Selective Hcn1 Channels Inhibition by Ivabradine in Mouse Rod Photoreceptors

Gian Carlo Demontis,1 Claudia Gargini,1 Timoteo Giacomo Paoli,1 and Luigi Cervetto2

PURPOSE. To evaluate in mammalian rod photoreceptors the different hyperpolarization-activated cyclic nucleotide (HCN)- selectivity for hyperpolarization-activated cyclic nucleotide- gated channel isoforms by pacing cells10,11 and retinal neu- gated (Hcn1, coded by Hcn1) over potassium-selective (Kir rons.12–14 However, all HCN isoforms have high structural 2.4, coded by Kcnj14) channels of ivabradine, a selective homology in the pore region, which includes the putative 15 inhibitor of the cardiac “funny” current (If). drug-binding pocket, suggesting that visual symptoms may METHODS. Rods were isolated from the mouse retina and volt- result from the suppression by If inhibitors of the hyperpolar- age clamped by the perforated-patch technique. The hyperpo- ization-activated currents (Ih) in retinal neurons. An additional action of these inhibitors on potassium currents may also play larization-activated current (Ih) was blocked by ivabradine during repetitive stimulation with activating/deactivating voltage a role in the generation of visual phenomena, as suggested by steps from Ϫ80 to Ϫ30 mV, from a holding of Ϫ35 mV. the observation that zatebradine, an If inhibitor structurally related to ivabradine, blocks potassium currents and Ih in RESULTS. Full inhibition was observed at a high concentration of 16 ␮ ␮ amphibian rods. ivabradine (30 M), with intermediate effects at 3 and 0.3 M. The aim of the present work was to investigate in mamma- Steady state activation and activation kinetics of the ivabradine- lian rods the inhibition of Ih by ivabradine and its selectivity and CsCl-blocked currents were similar, consistent with the over potassium currents. To this end we compared the effects block by ivabradine of ion permeation through Hcn1 channels. of ivabradine and CsCl on inward and outward currents of Hcn1 blockade was also consistent with the lack of current Ϫ mouse rod photoreceptors. CsCl is an inorganic blocker of reactivation during long steps at 110 mV. At doses that fully inward rectifier currents that has already been used to investi- block Ih, ivabradine does not affect the inward rectifier current gate properties and functional roles of voltage-dependent cur- through potassium-selective Kir 2.4 channels or the outward 17–19 Ϫ rents of rod photoreceptors. currents evoked by stepping up from 80 to 50 mV. Our results indicate that in mammalian rods, ivabradine is a CONCLUSIONS. In mammalian rods, ivabradine is a selective in- selective inhibitor of Hcn1 channels that does not affect the hibitor of Hcn1 channels. Phosphenes perception in response inward rectifier current through potassium-selective Kir 2.4 to abrupt changes in luminance, which has been transiently channels coded by Kcnj1420 or the outward currents. Phos- reported in a dose-dependent way by few patients treated with phenes perception in response to abrupt changes in lumi- ivabradine, was consistent with Hcn1 inhibition in rods. (In- nance, which has been transiently reported in a dose-depen- vest Ophthalmol Vis Sci. 2009;50:1948–1955) DOI:10.1167/ dent way by a few patients treated with ivabradine, is

iovs.08-2659 consistent with the inhibition by If inhibitors of Hcn1 channels in rods. he cardiac pacemaker current (If) in the sinus node plays a Tkey role in controlling heart rate,1 providing the rationale for the development of selective I inhibitors for the treatment f MATERIALS AND METHODS of stable angina (for a review see Ref. 2). Organic compounds such as zatebradine (ULFS 49), ivabradine (S16257), and cilo- Animals and Cell Dissociation bradine (DK-AH 269) inhibit cardiac If by a use-dependent mechanism that requires the drug to access the open pore Adult male C57BL/6 mice, 30 to 60 days old, were reared on a 12-hour from the intracellular side.3–6. dark/12-hour light cycle with ad libitum access to water and food. The most frequent side effects reported during clinical trials Rearing and handling complied with institutional guidelines and with

by healthy volunteers and patients treated with If inhibitors are the ARVO Statement for the Use of Animals in Ophthalmic and Vision luminous phenomena (primarily phosphenes).7,8 Molecular9 Research. From a mouse deeply anesthetized by intraperitoneal injec- and electrophysiological evidence indicates the expression of tion of pentothal sodium (35 mg/kg; Gellini, Aprilia, Italy), the retina was quickly isolated through a corneal slit by gentle squeezing of the eye with curved forceps and then was placed in cold Locke solution, pH 7.5.21 The animal was then killed by an intraperitoneal lethal dose From the 1Dipartimento di Psichiatria, Neurobiologia, Farmacolo- 2 of pentothal. Mouse rod photoreceptors dissociated by gentle mechan- gia e Biotecnologie and the Dipartimento di Fisiologia Umana, Uni- Ј versita` di Pisa, Pisa, Italy. ical trituration after enzymatic treatment (5 at 30° in 14–18 U/mL Supported by the Italian Ministry of University and Education Grants papain, 0.3 mg/mL hyaluronidase, and 100 U/mL DNase) were identi- MIUR 2004–2004057720_004 and MIUR 2006–2006053302_002. ﬁed by their characteristic morphology (Figs. 1A, 1B) and by rhodopsin Submitted for publication August 3, 2008; revised October 23, immunocytochemistry (Fig. 1C). 2008; accepted February 2, 2009. Disclosure: G.C. Demontis, Institute de Recherches Servier (F, C); C. Gargini, Institute de Recherches Servier (F, C); T.G. Paoli, Chemicals Institute de Recherches Servier (F); L. Cervetto, Institute de Recher- CsCl, proteolytic enzymes, amphotericin, and salts were from Sigma-Aldrich ches Servier (F, C) Italia. Ivabradine (S-16257), chemical name (S)-3-(3-(((3,4-dimethoxybi- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- cyclo(4.2.0)octa-1,3,5-trien-7-yl)methyl)methylamino)propyl)-1,3,4,5- ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. tetrahydro-7,8-dimethoxy-2H-3-benzazepin-2-one hydrochloride, was Corresponding author: Gian Carlo Demontis, Dipartimento di provided by Institut de Recherches Internationales Servier (France). Psichiatria e Neurobiologia, Universita` di Pisa, Via Bonanno, 6, I-56126 The drug was dissolved in ultrapure-grade water (MilliQ; Millipore, Pisa, Italy; [email protected]. Billerica, MA) as a 30-mM stock solution and stored up to 5 days at 4°C

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͑ ͒ ϭ ͕ ϩ ͓͑ Ϫ ͒ ͔͖ F V 1/ 1 exp V V1/2 /s (1)

where V1/2 is the half-activation voltage and s is the inverse slope factor of Ih. The time constants of activation were estimated by ﬁtting net currents by

͑ ͒ ϭ ϩ ͓Ϫ͑ Ϫ ͒ ␶͔ I t IMAX A0 exp t t0 / (2)

where t0 is the time of application of the activating step, IMAX is the ␶ maximum current amplitude, A0 is the initial current amplitude, and is the time constant of activation.

Statistical Analysis After assessing that current amplitudes were normally distributed (data not shown), parametric statistical analysis was applied to compare current amplitudes, before and after drug administration, obtained by averaging 10 individual sweeps. The effects of ivabradine concentra-

tion on Ih inhibition were analyzed by one-way ANOVA using the function implemented in data analysis software (Origin 6.0; Microcal, GE Healthcare, Chalfont, St. Giles, UK). Comparison between control and ivabradine concentrations was carried out by multiple t-tests, adopting the correction by Bonferroni. Student’s t-test was used to compare current amplitudes at Ϫ120 after block by either ivabradine 30 ␮M or CsCl 3 mM (see Figure 5C).

FIGURE 1. morphologic and functional properties of isolated rods. (A, RESULTS B) Living mouse rods, freshly isolated by enzymatic treatment followed by mechanical dissociation, are identified by their characteristic mor- In Figure 2A, an inward current of Ϫ9.6 pA (CNTR) activates in phology, with slender outer segments (OS) and inner segments (IS) and response to a hyperpolarizing voltage step to Ϫ80 mV. The a cell body (CB). (C) A rod fixed with 1% paraformaldehyde in phos- average value from 21 rods was Ϫ14.9 Ϯ 2.2 pA (range, phate-buffered saline for 5 minutes at 4°C was stained by the anti– Ϫ Ϫ rhodopsin antibody in the OS. A long tiny axon connects the CB to the 4.7/ 50.9 pA). Note that after block by CsCl, a known Ih rod spherule (RS) that represents the rod axon terminal. In most cases, inhibitor, the membrane current approaches the zero-current Ϫ however, the RS is lost during the dissociation. The small size of level, indicating that Ih is the main inward current at 80 mV. 5 mammalian rod IS and CB (diameter, 2–4 ␮m) makes it difficult to Ih inhibition by the use-dependent If inhibitor ivabradine obtain stable recordings from these cells. was assessed by repeated application of the activation/deactivation protocol, shown in Figure 2B. In preliminary experiments, a slower and less complete block was also obtained and diluted in Locke solution at the desired final concentration for cell with the use of shorter and more frequent stimuli (not shown) superfusion. and in the absence of repetitive stimulation (Fig. 2D). Data in

Figures 2C-H illustrate the time and dose dependence of Ih Patch-Clamp Recordings inhibition by ivabradine. Sweeps in Figures 2C, 2E, and 2G were acquired at selected times before and during application Unless specified, perforated-patch recordings were carried out in of ivabradine 30 (Fig. 2C), 3 (Fig. 2E), and 0.3 ␮M (Fig. 2G). Locke solution as previously reported for guinea pig rods,21 and volt- Note that in Figure 2C, at Ϫ30 mV, all sweeps superimpose, as age-dependent currents were measured using an amplifier (Axopatch expected for the selective block of a current reversing close to 1D; Axon Instruments, Sunnyvale, CA), low-pass filtered at 200 Hz by Ϫ30 mV. As shown in Figure 2D, full I inhibition by 30 ␮M a four-pole Bessel filter, and digitized online at 1 kHz (Digidata 1320 h ivabradine takes place in approximately 6 minutes, and the board driven by pClamp 8.0 software; Axon Instruments). current approaches the horizontal dashed line that marks the The temperature of the 1-mL recording chamber was set at 32°C by zero-current level expected for a cell with fully blocked I (Fig. a 51-W Peltier device (RS Components, Milan, Italy). The superfusing h 2A). Longer times are required for partial inhibition, as shown Locke solution (flow rate, 3 mL/min), was heated by a 1.2-W Peltier in (Figs. 2E, 2F) and (Figs. 2G, 2H) for 3 and 0.3 ␮M ivabradine, device (RS components). respectively. The block was occasionally observed to reverse on prolonged washout of the drug, as shown in Figure 2F, Analysis of Records although the time course of current recovery often exceeded

The fractional inhibition of Ih was estimated by the ratio of current the limited recording time allowed by the small size of mam- amplitudes at Ϫ80 in the presence and in the absence of the drug. To malian rods.

estimate Ih activation, net currents were computed by subtracting The inhibition illustrated in Figures 2C-H was observed voltage-clamp records in the presence of the blocker from those in during repetitive application of the stimulation protocol illus- control saline (difference records). Conductance values were com- trated in Figure 2B. The inhibition was not strictly use depen- puted from net currents amplitude during the last 100 ms of the dent, however, because a current reduction was also observed Ϫ 2-second step using the Ih reversal potential of 30 mV measured in when the repetitive stimulation was brieﬂy stopped (see hor- preliminary experiments, as previously reported for guinea pig rods in izontal dotted lines in Fig. 2D). Data in Figure 2H plot the 21 similar recording conditions. Conductance values for each step were average Ih inhibition, with the SEM, measured in response to normalized to those at Ϫ120 mV to generate fractional activation the application of three ivabradine concentrations and 3 mM values (F(V)). F(V) values were interpolated by CsCl.

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FIGURE 2. Time- and dose-depen-

dent Ih inhibition by ivabradine. (A) Sweeps plot the currents activated by a 2-second long hyperpolarizing step to Ϫ80, imposed from a holding voltage of Ϫ35 mV, close to the membrane potential of mammalian rods in darkness.35 CNTR and CsCl indicate sweeps recorded before and after application of 3 mM CsCl, re-

spectively. (B) The use-dependent Ih block by ivabradine was investigated by the application every 5 seconds of a stimulation protocol that alternated a 1.2-second activating step at Ϫ80 mV, with a deactivating step at Ϫ30

mV, close to the Ih reversal. (C, E, G) Voltage-clamp responses to the application of the stimulation protocol illustrated in (B). Horizontal dashed lines in (A, E, G) indicate the zero- current level. Lowercase letters close to the sweeps indicate records acquired at different times, either before or during the application of ivabradine 30 ␮M(C), 3 ␮M(E), and 0.3 ␮M(G). (C) Transient increase in inward in current (labeled b) was a perfusion artifact because it was not systematically observed with 30 ␮M ivabradine but was observed in some control records after switching to different perfusion lines. (D, F, H) Data points plot the amplitude of inward current measured at the end of the 1.2-second hyperpolarizing pulse at Ϫ80 mV. Dotted lines in (D) indicate current levels at the moment of transient interruption of the stimulation protocol to assess the use dependence of ivabradine blockade. (F) Ten sweeps were averaged to reduce the recording noise; each data point is therefore the mean value from 10 records. Note that on ivabradine washout, slow and partial recovery is present. (I) Columns plot the mean fractional current (with SEM) remain- ing after the application of ivabradine or CsCl. Three cells were used for each dose, and each cell was tested once with a single dose. Sig- niﬁcant differences (P Ͻ 0.001) were found between 0 ␮M ivabradine and both 3 and 30 ␮M ivabradine.

Use-independent block by ivabradine has been reported22 Ϫ60 and Ϫ70 mV, and difference records for these voltages in for Hcn1 homomeric channels. To evaluate the hypothesis that Figure 3C do not show clear evidence for slowly activating

ivabradine inhibits Ih carried through Hcn1 homomeric chan- inward currents. Average conductance values (see Materials nels, we compared the voltage dependence and time constants and Methods), with their SEM, are plotted in Figure 3D and of activation of ivabradine-blocked and of Cs-blocked currents; fitted with equation 1. Ivabradine- and Cs-blocked currents Ϫ the data are illustrated in Figure 3. Steady state Ih activation have half-activation voltages close to 75 mV, but the inverse was investigated by the stimulation protocol shown in Figure slope factor s is larger for ivabradine-blocked (7.6 mV) than for 3A. Difference records (see Materials and Methods) in Figures Cs-blocked (5.1 mV) currents. 3B and 3C plot currents blocked by ivabradine 30 ␮M and CsCl Dotted traces in Figures 3E and 3F plot on an expanded time 3 mM, respectively (when the current inhibition was com- scale the ivabradine (Fig. 3E) and Cs-blocked (Fig. 3F) currents plete). Data plotted in Figure 3B indicate that ivabradine inhib- at Ϫ120 mV and are fitted by equation 2 (continuous smooth its the slowly activating inward currents induced in response to lines). Differences between sweeps and fit curves are plotted voltage steps from Ϫ60 to Ϫ120 mV. On the other hand, Cs is in the upper part of the panels and stay close to the zero-

not very effective at blocking Ih activated by voltage steps at current level (dashed horizontal lines), indicating that a single

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FIGURE 3. Ivabradine inhibition of HCN1 channels. (A) Traces plot the stimulation protocol used for the study of Ih steady state activation. From a holding of Ϫ35 mV, 2-second long voltage steps, ranging from Ϫ60 to Ϫ120 mV in 10-mV steps, were followed by a 0.5-second step at Ϫ70 mV for tail current analysis. (B, C) Difference records obtained by subtracting records in the presence of 30 ␮M ivabradine (B) or 3 mM CsCl (C) from their corresponding control records. (D) Current amplitudes during the last 100 ms of each difference record were converted to conductances and normalized to the value at Ϫ120 mV to generate fractional activation curves. Average fractional activation plotted from ivabradine-sensitive ( filled circles) and Cs-sensitive (open circles) currents. Smooth curves through data points plot best fits by equation 1. Ivabradine data points were best ϭϪ Ϯ ϭ Ϯ fit by the continuous curve using the following parameters: V1/2 75.3 2.1 mV; s 7.6 0.7 mV. CsCl data points were best fit by the dotted ϭϪ Ϯ ϭ Ϯ curve using the following parameters: V1/2 75.8 2.3 mV; s 5.1 0.6 mV. (E, F) Dotted traces plot on an expanded scale time difference records at Ϫ120 mV from (B) and (C)in(E) and (F), respectively. The smooth continuous lines are best fits of equation 2 to difference records, with time constants of 38 and 33 ms for the ivabradine and the Cs-sensitive currents, respectively. Horizontal dotted traces were computed as the difference between fit and difference records. (G) Data points plot average activation time constants as a function of activating voltages from three independent experiments for ivabradine-blocked ( filled circles) and Cs-blocked (open circles) currents. (H, I) The response to a 18.75-second long Ϫ ␮ step at 110 (H) applied after full Ih inhibition by 30 M ivabradine is plotted (I).

␮ exponential decay provides a reasonably good fit to Ih activa- full Ih inhibition by 30 M ivabradine (note the difference in Ϫ tion. Average activation time constants are plotted in Figure 3G the holding currents at 80 mV between control and Ih- for ivabradine-blocked (filled circles) and Cs-blocked (open blocked sweeps). For comparison, the effect of 3 mM CsCl is circles) currents, indicating that both drugs block currents shown in Figure 4B. Data points in Figures 4C and 4D plot the with similar fast-activation kinetics. It has been reported22 that average amplitudes of difference records from three cells, with after ivabradine blockade, long hyperpolarizing pulses fail to each SEM, indicating that neither ivabradine nor CsCl affects reactivate the current through Hcn1 homomeric channels, in currents generated in response to voltages more positive than contrast to the behavior of the f-channels of pacing cells.23 As Ϫ50 mV. a further test for the selective inhibition of rod Hcn1 channels An additional outward current of rods is carried by anions ␮ by ivabradine, after attaining full Ih inhibition by 30 M ivabra- flowing through a voltage- and calcium-dependent conduc- dine (not shown), we applied an 18.75-second long step from tance.17 Activation of this conductance by membrane depolar- Ϫ35 to Ϫ110 mV. As shown in Figure 3I, no current reactiva- ization above Ϫ30 mV generates a slowly activating, noninac- tion was observed in response to the long step at Ϫ110 mV. tivating, outward currents, as shown in Figure 4E. Note that on ϩ We next addressed the selectivity toward Ih by testing stepping to 50, close to the calcium reversal potential, the ivabradine effects on other voltage-dependent currents of rod chloride current is suppressed. photoreceptors. Sweeps in Figures 4A and 4B plot currents Data in Figure 4F, recorded from a cell with a small outward activated by stepping from Ϫ80 mV to voltages ranging from chloride current, suggest that the drug does not block the Ϫ10 to ϩ50 mV in 20-mV steps. Data in Figure 4A show the current through voltage and calcium-dependent chloride chan- current recorded before (thin traces) and after (thick traces) nels. The expression of Kcnj14 in mouse rods has recently

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FIGURE 4. Selective inhibition of Ih by ivabradine. (A, B) Outward currents were generated in response to 800-ms long voltage-steps, ranging from Ϫ70 to ϩ50 mV, from a holding voltage of Ϫ80 mV. The step duration was selected to attain steady state deactivation of the transient outward current. Data in (A) and (B) plot currents recorded in response to voltage stimuli ranging from Ϫ10 to ϩ50 mV in 20-mV steps before (thin traces) and after (thick traces) application of 30 ␮M ivabradine (A) or 3 mM CsCl (B). Horizontal dashed lines indicate zero-current levels. (C, D) Data points plot average difference-record amplitudes from three independent experiments using 30 ␮M ivabradine (C) or 3 mM CsCl (D). Currents were measured at the end of the 0.8-second long voltage steps stimuli, ranging from Ϫ50 to ϩ50 mV in 10-mV steps. (E) Sweeps plot currents generated in response to voltage-clamp stimuli ranging from Ϫ30 to ϩ50 mV in 20-mV steps. Large inward tail currents were measured on stepping back to Ϫ35 mV from steps, indicating that the outward current is carried in part by chloride, whose (ءء) and ϩ30 mV (ء) ϩ10 reversal potential is close to zero. No inward tail was measured after stepping back from ϩ50 mV. Note that this chloride current was erratic, was expressed by a fraction of rods, and may in addition sponta- neously appear and fade out during recording in the absence of ivabradine. (F) Sweeps plot current generated in response to voltage stimuli ranging from Ϫ30 to ϩ30 mV in 20-mV steps before (thin traces)

and after (thick traces) Ih inhibition by 30 mM ivabradine (note the differences in the holding currents at Ϫ90 mV). *Records in ivabradine.

been reported.20 Considering that Kcnj14 codes for Kir 2.4 voltages negative to the potassium reversal potential (approx- channels, selective inhibition of Hcn1 channels is expected to imately Ϫ90 mV), current amplitudes increased in the ivabra- reveal an inward rectiﬁer current. The known sensitivity of Kir dine-treated cell compared with the Cs-blocked rod. Average channels to Cs predicts that the inward rectiﬁcation should not data from three experiments are plotted in the Figure 5C, be present in records acquired after CsCl application. Figures showing that for voltages negative to Ϫ80 mV, an inward

5A and 5B plot currents measured in response to voltage steps rectification is apparent after full Ih inhibition by ivabradine, ranging from Ϫ90 to Ϫ120 mV, in 10-mV steps, after complete whereas in the presence of CsCl, the rectification was reduced. ␮ Ϫ inhibition of the slowly activating Ih by ivabradine 30 M (Fig. Activation at 70 mV of a slowly inactivating calcium current 5A) or 3 mM CsCl (Fig. 5B). may contribute to the inward rectification after ivabradine. To Note that although these rods had similar membrane resis- evaluate this possibility, we investigated the properties of in- tance (compare current amplitudes at Ϫ35 and Ϫ70 mV in ward currents through calcium channels, isolated in the pres- Figs. 5A and 5B) and resting potential in bright light, for ence of CsCl and other potassium channel blockers, as shown

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FIGURE 5. Inward rectification in the presence of ivabradine. (A, B) Currents were recorded in response to voltage stimuli ranging from Ϫ90 to Ϫ120 mV in 10-mV steps after the application of 30 ␮M ivabradine (A) or 3 mM CsCl (B). Horizontal dashed lines indicate zero-current levels. (A) Vertical deflections are perfusion-related artifacts. (C) Data points plot average current amplitudes measured at the end of the 2-second steps, ranging from Ϫ60 to Ϫ120 mV in 10-mV steps, in the presence of ivabradine (open squares) and CsCl ( filled squares), respectively. (D) Sweeps plot current generated in response to a 225-ms long voltage ramp from Ϫ75 to ϩ50 mV in the presence of 20 mM TEA, 2.5 mM

BaCl2, and 3 mM CsCl (Ba/Cs/TEA solution) (thin trace) or in the pres- ␮ ence of 200 M CdCl2 in addition to the Ba/Cs/TEA solution (thick trace). NaCl was reduced to 120 mM to pre- vent cell shrinking, and records were low-pass ﬁltered at 5 kHz and sam- pled at 33 kHz. (E, F) Sweeps plot the inward currents activated by 2-second voltage steps at Ϫ80 and Ϫ120 mV for two different rods. Horizontal dashed lines in (E, F) plot zero-current level. The arrow in (E) points to the peak in the current evoked by the voltage step at Ϫ120 mV. Calibration bars in (E) apply to (E) and (F). (G) Thin and thick traces plot the outward currents evoked by a voltage step from Ϫ70 to ϩ40 mV for the cells in (E) and (F), respectively. (H) Sweeps plot on expanded scales the responses to the Ϫ120 mV voltage step in (A, thick trace) and (E, dotted trace).

␮ in Figure 5D (see legend). The block by 200 M CdCl2 (arrow) The current decays in Figures 5A and 5E have a similarly conﬁrmed that in these recording conditions, the current was slow time course, with a decrease of several pA within 2 indeed ﬂowing through calcium channels. As shown in Figure seconds, as shown in Figure 5H, which plots on expanded 5D, the current turns inward at approximately Ϫ50 mV, sug- scales data from the Ϫ120 mV step in Figures 5A and 5E. gesting that calcium current activation may not explain the

residual inward rectiﬁcation after complete Ih inhibition by ivabradine, which turns inward negatively to Ϫ80 mV. DISCUSSION In a fraction of rods (approximately 20%), we observed an inward peak of 2 to 5 pA (arrow) in the current activated by a The present results show that ivabradine, a use-dependent Ϫ 5,23 step to 120 mV, as shown in Figure 5E. For comparison, inhibitor of cardiac pacemaker If, is a selective inhibitor of Figure 5F plots the inward current at Ϫ80 and Ϫ120 mV for a Hcn1 channels in mouse rod photoreceptors. Ϫ second rod that did not display this transient peak at 120 mV. Ivabradine at a high dose that fully blocks Ih (see data in Note that rods in Figures 5E and 5F had similar access resis- Figs. 2 and 3) does not affect membrane currents that gate tance (approximately 100 M⍀) and outward currents (Fig- positively to Ϫ50 mV, as shown in Figures 4A to 4D. The ure 5G). absence of ivabradine effects on outward currents is in sharp

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contrast to the inhibition observed in amphibian rods with and shape the voltage response to dim light stimuli.14 The zatebradine,16 a molecule with structural analogies to ivabra- observation that in many cases phosphenes perception is trig- dine. Although species differences (amphibia vs. mammals) gered by an abrupt change in luminance suggests that the may contribute to the different selectivity of ivabradine and inhibition of Hcn2 channels open in darkness may not be zatebradine in rod photoreceptors, zatebradine was less selec- sufﬁcient to induce phosphenes. Data in Figure 3D indicate 24 Ϫ tive for If than ivabradine in mammalian pacing cells. that Hcn1 channels are closed at 40 mV, the rod membrane Analysis of the effects of ivabradine and CsCl (Figs. 2, 3) potential in darkness,35 and a 10- to 15-mV hyperpolarization is

indicates similar activation kinetics and half-activation voltages required for Ih to reach an intensity of 2 to 3 pA and to have for ivabradine and Cs-sensitive currents. These results are con- functional relevance. These observations are consistent with sistent with ion permeation through homomeric Hcn1 chan- the notion that phospenes are triggered by the block of Hcn1 nels,12,25–28 as expected from in situ hybridization9 and immu- channels in rods. nolabeling with an antibody anti-HCN1 in mouse retina.14 Phosphenes may disappear despite continued treatment, Furthermore, in general agreement with the effects of ivabra- suggesting that Hcn1 inhibition is required but not sufficient to dine on heterologously expressed homomeric Hcn1 chan- trigger phosphenes. In this context, it is tempting to speculate nels,22 in rods the blocked channels do not reopen during about the role Kir-mediated currents described in Figures 5E–H prolonged hyperpolarization (Fig. 3I). Last, ivabradine-induced may play in phosphene perception. In other sensory neurons, block is not strictly use dependent in rods (Fig. 2D), similar to a low ratio between Ih and Kir-mediated currents is associated homomeric Hcn1 channels and in contrast with the use-depen- with spontaneous low-frequency oscillations,36 which are typ- 37 dent inhibition of If in pacing tissue, when HCN4 is the main ical of amplified resonance. Considering the variability in Ih 10,11 isoform. These observations lend further support to the amplitude we have reported, in a fraction of rods If inhibitors notion that ivabradine blocks homomeric Hcn1 channels in may reduce the Ih/Kir ratio below a critical level, triggering an mouse rods. amplified resonance mode in response to large hyperpolariza- Some differences between CsCl and ivabradine were noted. tion. In these rods, the amplification of spontaneous voltage More negative activation thresholds (Figs. 3B, 3C) and steeper fluctuations may generate phosphenes until the Ih/Kir ratio inverse slope factors (Fig. 3D) were found for CsCl-subtracted rises above the critical level. than for ivabradine-subtracted records. Similar negative thresh- In conclusion, the demonstration of selective Ih inhibition old and steep slope factors for the activation of Cs-subtracted in rods by ivabradine paves the way to future work investigat- currents have been reported in salamander rods,19 suggesting ing the impact of Hcn1 and Kir 2.4 channel interaction on the

that ivabradine is a more effective Ih inhibitor than Cs at processing of visual signals by retinal networks. membrane potentials close to the Ih activation threshold. These effects in rods are consistent with a voltage-dependent Acknowledgments Ih block by Cs and in agreement with the known voltage 29 dependence of If block by Cs. The authors thank Dario DiFrancesco for helpful comments and critical Selective Ih inhibition by ivabradine revealed an inward reading of an earlier version of this manuscript. rectification that has never previously been reported in rods. The inward rectification observed in the presence of ivabradine (Figs. 5A–C) is consistent with the expression by rods of References the potassium-selective and Cs-sensitive inward rectifier Kir2.4 20 1. Brown H, DiFrancesco D. Voltage-clamp investigations of mem- (coded by Kcnj14). Our data suggest that in mouse rods, brane currents underlying pace-maker activity in rabbit sino-atrial ivabradine is a more selective inhibitor of Ih than CsCl, which node. J Physiol. 1980;308:331–351. blocks Ih and Kir2.4-mediated currents. 2. DiFrancesco D, Camm JA. Heart rate lowering by specific and Our results indicate considerable variability in amplitude selective I(f) current inhibition with ivabradine: a new therapeutic and kinetics of inward rectification. The presence in some rods perspective in cardiovascular disease. Drugs. 2004;64:1757–1765. (Fig. 5E) of a peak in the current activated by a voltage step to 3. Van Bogaert PP, Pittoors F. Use-dependent blockade of cardiac Ϫ120 mV suggests some variability in the voltage-dependent pacemaker current (If) by cilobradine and zatebradine. Eur J Phar- 30 macol. 2003;478:161–171. inactivation of Kir currents. Regarding Ih variability (Figs. 1, 4. DiFrancesco D. Some properties of the UL-FS 49 block of the 5), cAMP has been reported to increase Ih amplitude in rabbit rods,12 and the observed variability may result from changes in hyperpolarization-activated current (i(f)) in sino-atrial node myo- cytes. Pflugers Arch. 1994;427:64–70. cAMP levels. Although no data are available in rods, changes in 31 5. Bois P, Bescond J, Renaudon B, Lenfant J. Mode of action of phosphoinositides may also affect Ih gating. The transcrip- 20 bradycardic agent, S 16257, on ionic currents of rabbit sinoatrial tion factors coded by Nrl and Nr2e3 are required for Kcnj14 node cells. Br J Pharmacol. 1996;118:1051–1057. but dispensable for Hcn1 expression in mouse rods (see the 6. Van Bogaert PP, Goethals M, Simoens C. Use- and frequency- GEO database in Medline, http://www.ncbi.nlm.nih.gov/sites/ dependent blockade by UL-FS 49 of the if pacemaker current in entrez?dbϭgeo; use the search terms: Kcnj14 and Nrl or Hcn1 sheep cardiac Purkinje fibres. Eur J Pharmacol. 1990;187:241– and Nrl), and the uncoordinated expression of Kcnj14 and 256. Hcn1 may also contribute to the observed differences in the 7. Glasser SP, Michie DD, Thadani U, Baiker WM. Effects of zatebra- characteristics of inward rectification. dine (ULFS 49 CL), a sinus node inhibitor, on heart rate and Phosphene perception has been occasionally reported by exercise duration in chronic stable angina pectoris: Zatebradine 7,8 Investigators. Am J Cardiol. 1997;79:1401–1405. patients treated with If-inhibitors, and evidence based on 8. Borer JS, Fox K, Jaillon P, Lerebours G; Ivabradine Investigators ERG recordings indicate that If inhibitors interfere with signal processing across the rod to rod-bipolar synapse.32 Inhibition Group. Antianginal and antiischemic effects of ivabradine, an I(f) inhibitor, in stable angina: a randomized, double-blind, multicen- of Hcn1 and Hcn2 channels expressed by rods9,12 and rod- 9,13,14 tered, placebo-controlled trial. Circulation. 2003;107:817–823. bipolar cells, respectively, may increase the amplitude of 9. Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A. voltage responses to light and spontaneous voltage fluctuations Cellular expression and functional characterization of four hyper- (for a review see Ref. 33), thus preventing their rejection by polarization-activated pacemaker channels in cardiac and neuronal 34 the threshold filter implemented in rod bipolar cells. Recent tissues. Eur J Biochem. 2001;2681646–1652. evidence has shown that in rod bipolar cells, Hcn2 channels 10. Seifert R, Scholten A, Gauss R, Mincheva A, Lichter P, Kaupp UB. open in darkness may endow the cell with resonant behavior Molecular characterization of a slowly gating human hyperpolar-

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