, the Erev

of ACh-elicited currents in K9K10-expressing oocytes inthe presence of 5 mM Mg2þ was almost identical tothat obtained with 5 mM Ba2þ in the same oocytes(n=6 oocytes), thus suggesting that both cations havea similar permeability through the K9K10 receptor. TheErev data averaged from the di¡erent oocytes were plot-ted against the concentration of Ca2þ, Ba2þ or Mg2þ

(for this latter cation, only the 5 mM concentration wasplotted) in the extracellular medium (Fig. 2D). The PCa/PNa, PBa/PNa were 9S 1 (n=5 oocytes) and 9.7 S 1.3

Fig. 3. Modulation of the K9K10 nAChR by Ca2þ and Ba2þ. (A) Representative records of responses evoked by 10 WM ACh in an K9K10-ex-pressing oocyte superfused with NFS containing di¡erent Ca2þ concentrations (Vhold =390 mV). (B) Bar diagram illustrating the biphasic ef-fect, potentiation and block, the extracellular Ca2þ exerts on the K9K10 receptor. Current amplitudes obtained at the di¡erent Ca2þ concentra-tions in each oocyte were normalized with respect to that obtained at 1.8 mM in the same oocyte. Each bar represents the meanSS.E.M. ofthe normalized response obtained in di¡erent oocytes (n=6^11). (C,D) The same as A and B in NFS solutions in which BaCl2 substituted forCaCl2 (n=9^11 oocytes). Asterisks denote a signi¢cant di¡erence with respect to the value at 0.5 mM Ca2þ (P6 0.05).

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(n=6 oocytes), respectively, as estimated by the ex-tended GHK voltage equation (see Section 2). The rel-ative permeability of Mg2þ could only be evaluated inone oocyte and the value obtained (PMg/PNa = 10.3) didnot di¡er from those obtained for Ca2þ and Ba2þ. Eventhough caution should be taken concerning the perme-ability estimates, they suggest that Ca2þ, Ba2þ andMg2þ have a similar and high relative permeabilitythrough the K9K10 receptor.

3.2. Modulation of the recombinant K9K10 nAChR bydivalent cations

Extracellular Ca2þ ions modulate the activity of sev-eral nAChRs. It has been shown that external Ca2þ

reduces the single channel conductance through bothneuronal and muscle nicotinic receptors (Decker and

Dani, 1990; Mulle et al., 1992a,b). However, Ca2þ pos-itively modulates macroscopic currents through neuro-nal nAChRs due to an allosteric e¡ect of this cation atan extracellular site (Galzi et al., 1996; Mulle et al.,1992a,b; Vernino et al., 1992). The homomeric K9nAChR is strongly blocked by external Ca2þ (Katz etal., 2000), whereas preliminary data indicate that theheteromeric K9K10 receptor shows a more complex re-sponse upon variations of this divalent cation (Elgoy-hen et al., 2001). Given the key role Ca2þ plays at theolivocochlear synapse, we were interested in performinga detailed study of the e¡ects of Ca2þ and other diva-lent cations on the activity of the K9K10 receptor.Fig. 3A shows representative responses of K9K10-ex-

pressing oocytes to ACh (10 WM) in the presence ofincreasing concentrations of Ca2þ ions in the extracel-lular solution. Currents were negligible in a nominally

Fig. 4. Mg2þ does not potentiate but it blocks the K9K10 nAChR. (A) Representative traces of responses evoked by 10 WM ACh in an K9K10-expressing oocyte (n=4 oocytes) superfused with Ca2þ-free NFS solutions containing di¡erent concentrations of MgCl2. The record on the leftis a control response obtained in the same oocyte superfused with NFS (1.8 mM Ca2þ and 0 Mg2þ). Note that in the absence of Ca2þ, currentselicited by 10 WM ACh are negligible when compared to control ones. (B,C) Inhibition curves obtained in the presence of a ¢xed concentrationof Ca2þ (0.5 and 1.3 mM in B and C, respectively) and di¡erent concentrations of MgCl2. Current amplitudes evoked by 10 WM ACh at thedi¡erent MgCl2 concentrations were normalized with respect to the amplitude obtained in the same oocyte superfused with a NFS saline solu-tion containing 0 mM Mg2þ and either 0.5 (B) or 1.3 (C) mM Ca2þ. Each data point represents the meanSS.E.M. of three to seven oocytes.The IC50 values were 0.38S 0.18 mM, nH =1.06 and 0.33S 0.02 mM, nH =0.58 for 0.5 and 1.3 mM Ca2þ, respectively.

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Ca2þ-free solution, were potentiated by low millimolarconcentrations of Ca2þ and were inhibited by Ca2þ

concentrations above 0.5 mM. The bar diagram inFig. 3B illustrates the averaged data obtained from dif-ferent oocytes upon variation of external Ca2þ. Currentamplitudes at di¡erent Ca2þ concentrations in each oo-cyte were normalized with respect to those obtained at1.8 mM in the same oocyte. A one-way ANOVA indi-cated that di¡erences in mean current amplitudes be-tween 0, 0.1 and 3 mM Ca2þ were signi¢cant with re-spect to the mean amplitude obtained at 0.5 mM Ca2þ

(P6 0.05). When Ba2þ substituted for Ca2þ in the ex-ternal solution (Fig. 3C,D), the same dual e¡ect, poten-tiation and block, was observed. The one-way ANOVAindicated that di¡erences in mean current amplitudesbetween 0, 3 and 10 mM Ba2þ were signi¢cant withrespect to the mean amplitude obtained at 0.5 mMBa2þ (P6 0.05).In order to evaluate whether Mg2þ could substitute

for Ca2þ in both its potentiating and blocking e¡ects,the same type of experiments as those shown in Fig. 3were carried out in oocytes superfused with an externalsolution in which Ca2þ was substituted for Mg2þ. Fig. 4shows representative traces of the responses obtained inNFS (1.8 mM Ca2þ without Mg2þ) and those obtainedin a nominally Ca2þ-free solution containing increasingconcentrations of Mg2þ. As illustrated in Fig. 4A, verysmall currents were obtained in the absence of Ca2þ

and low (up to 0.5 mM) Mg2þ as compared to thoseobtained in NFS. Upon increasing the concentration ofMg2þ to 1.8 and 3 mM, responses to 10 WM ACh wereundetectable. This result indicated that currentsthrough the K9K10 receptor were not potentiated byMg2þ but suggested that this divalent cation was exert-ing a blocking e¡ect. In order to study the potency ofthe Mg2þ block, oocytes were superfused with a salinesolution containing a ¢xed concentration of Ca2þ

(0.5 mM) and increasing concentrations of Mg2þ

(Fig. 4B). Under this condition the IC50 for the Mg2þ

block was 0.38 S 0.18 mM, nH = 1.06. Block by Mg2þ

was also analyzed at 1.3 mM Ca2þ, a concentrationsimilar to that observed in the perilymph bathing thebasolateral membrane of mammalian OHCs. An IC50of 0.33 S 0.02 mM with an nH of 0.58 was observed(Fig. 4C).In order to further investigate the e¡ect of divalent

cations on the ionic currents through the K9K10 recep-tor, the following experiments were carried out inNMG-based solutions in which either Ca2þ (ICa) orBa2þ (IBa) were the only inward charge carriers throughthe channel. In NFS solutions, inward currents are car-ried by Naþ plus Ca2þ or Ba2þ (ITotal). As illustrated inthe representative traces of Fig. 5A (upper panel), cur-rents elicited by ACh increased when the concentrationof Ca2þ was raised from 0.2 to 2 and 5 mM. This result

contrasts with the strong blocking e¡ect of millimolarCa2þ concentrations (s 1.8 mM) on ACh-elicited cur-rents in K9K10-expressing oocytes superfused with nor-mal saline solutions (see Fig. 3). The same experimentwas carried out substituting Ca2þ with Ba2þ and, asobserved with Ca2þ, the higher the Ba2þ concentration(between 0.2 and 5 mM) the greater the amplitude ofthe ACh-elicited currents (Fig. 5A, lower panel). Uponincreasing the Ba2þ concentration from 5 to 10 mM, nofurther signi¢cant increase in current was observed.Fig. 5B summarizes the e¡ects of increasing the concen-tration of either Ca2þ or Ba2þ in NMG and NFS solu-tions. Increments in the external concentration of Ca2þ

or Ba2þ (0.2 to 2 and 5 mM) in the NMG-based solu-tions caused signi¢cant increments in ICa and IBa,respectively. Conversely, the increase in the externaldivalent cation concentration in the NFS solutionscaused a strong reduction of ITotal. This result is similarto that reported for the homomeric K9 receptor (Katzet al., 2000) and suggests that block of ITotal by Ca2þ

and Ba2þ might be due in part to the fact that thesecations hinder the passage of Naþ through the channelas proposed for other nAChRs (Decker and Dani,1990).

3.3. Voltage sensitivity of Ca2+ potentiation and block

Ca2þ potentiation of several nAChR has been shownto be voltage-independent (Mulle et al., 1992a,b),whereas Ca2þ block has been shown to be voltage-de-pendent and mainly due to interactions of divalent cat-ions within the channel pore (Decker and Dani, 1990;Katz et al., 2000; Mulle et al., 1992a,b).In order to evaluate the voltage sensitivity of Ca2þ

potentiation and block, voltage ramps (3120 to+50 mV) were performed 10 s after the peak responseto 10 WM ACh in oocytes voltage-clamped at 370 mV,superfused with NFS solutions containing di¡erentCa2þ concentrations. Fig. 6A shows representativeI^V curves obtained in the same oocyte upon varyingthe Ca2þ concentration between 0.1 and 0.5 mM (con-centration range at which currents at hyperpolarizedpotentials, at either 370 or 390 mV, were potentiatedupon increasing external Ca2þ, see Fig. 3A). Compar-ison of current amplitudes obtained at 0.2 and 0.5 mMCa2þ with respect to those at 0.1 mM Ca2þ indicatedthat responses were potentiated to the same extent atboth hyperpolarized and depolarized membrane hold-ing potentials (Fig. 6A). This indicates that, as reportedfor other nAChRs (Mulle et al., 1992a,b), potentiationof the K9K10 receptor by Ca2þ is voltage-independent.Conversely, as illustrated in Fig. 6B,C, block by Ca2þ

was sensitive to membrane voltage, being more pro-nounced at hyperpolarized than at depolarized poten-tials. Fig. 6B shows representative I^V curves obtained

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in the same oocyte upon varying the Ca2þ concentra-tion between 0.5 and 3 mM (concentration range atwhich currents at hyperpolarized potentials, at either370 or 390 mV, were blocked upon increasing externalCa2þ, see Fig. 3A). Voltage-dependent block by Ca2þ isalso evident when comparing I^V curves obtained inthe presence of low Ca2þ (0.1 mM) to those obtained

in 3 mM external Ca2þ (Fig. 6C). The bar graph inFig. 6D shows that at 3 mM Ca2þ recti¢cation is out-ward whereas at low external Ca2þ recti¢cation is in-ward, as evaluated by measuring the ratio of currentamplitudes at +40 and 390 mV. This result suggestsa more e⁄cient blockage of the K9K10 receptor byCa2þ ions at hyperpolarized potentials.

Fig. 5. Currents carried by divalent cations. (A) Representative traces of currents elicited by 100 WM ACh in K9K10-expressing oocytes super-fused with a NMG-based saline solution containing di¡erent concentrations of either Ca2þ (upper panel) or Ba2þ (lower panel), Vhold =390mV. ACh-evoked currents increase as the concentration of either Ca2þ or Ba2þ (only permeant ions in the external medium) is increased. (B)Variations in current amplitude as a function of divalent cation concentration in the external medium in the NMG-based solutions (¢lled circlesand triangles) and in NFS solutions (clear circles and triangles). Amplitude values obtained at the di¡erent Ca2þ and Ba2þ concentrations inthe NMG-based solutions were normalized with respect to those obtained at 2 mM Ca2þ (ICa) or 2 mM Ba2þ (IBa), respectively. Each datapoint represents the meanSS.E.M. of the normalized response averaged from six to eight oocytes. ITotal data points were taken from the ex-periments illustrated in Fig. 2B,D. Note that when the divalent cation concentration in the external solution is increased from 0.2 to 5 mM,ITotal decreases whereas ICa and IBa increase.

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3.4. E¡ects of varying external Ca2+ on the apparenta⁄nity of the K9K10 nAChR for ACh

In order to evaluate whether changes in the AChapparent a⁄nity for the K9K10 receptor could accountfor the modulation of the K9K10 receptor by Ca2þ,concentration^response curves to ACh were performedat di¡erent Ca2þ concentrations. Fig. 7 illustrates theconcentration^response curves obtained in K9K10-ex-pressing oocytes superfused with a NFS solution con-taining either 1.8 mM or nominally zero Ca2þ. Underthese conditions the EC50 values were 49.2 S 5.4 WMand 13.6 S 1.5 WM for 0 and 1.8 mM Ca2þ, respectively.These di¡erences in the EC50 show that in a nominallyCa2þ-free saline solution the a⁄nity of the K9K10 re-ceptor for the agonist was lower than that observed inthe presence of 1.8 mM Ca2þ. The maximal responseobtained in zero Ca2þ was not signi¢cantly di¡erentfrom that obtained in 1.8 mM Ca2þ, indicating that

at high ACh concentrations, the activity of the K9K10receptor becomes independent of the presence of Ca2þ

in the external medium. These results suggest that Ca2þ

potentiation can be accounted for, at least in part, bychanges in the apparent a⁄nity of the K9K10 receptorfor ACh.

4. Discussion

In this work we report three major functional char-acteristics of the recombinant K9K10 nAChR receptor.First, it is highly permeable to Ca2þ and also to otherdivalent cations like Ba2þ and Mg2þ. Second, it is po-tentiated and blocked, through di¡erent mechanisms,by external Ca2þ in the physiological range. Third,both Ba2þ and Mg2þ are able to block this receptor,whereas only Ba2þ is able to substitute for Ca2þ in itspotentiating e¡ect.

Fig. 6. Voltage sensitivity of Ca2þ modulation. (A) Representative I^V curves obtained by application of a voltage ramp protocol (3120 to+50 mV, 2 s) 10 s after the peak response to 10 WM ACh in an K9K10-expressing oocyte voltage-clamped at 370 mV and superfused withNFS containing 0.1, 0.2 and 0.5 mM CaCl2. Note that potentiation of current amplitudes between 0.2 and 0.5 mM Ca2þ with respect to0.1 mM is similar at hyperpolarized and depolarized potentials (n=6 oocytes). (B) I^V curves obtained by the same protocol as in A, carriedout in oocytes superfused with NFS containing 0.5, 1.8 or 3 mM CaCl2. Note that Ca2þ block is more evident at hyperpolarized than at depo-larized potentials (n=6 oocytes). (C) I^V curves obtained by the same protocol as in A, carried out in oocytes superfused with NFS containingeither 0.1 or 3 mM Ca2þ. (D) Each bar represents the meanSS.E.M. of the ratio of current amplitudes obtained at +40 mV and 390 mV at0.1 and 3 mM Ca2þ (n=6 oocytes).

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4.1. Permeability of the K9K10 nAChR to divalentcations

Ligand-gated channels constitute an important path-way for calcium in£ux into cells at voltages close totheir resting potentials. Entry of Ca2þ through nAChRshas been shown to be involved in the modulation oftransmitter release in the central nervous system (Roleand Berg, 1996) and in the activation of secondary cal-cium-dependent conductances, as is the case of the IKCa

following activation of the cholinergic hair cell receptorboth in chick (Fuchs and Murrow, 1992) and mammals(Blanchet et al., 1996; Dulon et al., 1998; Erostegui etal., 1994a,b; Evans, 1996; Glowatzki and Fuchs, 2000;Nenov et al., 1996; Oliver et al., 2000). Based on theirrelative Ca2þ to Naþ permeability (PCa/PNa), nAChRshave been subdivided into three categories. The muscletype is the least permeable of all nAChRs (PCa/PNaW0.2^0.4, Adams et al., 1980; Decker and Dani,1990; Vernino et al., 1994), heteromeric neuronal recep-tors have a higher Ca2þ permeability (PCa/PNaW1.5,Fieber and Adams, 1991; Sands and Barish, 1991; Ver-nino et al., 1992, 1994) and the homomeric K7, K8 andK9 nAChR have the highest relative Ca2þ permeability(PCa/PNaW10^20, Bertrand et al., 1993; Katz et al.,2000; Sands and Barish, 1991; Se¤gue¤la et al., 1993).This latter group of nAChRs is similar to those li-gand-gated channels bearing the highest Ca2þ perme-ability, such as the N-methyl-D-aspartate receptors

and the cyclic nucleotide-gated channels from bovineretinal cones and olfactory sensory neurons (Frings etal., 1995; Mayer and Westbrook, 1987). The permeabil-ity to Ca2þ of the K9K10 heteromeric receptor estimatedin the present study is similar to that reported for thehomomeric K9 nAChR (PCa/PNaW9, Katz et al., 2000).Ca2þ as well as Ba2þ and Mg2þ block the Naþ currentthrough several ligand-gated channels, including theK9K10 receptor. As discussed in our previous paper(Katz et al., 2000), one should not disregard that thisinteraction violates the principle of ionic independenceassumed by the GHK formalism that was used to eval-uate the divalent cation permeability of all these chan-nels. Therefore, although informative, the absolute val-ues obtained for relative permeability by applying theGHK equation should only be taken as estimates.Even though the model of ion permeation repre-

sented by the GHK equation does not accurately pre-dict the amount of Ca2þ ions £owing through nAChRs(Lewis, 1979; Vernino et al., 1994), the fact that inX. laevis oocytes approximately 85% of the current ob-served upon activation of the K9K10 receptor is due tothe secondary activation of the native IClCa (see alsoElgoyhen et al., 2001) suggests that, as reported for therecombinant homomeric K9 (Katz et al., 2000) and K7(Se¤gue¤la et al., 1993) nAChRs, the fraction of Ca2þ

entering through this channel is signi¢cant. A signi¢-cant Ca2þ in£ux through this receptor is consistentwith the physiological function of the cochlear OHCreceptor, namely, to allow Ca2þ entry to the cell forthe subsequent activation of a hyperpolarizing currentthrough a calcium-dependent KCa channel (Blanchet etal., 1996; Dulon et al., 1998; Erostegui et al., 1994a;Evans, 1996; Fuchs and Murrow, 1992; Nenov et al.,1996; Oliver et al., 2000).

4.2. Modulation of the K9K10 nAChR by divalentcations

Muscle and neuronal nAChRs, both recombinantand native, are modulated by external Ca2þ. Ca2þ po-tentiates macroscopic currents through neuronal recep-tors in the physiological range whereas single channelconductances of these same channels are reduced byCa2þ (Mulle et al., 1992a; Vernino et al., 1992). Ca2þ,on the other hand, blocks muscle receptors both at thesingle channel and at the macroscopic level (Decker andDani, 1990; Vernino et al., 1992). In both types ofreceptors, blockage has been postulated to be due toa reduction in the Naþ £ux due to interactions ofCa2þ at the pore region, whereas Ca2þ potentiation ofneuronal nAChRs has been shown to result from anallosteric e¡ect of this cation at an external site (Deckerand Dani, 1990; Galzi et al., 1996; Mulle et al., 1992a;Vernino et al., 1992). The homomeric K9 receptor re-

Fig. 7. Potentiation by Ca2þ involves changes in ACh apparent af-¢nity. Concentration^response curves to ACh in oocytes superfusedwith NFS containing 0 and 1.8 mM Ca2þ (Vhold =370 mV). Eachdata point represents the meanSS.E.M. of current amplitudes ob-tained at the di¡erent ACh concentrations normalized to the ampli-tude obtained at 1 mM ACh in a NFS solution containing 1.8 mMCa2þ in the same oocyte (n=3 oocytes). EC50 values were 49.2S7 WM, nH =1.67 and 13.6S 1.5 WM, nH =1.13 for 0 and 1.8 mMCa2þ, respectively.

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sembles the muscle receptor in that it is not potentiatedbut is blocked, in a voltage-dependent manner (IC50100 WM at 370 mV), by Ca2þ at physiological concen-trations. As reported for the muscle nAChR, we havesuggested that this block involves hindering the passageof Naþ through the channel in the presence of Ca2þ

(Katz et al., 2000). Distinct to that described for K9receptors (Katz et al., 2000), macroscopic currentsthrough the K9K10 receptors were potentiated byCa2þ in the micromolar range and were reduced bythis cation at concentrations greater than 0.5 mM. Asimilar biphasic e¡ect of Ca2þ on macroscopic currentshas been reported for the native K7 receptors present inchick ciliary ganglion neurons (Liu and Berg, 1999).The fact that both processes, potentiation and block,are active within a similar range of concentrationsdoes not allow the estimation, from macroscopic cur-rents, of either the sensitivity or the e⁄cacy of Ca2þ topositively or negatively regulate this channel. However,the fact that at concentrations as low as 0.8 mM block-age starts to predominate, suggests that as reported forthe K9 receptor (Katz et al., 2000), the K9K10 receptoris more sensitive to Ca2þ block than the neuronal andmuscle nAChRs (Liu and Berg, 1999; Mulle et al.,1992a,b; Vernino et al., 1992). The fact that Ba2þ exertsthe same biphasic e¡ect on the ACh-evoked currentsthrough the K9K10 receptor whereas Mg2þ only blocksthese currents, is consistent with what has been re-ported for other nAChRs receptors (Booker et al.,1998; Ifune and Steinbach, 1991; Katz et al., 2000;Liu and Berg, 1999; Mulle et al., 1992a,b).The voltage dependence of Ca2þ block suggests that,

as reported for the recombinant homomeric K9 receptor(Katz et al., 2000), the neuronal and muscle nAChRs(Decker and Dani, 1990; Mulle et al., 1992a,b), as wellas for other ligand-gated and mechanosensitive chan-nels (Frings et al., 1995; Ricci and Fettiplace, 1998),the site of action of this ion might lie within the channelpore. Moreover, Ca2þ (as well as Ba2þ) is able to po-tently block ACh-evoked currents at physiological con-centrations when both Naþ and Ca2þ are the chargecarriers in the external solution (ITotal), but this blockis not observed when Ca2þ (or Ba2þ) is the sole chargecarrier in the external solution (ICa). We cannot disre-gard, however, the possibility that aside from this per-meation-mediated mechanism of block, divalent cationscould bind to sites within the pore and inhibit macro-scopic currents by altering channel gating or desensiti-zation kinetics. This hypothesis emerges from the factthat in the NMG-based saline solutions (with Ba2þ asthe sole permeant ion in the external medium), whenthe external Ba2þ concentration was raised from 5 to10 mM, IBa did not increase as one would expect ifblockage were only due to reduction of the monovalentcurrent.

Previous work on the modulation by Ca2þ of othernAChRs has shown that potentiation is independent ofboth Ca2þ in£ux and membrane potential (Mulle et al.,1992a,b; Vernino et al., 1992). Even though Ca2þ ishighly permeable through the K9K10 channel, potentia-tion was observed both in the presence of the fast Ca2þ

chelator BAPTA and at very depolarized voltages, atwhich Ca2þ entry would be negligible. Potentiation athyperpolarized potentials could be accounted for by anincrease in Ca2þ currents due to the rightward shifts inthe Erev, as expected for a permeant divalent cationupon raising its concentration. However, if this wasthe case, this positive shift should have a¡ected currentsin the opposite direction at depolarized potentials, i.e.inhibition instead of potentiation. As Ca2þ enhancedthe ACh-evoked currents through the K9K10 receptorto the same extent at both negative and positive volt-ages, we postulate that potentiation is due to an e¡ectthat is independent of Ca2þ in£ux and membrane po-tential. As reported for other nAChRs, the fact thatCa2þ caused a leftward shift in the ACh concentra-tion^response curve indicates that potentiation byCa2þ involves changes in the apparent a⁄nity of theK9K10 receptor for ACh. Previous studies have sug-gested that Ca2þ modulates some nAChRs by increas-ing the probability (Amador and Dani, 1995) or thefrequency of channel opening (Mulle et al., 1992a,b).However, single channel analysis is required in orderto postulate a mechanism for Ca2þ potentiation ofK9K10 nAChRs, which could include, apart fromchanges in binding a⁄nity, alterations in the kineticproperties of these channels.

4.3. The recombinant K9K10 nAChR and the native haircell cholinergic receptor

The high Ca2þ permeability of the recombinantK9K10 receptor is consistent with the physiologicalfunction of the native receptors, namely to allow Ca2þ

entry to the cell for the subsequent activation of anIKCa (Blanchet et al., 1996; Dulon et al., 1998; Eroste-gui et al., 1994a,b; Evans, 1996; Glowatzki and Fuchs,2000; McNiven et al., 1996; Nenov et al., 1996; Oliveret al., 2000). Even though Ca2þ permeability of the haircell receptor has not been studied in detail, a report onchick short hair cells (equivalent to mammalian OHCs)shows the Erev becomes 17 mV more depolarized whenthe Ca2þ concentration is raised from 1 to 47 mM,suggesting that the avian cholinergic receptor has asubstantial Ca2þ permeability (McNiven et al., 1996).The combination of calcium imaging with confocal mi-croscopy and electrophysiological techniques has re-vealed that concomitant to the activation of the IKCa,ACh elicits a Ca2þ response restricted to the synapticregion of OHCs (Blanchet et al., 1996). Moreover, stud-

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ies of Ca2þ relative permeability performed on hair cellprecursors from an immortalized cell line derived fromthe organ of Corti show that the Ca2þ permeability ofthe cholinergic receptor of those cells is very high (PCa/PNaW80, Jagger et al., 2000). This high Ca2þ perme-ability reinforces the hypothesis that the cholinergicOHC receptor would serve as a pathway to increaseinternal Ca2þ, rather than to depolarize the membranedue to Naþ entry to the cell.The biphasic modulation, potentiation and block,

that Ca2þ exerts on the K9K10 recombinant receptorhas also been reported for the chick short hair cellreceptor (McNiven et al., 1996). Moreover, the removalof Ca2þ reduces the early (cholinergic) current evokedby ACh in OHCs (Blanchet et al., 1996; Evans, 1996).In both preparations, however, Ca2þ was replaced byan equal amount of Mg2þ, invalidating any assumptionregarding the e¡ects of Ca2þ itself, since Mg2þ blocksthe ACh receptor with an IC50 of 0.38 mM (presentresults), which is lower than the concentration used inthose studies. Block by Mg2þ of ACh-evoked Kþ cur-rents in OHCs has been described (Nenov et al., 1996).However, in order to compare the e¡ects of divalentcations on the K9K10 recombinant receptor with thoseon the native OHC receptor, further studies on Ca2þ

permeability and divalent cation modulation should becarried out in OHCs in order to discriminate the directe¡ects of these ions on the ACh receptor from thoseover the secondary calcium-activated Kþ channelspresent on those cells.

4.4. Conclusions

We have shown that the K9K10 nAChR is highlypermeable to Ca2þ and that it is modulated by physio-logical concentrations of this cation. According to ourresults, the higher the Ca2þ concentration in the synap-tic cleft, the higher the fraction of the synaptic currentthrough the cholinergic receptor that will be carried byCa2þ and the smaller the current carried by monovalentcations. Thus, the function of this receptor would be tomaximize the activation of the hyperpolarizing IKCa byCa2þ, rather than to produce a membrane potentialshift due to entry of a depolarizing Naþ current.

Acknowledgements

This work was supported by an International Re-search Scholar grant from the Howard Hughes MedicalInstitute, a grant from Agencia Nacional de Promo-ciones Cient|¤¢cas y Te¤cnicas Argentina and the Re-search Fellowship Ramo¤n Carrillo-Arturo On‹ativia(Argentina) to A.B.E.

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