Laura Sartiani

Pascal Bochet

Elisabetta Cerbai

Alessandro Mugelli

Pascal Bochet #

Dr Rodolphe Fischmeister

Functional expression of the hyperpolarization-activated non selective cation current I f in immortalized HL-1 cardiomyocytes

INTRODUCTION

A variety of tissue cultures are used by cellular cardiologists to study cardiac function at the cellular level. Most of them use cardiomyocytes isolated from immature hearts, typically from chick embryonic or rat neonatal hearts, which retain their capacity to divide and maintain a cardiac phenotype in culture. However, in spite of a number of efforts (see e.g. [START_REF] Jacobson | Cell cultures of adult cardiomyocytes as models of the myocardium[END_REF][START_REF] Bugaisky | Differentiation of adult rat cardiac myocytes in cell culture[END_REF], long-term cultures of cardiomyocytes isolated from adult hearts lead to a progressive change in phenotype, as the cells move through dedifferentiated and redifferentiated states. Therefore, it has been a challenge for a number of investigators to obtain a stable adult cardiac cell line which would retain a differentiated cardiac phenotype while proliferating in culture.

Recently, one such effort gave rise to HL-1 cells [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]. These cells are derived from the atria of a transgenic mouse expressing the SV40 big T antigen under the control of the atrial natriuretic factor (ANF) promoter. The oncoprotein is responsible for the unique feature of these cells to proliferate indefinitely in vitro [START_REF] Lanson | The MRE11-NBS1-RAD50 pathway is perturbed in SV40 large T antigen-immortalized AT-1, AT-2 and HL-1 cardiomyocytes[END_REF], in contrast to other cardiac cell lines [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]. Under suitable culture conditions, HL-1 cells show spontaneous contractile activity and express many of the cardiac-specific genes, typical of a differentiated adult cardiac phenotype [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]. These include type (adult isoform) myosin heavy chain, -type-cardiac actin, ANF and connexin 43, the most abundant protein of cardiac gap junctions. Moreover, immunofluorescence methods showed the existence of perinuclear granules containing ANF, cardiac-specific muscle desmin filaments and sarcomeric myosin. Compared to adult cardiomyocytes and like mitotically active embryonic myoblasts, HL-1 cells show a less organised ultrastructure, their cytoplasm containing a lot of developing myofibrils and glycogen rich areas [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF].

With regard to signalling pathways, HL-1 cells express 1-adrenergic receptors which, as in rat cardiac myocytes (McWhinney et al. 2000b), couple to phosphatidyl inositol hydrolysis and mitogen-activated-phosphorylated-kinase signalling pathway (McWhinney et al. 2000a).

Moreover, [START_REF] Neilan | An immortalized myocyte cell line, HL-1, expresses a functional -opioid receptor[END_REF] showed the presence of functional -opioid receptors coupled to phosphatidyl inositol hydrolysis, in analogy with several studies performed in rat cardiomyocytes. However, up to now, there is no available information about the cAMP pathway in HL-1 cells.

Although confluent HL-1 cells typically show a spontaneous contractile activity, which suggests either a functional excitation-contraction coupling or the presence of spontaneous release of Ca 2+ from sarcoplasmic reticulum stores [START_REF] Viatchenko-Karpinski | Intracellular Ca 2+ oscillations drive spontaneous contractions in cardiomyocytes during early development[END_REF], little is known about the electrophysiological properties of these cells. Only the presence of the inward delayed rectifier K + current (IKr) has been shown [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]. This current is typical of cardiac tissue and is sensitive to dofetilide, an antiarrhythmic drug used clinically in atrial fibrillation and other arrhythmias. However, several reports [START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF][START_REF] Brochu | Pacemaker current in single cells and in aggregates of cells dissociated from the embryonic chick heart[END_REF][START_REF] Robinson | Developmental change in the voltage-dependence of the pacemaker current, If, in rat ventricle cells[END_REF][START_REF] Cerbai | Influence of postnatal-development on If occurrence and properties in neonatal rat ventricular myocytes[END_REF][START_REF] Abi-Gerges | Functional expression and regulation of the hyperpolarization activated nonselective cation current in embryonic stem cell-derived cardiomyocytes[END_REF][START_REF] Yasui | If current and spontaneous activity in mouse embryonic ventricular myocytes[END_REF]) demonstrated that spontaneously beating cardiac myocytes possess a hyperpolarization activated If current [START_REF] Difrancesco | Properties of the hyperpolarizing-activated current (If) in cells isolated from the rabbit sino-atrial node[END_REF]. Moreover, at least in human [START_REF] Porciatti | The pacemaker current If in single human atrial myocytes and the effect of -adrenoceptor and A1-adenosine receptor stimulation[END_REF], If current has been shown to be present in atrial myocytes, from which HL-1 cells are derived. Therefore, our aim in this study was to search for the presence of If current in HL-1 cells and to characterize some of its basic properties.

A preliminary report of this work has appeared elsewhere [START_REF] Sartiani | Voltage-dependent inward ion channel currents in immortalized HL-1 cardiomyocytes[END_REF].

METHODS

Culture and isolation of HL-1 cells

HL-1 cells at passage 71 were obtained from Dr. Claycomb, who first established and characterized the cell line [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]. Cells were grown in T25 coated flasks, as previously described [START_REF] Claycomb | HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte[END_REF]). Cells were maintained in "Claycomb Medium" (JRH Biosciences, Lenexa, Kansas, USA), supplemented with 10% foetal bovine serum (Life Technologies, Cergy Pontoise, France), 4 mM L-Glutamine (Life Technologies), 10 µM norepinephrine (Sigma Aldrich, L'Isle d'Abeau Chesnes, France) and penicillin-streptomycin (Life Technologies). The cultures were grown at 37°C, in an atmosphere of 5% CO2 and 95% air at a relative humidity of approximately 95%. The medium was changed every 24 hr.

Single HL-1 cells were isolated from cultures at different passages (from 74 to 93), in condition of confluence or 24 hr and 48 hr after splitting. To do this, the cells were detached using a 5 min enzymatic dissociation with trypsin-EDTA (Life Technologies). Digestion was stopped by adding medium and the sedimented cells were either re-plated at a 1 to 3 dilution (new "passage") or used for daily measurements.

Intracellular Ca 2+ measurements

After dissociation from the culture flask, cells were plated on gelatin/fibronectin coated coverslips. After reaching confluence they were loaded with Fluo-3 AM (Molecular Probes, Eugene, Oregon, USA) by incubation in 3 µM Fluo-3 AM in normal Tyrode's solution at 37°C for at least 30 min. They were put on the stage of an inverted confocal microscope (LSM510, Zeiss France, Le Pecq, France) and maintained at 37°C. Images were acquired with a Plan-Apochromat 63x/1.4 oil objective (Zeiss). Excitation was at 488 nm and fluorescence detection between 530 and 600 nm. Solutions were applied by gravity perfusion (1-2 ml/min) and the excess liquid was aspirated with a vacuum pump (Medcalf Bros, Potters Bar, UK); in these conditions, the time for exchanging solution near the cells (chosen in the centre of the dish to avoid turbulence) was approximately 17 s. A 64 x 64 pixels zone was scanned repeatedly at intervals of 50 ms. Finally, the signal was stored on a computer and the analysis was performed with the LSM program and Origin (Microcal Software, Inc., Northampton, MA, USA). The spontaneous beating frequency was determined by manual count of the number of oscillations in 10 sec intervals.

Electrophysiological recordings

Electrophysiological recordings were performed using the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981;[START_REF] Cerbai | Characterization of the hyperpolarizationactivated current, If, in ventricular myocytes isolated from hypertensive rats[END_REF][START_REF] Cerbai | Occurrence and properties of the hyperpolarization-activated current If in ventricular myocytes from normotensive and hypertensive rats during aging[END_REF] in isolated cells enzymatically dissociated from the culture at confluence or 24 and 48 hr after splitting. The experimental set-up consisted of an inverted microscope (Nikon, Champigny sur Marne, France), a home-made perfusion system, which allowed changes of the extracellular solution by gravity, a patch-clamp amplifier (Visual-Patch 500, Biologic, Claix, France) and a PC compatible computer. Data acquisition was performed by means of the Visual-Patch 500 V 1.31B software (Biologic) and data analysis by means of Biotools (Biologic) and Origin software.

After dissociation, the cells were re-suspended in normal Tyrode's solution and kept at room temperature 4-5 hours prior to experiments. During recordings, the cells were superfused with normal Tyrode´s solution (to measure action potentials, APs) or modified Tyrode´s solution (to measure If). Experiments were performed at room temperature. Patch-clamp pipettes, prepared from borosilicate glass capillary tubes (GC150-15, Clark Electromedical Instruments, Reading, UK) by means of a two-stage vertical puller (Narishige, Kyoto, Japan), had a resistance of 2-5 M, when filled with the internal solution. APs were measured in current-clamp conditions, elicited at a rate of 0.2 Hz by 2-ms square current pulses and sampled at 2 kHz. If current was evoked by 1.5 sec hyperpolarizing steps to potentials ranging from -60 to -130 mV from a holding potential of -40 mV. A mono-exponential fit of the current traces evoked at different potentials allowed to derive the time constant,  (s), of current activation which was analysed as a function of the membrane potential. The initial delay (maximally 22 ms) of the rising phase of the current was excluded from the fitting.

Since the protocol used to activate If current was not long enough to reach steady-state at all potentials, If amplitude was measured from the fit as the difference between the extrapolated value at steady state and that at the beginning of the test pulse, after the initial delay. These values were subsequently used to generate activation curves.

Current amplitudes were normalized with respect to cell membrane capacitance (Cm) in order to obtain current densities. Cm was automatically evaluated by the recording software (Visual-Patch, Biologic). Cm averaged 19.2 ± 1.7 pF (n=34), 18.3 ± 2.8 pF (n=6), and 17.1 ± 3.1 pF (n=10) in HL-1 cells at confluence, 24 hr and 48 hr after splitting, respectively (data not shown). These values were not significantly different and a similar dispersion of values, ranging from 5 to 53 pF, was present in the different passages of the cultures.

The reversal potential of If was evaluated by tail currents analysis. Briefly, tail currents were recorded by 1.5 s 'tail' steps to membrane potentials ranging from -70 mV to 0 mV in 5 or 10 mV step intervals, preceded by a 1 s conditioning potential step to -120 mV (holding potential -40 mV) at the end of which If was fully activated. To compensate for the presence of interfering currents and correct for the time-independent component (mainly leakage), the data were fitted to an exponential decay starting 30 to 50 ms after the beginning of the tail step. Current amplitudes were measured as the difference between the extrapolated value at steady state and that at the beginning of the tail step. Tail amplitudes were then plotted as a function of the test membrane potential to obtain the instantaneous current-voltage (I-V) relationship, which was then fitted by a linear function which intersected the X-axis at the reversal potential of If. Specific conductance was determined following the equation:

Vrev Vm I g f  
where gf (pS/pF) is the conductance calculated at the membrane potential Vm (mV), I (pA/pF) the current density and Vrev (mV), the reversal potential, derived from the analysis of the tail currents of the fully activated If current. Activation data were fitted by a Boltzmann function expressed by the equation:

k Vh Vm e Y / ) ( 1 1   
where Vm (mV) is the membrane test potential, Vh (mV) the fitted potential for half maximal activation and k (mV) the slope factor of the activation curve.

All data are expressed as mean ± standard error to the mean (S.E.M.). Statistical analysis was performed by means of Student's t-test. A p value of less than 0.05 was considered as significant.

Solutions and chemicals

Normal Tyrode's solution contained (in mM): NaCl 140; KCl 5.4; CaCl2 1.8; MgCl2 1.2; Dglucose 10; HEPES 5 (pH 7.35 with NaOH). Modified Tyrode's solution for If current was obtained from normal Tyrode's solution supplemented by (mM): NiCl2 2; BaCl2 2; 4aminopyridine 0.5 to eliminate, respectively, Ca 2+ current (T-and L-type), inward rectifier K + current, IK1, and transient outward K + current, Ito. KCl was increased to 25 mM to amplify If current.

Pipette solution contained (in mM): K-Aspartate 120; TEACl 10; Na2GTP 0.4; Na2ATP 5; MgCl2 2; EGTA (acid form) 11; CaCl2 5 (pCa 6.9); HEPES 10 (pH 7.2 with KOH).

L858051 was from Calbiochem (Meudon, France) and 8-bromo-cAMP from Sigma Aldrich.

Reverse Transcription-Polymerase Chain Reaction Amplification (RT-PCR)

Total RNA was isolated from HL-1 cells culture and mouse brain by the combined acid phenol-guanidinium thiocyanate-chloroform method, as used in the Extract-All protocol (Eurobio, Les Ulis, France). Prior to the reverse transcription (RT), RNA was treated with DNAse 1 (Life Technologies). cDNA were synthesised using Mouse Moloney Leukemia Virus Reverse Transcriptase (Life Technologies) following the instructions of the manufacturer and using poly dT oligonucleotide (12-18 bp) as primer. At the end of the RT the cDNA was aliquoted in water and kept at -80°C. Amplifications were performed on fresh aliquots.

PCR for hyperpolarization-activated, cyclic nucleotide-gated channel isoforms (HCN) was performed using primer constructions chosen on the basis of the mouse mRNA sequences I). Multiple alignment of the four sequences was generated with ClustalW [START_REF] Thompson | The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]. Selectivity of the primers was assured by the presence of mismatches at the 3'end with their not target sequences. Based on the human gene organization [START_REF] Ludwig | Two pacemaker channels from human heart with profoundly different activation kinetics[END_REF], primers were chosen on each side of the putative exons. The software Oligo [START_REF] Rychlik | A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA[END_REF] provided the thermodynamic characteristics relative to the compatibility of the primer pairs as well as an indication of the annealing temperature. PCR was conducted on 5 µl of the RT products, corresponding to 100 ng of initial total RNA, using 2.5 U Eurobiotaq® DNA polymerase (Thermus acquaticus YT1, Eurobio), 10 pmol of each primers (Life Technologies), 1.5 mM MgCl2 (Eurobio) in a final volume of 50 µl. The reaction was conducted in a thermal cycler (Gene Amp® PCR System 2400, Perkin Elmer, Courtabeuf, France), 3 min at 94°C, followed by 40 cycles consisting of 30 s at 94°C, 30 s at 52°C (HCN1 and HCN2) or 59°C (HCN3) or 54°C (HCN4), 90 s at 72°C and a final elongation at 72°C for 5 min. After amplification, 10 µl of PCR product were analysed on a 1.5% agarose gel stained with ethidium bromide.

(Table I near here) -actin PCR was carried out in the reverse transcription products both from HL-1 cells and mouse brain in order to test the efficiency of the reverse transcription reaction (RT+ products) and the deoxyribonuclease treatment quality (RT-product). The primers (Life Technologies) have sequences chosen on the basis of the mouse cytoskeletal -actin (AC: X03672) (Table I).

The PCR protocol was as described above, except for the annealing temperature, which was 54°C and the number of cycles, which was 35.

In order to determine the isoform identity of the amplified products, the PCR products were digested by specific restriction enzymes (Table I). The enzymatic digestion was performed as recommended by the manufacturer (New England Biolabs, Beverly, MA,USA).

RESULTS

Rhythmic oscillations of cytosolic Ca 2+ in HL-1 cells

During cardiac contraction, a transient increase in cytosolic Ca 2+ (Cai) occurs due to the sequential activity of voltage dependent Ca 2+ channels, intracellular Ca 2+ stores and Ca 2+ extrusion mechanisms [START_REF] Fabiato | Time and calcium dependence of activation and inactivation of calciuminduced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell[END_REF][START_REF] Dubell | A comparison of cytosolic free Ca 2+ in resting feline and rat ventricular myocytes[END_REF][START_REF] Nabauer | Regulation of calcium release is gated by calcium current, not gating charge, in cardiac myocytes[END_REF][START_REF] Barcenas-Ruiz | Voltage dependence of intracellular [Ca 2+ ]i transients in guinea pig ventricular myocytes[END_REF][START_REF] Bers | Ca transport during contraction and relaxation in mammalian ventricular muscle[END_REF]. In spontaneously beating cells, the rapid increase in Cai coincides with the occurrence of action potential (Huser et al. 2000), allowing Cai transients to be used as measures of the beating frequency. As shown in Fig. 2, the rhythmic oscillation of Cai of a spontaneously beating spot of cells was strongly affected by application of 4 mM extracellular Cs + , which completely blocked the spontaneous Cai rhythm (seen in 3 other experiments). After washout of Cs + , spontaneous activity readily recovered (Fig. 2). Since Cs + is a well known blocker of the hyperpolarization activated cationic (If) channel, which has been shown to be associated with cardiac pacemaker centres [START_REF] Difrancesco | Pacemaker mechanisms in cardiac tissue[END_REF], the blocking action of Cs + on spontaneous beating activity of HL-1 cells suggests a possible contribution of If current in the activity of these cells.

(Figure 2 near here)

Action potential characteristics of HL-1 cells

As it is generally accepted that If current contributes to the diastolic depolarisation phase of the action potential (AP) in spontaneously beating cells [START_REF] Difrancesco | Pacemaker mechanisms in cardiac tissue[END_REF], we recorded APs from HL-1 cells under whole-cell current-clamp conditions. Figure 3A andB show two representative APs recorded from two different isolated HL-1 cells. On average, the APs had a triangular shape, with an overshoot at +15.3  1.9 mV and a maximal diastolic potential of -68.8  1.6 mV (n=19). Action potential duration was 35.3  4.7 ms when measured at -20 mV and 69.2  8.9 ms at -50 mV (n=19). The two APs in Fig. 3 however differ in that the one in Fig. 3A exhibits a clear spontaneous diastolic depolarization phase, while in Fig. 3B, the diastolic potential was almost flat. Voltage-clamp experiments were performed on these two cells to check for the presence of a hyperpolarization-activated inward current. When the cells were voltage-clamped at -40 mV and hyperpolarizing steps to membrane potentials ranging from -60 to -130 mV were applied, a hyperpolarization-activated inward current was observed in the cell with a spontaneous diastolic depolarization phase (Fig. 3C), but not in the other cell (Fig. 3D). Out of 19 cells in which APs were successfully recorded, only 3 cells developed a spontaneous diastolic depolarization. Unfortunately, only the cell illustrated in

Characteristics of a hyperpolarization-activated inward current in HL-1 cells

In the experiments below, the cells were voltage-clamped right after patch-break, in order to make a more systematic assessment of the presence of If and to determine its characteristics.

In 30% of cells, hyperpolarization steps (1.5 sec long) ranging from -60 mV to -130 mV (10 mV intervals) elicited a time-dependent, non inactivating inward current, with an amplitude which increased as the potential became more negative. Representative current traces recorded in a single HL-1 cell are shown in Fig. 4A. Specific conductance was calculated as reported in Methods and normalized with respect to the maximal conductance value for each cell. Mean normalized conductance from 50 cells is plotted in Fig. 4B as a function of membrane potential. Activation curve was obtained by fitting the experimental data points to a Boltzmann function (see Methods). This yielded an activation threshold for the current between -50 and -60 mV, a half maximal activation potential (Vh) of -83.1  0.7 mV and a slope factor (k) of 9.0  0.6 mV. Time constants (, s) of activating currents were determined by mono-exponential fitting of individual current traces. Mean  values from 50 cells are plotted in Fig. 4C as a function of membrane potential. As the voltage becomes more negative, the activation of the current becomes progressively faster, the time constant going from 7.2  0.7 s at -60 mV to 0.20  0.03 s at -130 mV.

(Figure 4 near here)

The measurement of tail currents amplitude was used to evaluate the reversal potential (Vrev) of the current. Tail currents traces displayed in Fig. 5A were elicited in a 39 pF cell by depolarizing steps ranging from -60 to 0 mV in 5 mV increments, following a hyperpolarizing pulse to -120 mV, sufficient to maximally activate the current. Tail current amplitudes were measured as described in Methods and normalized with respect to Cm. The fully activated I-V relationship shown in Fig. 5B [START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF], atrial [START_REF] Porciatti | The pacemaker current If in single human atrial myocytes and the effect of -adrenoceptor and A1-adenosine receptor stimulation[END_REF]) and ventricular cells [START_REF] Cerbai | Characterization of the hyperpolarizationactivated current, If, in ventricular myocytes isolated from hypertensive rats[END_REF], as well as Purkinje fibers [START_REF] Difrancesco | Block and activation of the pace-maker channel in calf Purkinje fibres: effects of potassium, caesium and rubidium[END_REF] and cells (Callewaert et al. 1984). The block is voltage-dependent as the current is reduced only at potentials negative to its reversal potential, Vrev.

Figure 6 shows that the hyperpolarization-activated inward current recorded in HL-1 cells was also sensitive to Cs + . Tail currents were elicited as described in Methods in the absence or presence of 4 mM Cs + added to the extracellular solution. As shown by the individual current traces in Fig. 6A, application of Cs + resulted in a complete block of the current elicited during the conditioning pulse at -120 mV. Moreover, the I-V relationship in 4 mM Cs + (Fig. 6B) shows a clear reduction of the inward currents at potentials negative to Vrev, while the outward currents were unaffected. Similar voltage dependent block for f-channel by Cs + has been reported in several other studies [START_REF] Difrancesco | Block and activation of the pace-maker channel in calf Purkinje fibres: effects of potassium, caesium and rubidium[END_REF][START_REF] Difrancesco | Properties of the hyperpolarizing-activated current (If) in cells isolated from the rabbit sino-atrial node[END_REF][START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF]. As shown in the activation curves of Fig. 6C, 4 mM Cs + blocked the hyperpolarization-activated inward current in HL-1 cells at all membrane potentials ranging from -60 to -130 mV, and its effect was mostly reversible upon washout. At -120 mV, 4 mM Cs + blocked 92.0 ± 7.9% (n=4) of the current.

Thus, from the voltage-dependence, the activation curve and the sensitivity to extracellular Cs + , we conclude that the hyperpolarization-activated inward current measured in HL-1 cells is carried by f-channels. This current will be denominated If from now on.

(Figure 6 near here)

Presence of I f throughout the cultures in HL-1 cells

As shown in Fig. 3, If was not found to be present in every cell. Therefore, we tested whether the state of the cultures might affect the occurrence of the current. At confluence, 31% of cells (34 out of 109 tested cells) exhibited an If current; when examined 24 hr after splitting, If was observed in 32% of cells (6 out of 19 tested cells) and in 23% of cells (10 out of 44 tested cells) 48 hr after splitting. Thus, the degree of confluence was not a determinant factor for the occurrence of If in HL-1 cells.

Figure 7A shows the scatter of If current densities (pA/pF) elicited at -120 mV in a large number of individual experiments and their mean values in each of the three culture conditions. The relative time constants of If current activation (, s) are reported in a similar manner in Fig. 7B. No significant difference in current density and time constants of current activation was found in the different culture conditions. Moreover, the activation curves obtained by fitting the experimental data did also not show a significant difference. Indeed, Vh values were -82.6 ± 0.6 mV (n=34), -80.9 ± 1.1 mV (n=6) and -83.9 ± 0.9 mV (n=10) for cells at confluence, 24 hr and 48 hr after splitting, respectively. In the three same conditions, the slope factors k was 9.3 ± 0.7 mV, 8.9 ± 1.0 mV and 9.5 ± 1.0 mV.

In conclusion, If current is present in about 30% of HL-1 cells and its characteristics are rather constant over the course of the culture. Thus, all data obtained at different degrees of confluence were pooled for subsequent analysis.

(Figure 7 near here)

Effect of adenylyl cyclase stimulation on I f in HL-1 cells

A classical hallmark of the cardiac If current is its stimulation by intracellular cAMP (for reviews, see e.g. [START_REF] Difrancesco | Pacemaker mechanisms in cardiac tissue[END_REF][START_REF] Kaupp | Molecular diversity of pacemaker ion channels[END_REF][START_REF] Accili | From funny current to HCN channels: 20 years of excitation[END_REF]. Thus, the direct binding of cAMP to the intracellular cyclic nucleotide-binding domain of f-channels leads to a positive shift in the activation curve of If current (DiFrancesco & Tortora, 1991; Ludwig et al. 1998; Santoro et al. 2000; Wainger et al. 2001; Moroni et al. 2001; Viscomi et al. 2001). Moreover, a cAMP dependent phosphorylation via cAMP-dependent protein kinase (PKA) has been reported to modulate If current [START_REF] Chang | Effects of protein kinase inhibitors on canine Purkinje fibre pacemaker depolarization and the pacemaker current If[END_REF][START_REF] Abi-Gerges | Functional expression and regulation of the hyperpolarization activated nonselective cation current in embryonic stem cell-derived cardiomyocytes[END_REF] producing an increase of the fully-activated current [START_REF] Accili | Differential control of the hyperpolarization activated current (if) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes[END_REF][START_REF] Proenza | Modulation of mHCN2 by cAMP[END_REF].

To examine whether If current is also regulated by cAMP in HL-1 cells, we tested the effect of L858051, a water-soluble and stable analogue of forskolin. Forskolin is known to directly stimulate adenylyl cyclase activity and increase intracellular cAMP. Like forskolin, L858051 has been shown to increase the L-type Ca 2+ current by a cAMP dependent mechanism (Hartzell & Budnitz, 1992). Figure 8 shows the mean fractional activation of If current in HL-1 cells plotted as a function of membrane potential, both in the absence and presence of 20 µM L858051. A fit of the data points to the Boltzmann function revealed a shift of the activation curve towards less negative potentials in the presence of L858051. Indeed, Vh varied from -96.0  0.9 mV to -90.6  1.4 mV (n=4, P<0.05). In the presence of 20 µM L858051, the maximally activated conductance was increased by 37  4% (n=4, P<0.001) compared to the control solution in the same cells. Thus, activation of the cAMP pathway in HL-1 cells leads to both a positive shift in the If activation curve and an increase of the fullyactivated If current.

(Figure 8 near here)

Molecular characterization of HCN isoforms present in HL-1 cells

So far, our results indicate that a time dependent inward current is present in HL-1 cells with the typical characteristics of an If current as found in cardiac pace-maker cells [START_REF] Porciatti | The pacemaker current If in single human atrial myocytes and the effect of -adrenoceptor and A1-adenosine receptor stimulation[END_REF][START_REF] Difrancesco | Pacemaker mechanisms in cardiac tissue[END_REF]. In the following, our aim was to pin-down the molecular identity of the f-channel(s) expressed in HL-1 cells.

Until now, four different isoforms of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels have been described to possibly underlie the If current [START_REF] Kaupp | Molecular diversity of pacemaker ion channels[END_REF]. Since several reports show the presence of the four isoforms (HCN1, HCN2, HCN3 and HCN4) in mouse brain [START_REF] Ludwig | A family of hyperpolarization-activated mammalian cation channels[END_REF][START_REF] Ludwig | Two pacemaker channels from human heart with profoundly different activation kinetics[END_REF][START_REF] Santoro | Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain[END_REF][START_REF] Santoro | Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS[END_REF], we used this tissue as a positive control to test the efficiency of the primer pairs, each being designed to amplify selectively one isoform (see Methods). Products obtained by PCR amplification had the size expected from the published sequences [START_REF] Ludwig | A family of hyperpolarization-activated mammalian cation channels[END_REF][START_REF] Santoro | Molecular and functional heterogeneity of hyperpolarization-activated pacemaker channels in the mouse CNS[END_REF] see Table I). Figure 9A shows an agarose gel of the PCR amplification products obtained from mouse brain using the RT-PCR amplification protocol described in Methods. Four fragments of the expected size are clearly detectable, thus verifying the efficiency of the four primer pairs. RT-PCR from HL-1 cells was performed with the same primers and the same thermal cycle conditions as in brain and produced a pattern of four bands (Fig. 9B), revealing the presence of all four isoforms. The comparison of the signals obtained from HL-1 cells and mouse brain clearly shows that HCN1 and HCN2 isoforms are detectable as strong bands both in HL-1 cells and in brain. Conversely, while the used PCR conditions allowed us to obtain rather strong HCN3 and HCN4 bands from brain, the same conditions performed in HL-1 cells yielded weak bands, just above the detectable level. Therefore, we expect HCN1 and HCN2 isoforms to be expressed in a larger amount with respect to HCN3 and HCN4 isoforms and to contribute predominantly to the functional If current in HL-1 cells.

(Figure 9 near here)

To check the identity of each amplified fragment, the products were digested using specific restriction endonucleases (Table I). Figure 10 shows the results obtained both from brain and HL-1 cells. Enzymatic digestion of HCN1 and HCN2 products both from brain and HL-1 cells were as expected from the sequences (Table I). Due to the weak signal obtained in HL-1 cells for HCN3 and HCN4 isoforms, the identity of the amplified products was assessed only in the brain products. There, the amplified fragments were abundant enough to produce detectable signals after the digestion. In both cases the size of the fragments was in accordance with a specific amplification of the right isoform sequences.

(Figure 10 near here)

DISCUSSION

In this study, we described the presence of a hyperpolarization-activated inward current in immortalized HL-1 cardiomyocytes. The current has many of the electrophysiological characteristics of If current present in pacemaker cardiac cells [START_REF] Difrancesco | Pacemaker mechanisms in cardiac tissue[END_REF]: 1) it is typically activated upon a hyperpolarization to membrane potentials <-50 mV; 2) its activation is time dependent and its amplitude increases as the potential becomes more negative; 3) it is blocked by 4 mM Cs + and is insensitive to Ba 2+ ; 4) it is activated by cAMP.

If current was present in only about 30% of HL-1 cells. Several studies have reported a cellular heterogeneity with respect to the presence of If current in myocytes isolated from native cardiac tissues, such as sino-atrial node (Denyer & Brown, 1990a;[START_REF] Belardinelli | Ionic mechanisms of adenosine actions in pacemaker cells from rabbit heart[END_REF]) and atrial tissue [START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF]. However, the morphology of HL-1 cells was rather homogeneous, characterized by a flat, triangular-spindle-like shape. After enzymatic dissociation, all HL-1 cells rounded up so that it was impossible by looking at the cell to predict which cell would express If current and which would not. We examined the possibility that f-channel density might increase together with an increase in cell size, as has been reported in dedifferentiated adult rat ventricular cells in primary culture [START_REF] Fares | Characterization of a hyperpolarization-activated current in dedifferentiated adult rat ventricular cells in primary culture[END_REF] and in rabbit sino-atrial node cells (Honjo et al. 1996). However, we found no significant difference in cell membrane capacitance between cells which expressed an If current (18.7 ± 1.3 pF, n=50) and those which did not (16.7 ± 0.8 pF, n=145), which is rather consistent with an independent regulation of f-channel expression and cell dimension [START_REF] Cerbai | Occurrence and properties of the hyperpolarization-activated current If in ventricular myocytes from normotensive and hypertensive rats during aging[END_REF][START_REF] Cerbai | Influence of postnatal-development on If occurrence and properties in neonatal rat ventricular myocytes[END_REF]. As shown in Fig. 7, the passage number or the degree of confluence of the cell culture was also not a determinant factor for the presence of If current. A possible hint for the limited presence of If current in HL-1 cells might come from the spontaneous activity of the cells. Indeed, prior to enzymatic dissociation, when the cells were attached to the protein matrix of the flask, there were regions where the cells showed a spontaneous contractile activity and other regions where the cells were quiet (Fig. 1). As shown in Fig. 2, these beating regions stopped their spontaneous activity in the presence of Cs + which strongly suggests the presence of If current in these cells as well as its contribution to spontaneous activity.

The I-V relationship of the fully activated If current in HL-1 cells is linear with a reversal potential of -21 mV, as determined in 25 mM K + external solution. This value is consistent with a selectivity of the channel for Na + and K + [START_REF] Difrancesco | A study of the ionic nature of the pace-maker current in calf Purkinje fibres[END_REF][START_REF] Difrancesco | Properties of the hyperpolarizing-activated current (If) in cells isolated from the rabbit sino-atrial node[END_REF]Ho et al. 1994). The permeability ratio PNa/K, as calculated using the Goldman-Hodgkin-Katz related equation (Frace et al. 1992), is 0.37 ± 0.02, a value in the range of those reported for If channels in cardiac myocytes (Frace et al. 1992;[START_REF] Irisawa | Cardiac pacemaking in the sinoatrial node[END_REF][START_REF] Cerbai | Characterization of the hyperpolarizationactivated current, If, in ventricular myocytes isolated from hypertensive rats[END_REF][START_REF] Cerbai | Characterization of the hyperpolarization activated current, If, in ventricular myocytes from human failing heart[END_REF][START_REF] Hoppe | Characterization of the hyperpolarizationactivated inward current in isolated human atrial myocytes[END_REF].

Analysis of the voltage dependence of If current activation in HL-1 cells allowed to determine a potential for half maximal activation (Vh) of -83.1  0.7 mV and a slope factor of the activation curve (k) of 9.0  0.6 mV which are similar to the values reported for If current in native human atrial myocytes [START_REF] Porciatti | The pacemaker current If in single human atrial myocytes and the effect of -adrenoceptor and A1-adenosine receptor stimulation[END_REF][START_REF] Hoppe | Characterization of the hyperpolarizationactivated inward current in isolated human atrial myocytes[END_REF] and cat atrial pacemaker cells [START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF]. Moreover, a recent study by Ulens & Tytgat (2001a) showed that the functional expression of concatenated heteromers of HCN1 and HCN2 isoforms in Xenopus oocytes display a current with similar activation parameters as those reported in our study. Finally, the hyperpolarization-activated Ih current in CA1 hippocampal neurons, where coexpression of HCN1 and HCN2 isoforms has been detected at the singlecell level, shows activation parameters that closely resemble those found in HL-1 cells (Franz et al. 2000).

Likewise, the time constant of activation of If current in HL-1 cells () varied as a function of voltage, from approximately 7.2  0.7 s at -60 mV to 0.20  0.03 s at -130 mV, in a similar manner as reported for If current in native human atrial myocytes [START_REF] Hoppe | Characterization of the hyperpolarizationactivated inward current in isolated human atrial myocytes[END_REF]) and cat atrial pacemaker cells [START_REF] Zhou | Properties of the pacemaker current (If) in latent pacemaker cells isolated from cat right atrium[END_REF]. At -120 mV,  was 0.29  0.04 s in HL-1 cells, which corresponds to an intermediate value between the slow and fast time constants reported for the current obtained at this potential upon expression of concatenated HCN1-HCN2 channels (Ulens & Tytgat, 2001a). For a closer comparison with the latter study, we performed a bi-exponential fit of If current traces evoked at -120 mV in HL-1 cells: this analysis yielded a fast and a slow time constants of 0.11 ± 0.02 s (n=10) and 0.58 ± 0.11 s (n=10), respectively, which are quite similar to the values reported by Ulens & Tytgat (2001a) and those obtained for Ih current in CA1 hippocampal neurons (Franz et al. 2000). These strong similarities between the functional properties of If current in HL-1 cells and the current induced by expression of a concatenated HCN1-HCN2 channel (Ulens & Tytgat, 2001a) or the Ih current in CA1 hippocampal neurons (Franz et al. 2000), strongly suggest that HCN1 and HCN2 channel isoforms also account for the If current in HL-1 cells. Further support for this hypothesis comes from our RT-PCR experiments, which showed that HCN1 and HCN2 channel mRNAs are expressed in larger amounts as compared to HCN3 and HCN4 isoforms in HL-1 cells. Thus, HL-1 cells differ from sino-atrial myocytes where the If current is carried mainly by HCN1 and HCN4 channel isoforms [START_REF] Ishii | Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node[END_REF][START_REF] Moroni | Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current If[END_REF].

Activation of the cAMP pathway by the forskolin analogue L858051 showed a small but significant shift of the If current activation potential Vh towards more positive values.

Although this effect was observed in a low portion of cells (4 out of 8 cells), several reasons make us confident that this phenomenon was real: 1) the effect of L858051 was mimicked by 8-bromo-cAMP (400 µM, 2 out of 10 cells), a membrane-permeant cAMP analogue which also activates PKA (data not shown); 2) there was no spontaneous shift of Vh in control conditions, after an equivalent amount of time (11  3 min) as when measuring the effect of L858051 (data not shown); 3) the shift in Vh was quantitatively similar to that reported by others for the heteromeric HCN1-HCN2 current (Ulens & Tytgat, 2001a), the homomeric HCN2 current (Ulens & Tytgat, 2001b), and the native Ih current in CA1 hippocampal neurons (Franz et al. 2000). Why then was the effect of cAMP on If current so rare? One likely reason is that the culture medium contained 10 M norepinephrine, thus providing a chronic stimulation of cAMP synthesis which is likely to maximally shift Vh towards positive voltages (Graf et al. 2001). In support of this hypothesis is the finding that Vh was -96.0  0.9 mV (n=4) under basal conditions in the cells responding to L858051, while Vh was -83.2  1.5 mV (n=4) in the cells not responding to the forskolin analogue.

As a consequence of the relative low occurrence of If current in HL-1 cells and its low responsiveness to cAMP, it was rather difficult to study in detail the intracellular mechanisms involved in this regulation. However, our data demonstrate that in addition to a positive shift of the If current activation, L858051 also increased significantly the If maximal conductance in HL-1 cells. This effect resembles that of 8-bromo-cAMP on Ih maximal conductance in CA1 hippocampal neurons (Gasparini & DiFrancesco, 1999) and of calyculin A, a phosphatase inhibitor, on If maximal conductance in rabbit sino-atrial node myocytes [START_REF] Accili | Differential control of the hyperpolarization activated current (if) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes[END_REF]. According to the latter study, the increase in If maximal conductance might result from phosphorylation via PKA of the f-channels [START_REF] Accili | Differential control of the hyperpolarization activated current (if) by cAMP gating and phosphatase inhibition in rabbit sino-atrial node myocytes[END_REF], which clearly differs from the direct binding of cAMP to the HCN channels leading to the positive shift of the activation curve (see e.g. [START_REF] Moroni | Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current If[END_REF][START_REF] Viscomi | C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels[END_REF]. Preliminary data by [START_REF] Proenza | Modulation of mHCN2 by cAMP[END_REF] show that homomeric mouse HCN2 channel displays an increase in maximal current due to PKA activation. This behaviour may be specific to the HCN2 channel isoform, since no effect of cAMP on maximal conductance was found when expressing the HCN1 [START_REF] Moroni | Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current If[END_REF] or HCN4 isoform [START_REF] Ishii | Molecular characterization of the hyperpolarization-activated cation channel in rabbit heart sinoatrial node[END_REF]. This suggests that, in HL-1 cells, modulation of the maximal conductance of f-channels is dominated by the HCN2 isoform.

Although the precise relationship between If activation and HL-1 automaticity was beyond the scope of this study, action potential recordings demonstrated that i) the maximal diastolic potential of HL-1 cells was more negative than the threshold potential for If activation, and ii)

If was always absent in cells lacking a spontaneous diastolic depolarization. Moreover, the spontaneous Cai oscillations were blocked by Cs + at the same concentration at which it fully blocked If. While this may indicate a possible participation of If current to the spontaneous contractile activity of these cells, a more precise pharmacological dissection of the different potential pacemaker currents is needed to evaluate their respective contribution to the electrical activity of these cells. For instance, in proliferating immature or early developed mouse myocytes which also express a functional If channel [START_REF] Abi-Gerges | Functional expression and regulation of the hyperpolarization activated nonselective cation current in embryonic stem cell-derived cardiomyocytes[END_REF], electrical activity can be generated by spontaneous Ca 2+ release from sarcoplasmic reticulum [START_REF] Viatchenko-Karpinski | Intracellular Ca 2+ oscillations drive spontaneous contractions in cardiomyocytes during early development[END_REF]. This indicates that the presence of If may not necessarily imply that this current is necessary or sufficient to generate spontaneous activity. HONJO, H., BOYETT, M. R., KODAMA, I. & TOYAMA, J. (1996). Correlation between electrical After washout of Cs + , spontaneous activity restored progressively (detail in lower panel, B) and reached a stable frequency of 3.8 Hz.

In conclusion HL-1 cells display a hyperpolarization-activated

  coding for HCN1 (Accession Number (AC): AJ225123; Ludwig et al. 1998), HCN2 (AC: AJ225122; Ludwig et al. 1998), HCN3 (AC: AJ225124; Ludwig et al. 1998), and HCN4 (AC: AF064875, partial cds; Santoro et al. 1998) isoforms (Table

  Figure 1 (lower panel, trace A) shows the rhythmic variation of fluorescence intensity of Fluo-3 AM loaded cells measured in a spontaneously beating region of a HL-1 cell culture (region A, upper panel). As a control, the variations of fluorescence intensity (lower panel, traces B and C) relative to two non beating zones (regions B and C, upper panel) are reported. The spontaneous beating frequency of different contracting spots of cells was calculated as reported in Methods, and ranged between 1.3 and 5 Hz (n=8). (Figure 1 near here)

Fig

  Fig. 3A and C supported the additional voltage-clamp protocol and elicited a hyperpolarization-activated inward current. In the 16 other cells, 11 were voltage-clamped with success and none of them developed a hyperpolarization-activated inward current.

  was obtained by plotting the mean values of normalized tail currents from 10 cells as a function of membrane potential. Best fit through the data points gave a linear relationship that intersects with the voltage axis (Vrev) at -20.8  1.5 mV and has a slope maximal conductance of 89.7  0.4 pS/pF. (Figure 5 near here) Sensitivity of the hyperpolarization-activated inward current to extracellular Cs + Blockade by extracellular Cs + is a hallmark of If current, since millimolar concentrations of Cs + were shown to inhibit If in sino-atrial node cells (DiFrancesco et al. 1986), latent atrial pacemaker cells

  If current which shares several functional properties with native cardiac f-channels. Molecular characterization of the HCN channels responsible for If current demonstrated a dominant contribution of HCN1 and HCN2 channel isoforms. HL-1 cells may thus provide a useful tool for testing pharmacological drugs acting on If current as well as identifying key factors in the cellular environment acting on HCN channel expression. FRACE, A. M., MARUOKA, F., NOMA, A. (1992). External K + increases Na + conductance of the hyperpolarization-activated current in rabbit cardiac pacemaker cells. Pflügers Archiv 421, 97-99. FRANZ, O., LISS, B., NEU, A. & ROEPER, J. (2000). Single-cell mRNA expression of HCN1 correlates well with a fast gating phenotype of hyperpolarization-activated cyclic nucleotide-gated ion channel (Ih) in central neuron. European Journal of Neuroscience 12, 2685-2693. GASPARINI, S. & DIFRANCESCO, D. (1999). Action of serotonin on the hyperpolarizationactivated cation current (Ih) in rat CA1 hippocampal neurons. European Journal of Neuroscience 11, 3093-3100. GRAF, E. M., HEUBACH, J. F. & RAVENS, U. (2001). The hyperpolarization-activated current If in ventricular myocytes of non-transgenic and 2-adrenoceptor overexpressing mice. Naunyn-Schmiedeberg's Archives of Pharmacology 364, 131-139. HAMILL, O. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGWORTH, F. J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv 391, 85-100. HARTZELL, H. C. & BUDNITZ, D. (1992). Differences in effects of forskolin and an analog on calcium currents in cardiac myocytes suggest intra-and extracellular sites of action. Molecular Pharmacology. 41, 880-888. HO, W. K., BROWN, H. F. & NOBLE, D. (1994). High selectivity of the If channel to Na + and K + in rabbit sinoatrial node cells. Pflügers Archiv 426, 68-74.

Figure 1 .

 1 Figure 1. Spontaneous activity measured by fluorescence intensity oscillations in Fluo-3 AM loaded HL-1 cells HL-1 cells at confluence were incubated in 3 M Fluo-3 AM in normal Tyrode's solution at

Figure 2 .

 2 Figure 2. Blockade of spontaneous beating of HL-1 cells by Cs +Fluorescence emission was measured from a group of HL-1 cells which were spontaneously beating. As shown in the upper panel, the cell preparation was first superfused with normal Tyrode's solution and spontaneous oscillations developed with a frequency of 2.7 Hz. When 4 mM Cs + was added to the solution, spontaneous activity stopped (detail in lower panel, A).

Figure 3 .

 3 Figure 3. Action potential characteristics of HL-1 cells

Figure 4 .

 4 Figure 4. Voltage-dependence of a hyperpolarization-activated I f current in HL-1 cells Isolated HL-1 cells were hyperpolarized during 1.5 sec from a -40 mV holding potential to various potentials ranging from -60 mV to -130 mV (in 10 mV intervals) as shown by the voltage-protocol in the bottom part of A. Tail currents were obtained by a subsequent depolarization to +40 mV as indicated. The top part in A shows representative activating and tail current traces obtained with this protocol. Mean If fractional activation (B) and If activation time constant (C) were obtained at each membrane potential from the fit of the activating current traces as described in Methods. Each symbol shows the mean  S.E.M. of 50

Figure 5 .

 5 Figure 5. Steady-state current-voltage relationship of I f current in HL-1 cells Isolated HL-1 cells were hyperpolarized during 1.5 sec to -120 mV from a -40 mV holding potential to fully activate If current. Tail currents were obtained by subsequent depolarizations to potentials ranging from -70 to 0 mV (in 5 or 10 mV steps) as indicated in the voltageprotocol in the bottom part of A. The top part in A shows representative current traces obtained with such protocol. Mean peak tail current amplitudes were normalized to cell membrane capacitance to give current densities and are shown in B as a function of membrane potential. Each symbol shows the mean  S.E.M. of 10 similar experiments as in A.

Figure 6 .

 6 Figure 6. Blockade of I f current by Cs + A, The same protocol as in Fig. 5 was applied to a HL-1 cell in a modified Tyrode's solution (control) and after application of 4 mM Cs + . Representative current traces are shown in A. The current-potential (I-V) relationship of the corresponding tail current density is shown in B under control conditions () and in the presence of 4 mM Cs + (). Specific If current conductance (gf) was calculated as described in Methods and is plotted in C as a function of membrane potential in control conditions (), in the presence of 4 mM Cs + () and after washout of Cs + ().

Figure 7 .

 7 Figure 7. Influence of the culture condition on I f current density and activation time constant If current densities (A) and activation time constants (B) were measured at -120 mV membrane potential in HL-1 cells which were either at confluence, 24 hr or 48 hr after splitting. Values from individual experiments () and means  S.E.M. (, with the number of

Figure 8 .

 8 Figure 8. Effect of the forskolin analogue L858051 on I f current in HL-1 cells Mean activation curve of If current was obtained in modified Tyrode's solution in the absence () and presence of 20 µM L858051 (). Each symbol shows the mean  S.E.M. of 4 cells.

Figure 9 .

 9 Figure 9. PCR amplification of hyperpolarization-activated channels cDNAs

-850 -650 -500 -400 -300 -200 -

Acknowledgements

We thank Patrick Lechêne for skilful technical help and Valérie Nicolas from the Service Commun d'Imagerie Cellulaire of the Institut de Signalisation et Innovation Thérapeutique (IFR-75 ISIT) for performing the experiments using the confocal microscope. L. Sartiani was supported by a research grant from the Italian Pharmacological Society. This work was supported by an INSERM-CNR exchange program.

A, amplification from mouse brain cDNA. B, amplification from HL-1 cells cDNA. Molecular weights (in bp) for the size markers are indicated on the left side. The primers and restriction enzymes used are listed in Table I. Note that all four HCN isoforms are equally detected in mouse brain whereas HCN1 and HCN2 are predominant in HL-1 cells.