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Cardiovascular Research Advance Access first published online on February 5, 2008
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Cardiovascular Research, doi:10.1093/cvr/cvn032
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

Guido Michels1,{dagger}, Mathias C. Brandt1,{dagger}, Naufal Zagidullin1, Ismail F. Khan1, Robert Larbig1, Sebastian van Aaken1, Jens Wippermann2 and Uta C. Hoppe1,3,*

1 Department of Internal Medicine III, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany
2 Department of Cardiothoracic Surgery, University of Cologne, Germany
3 Center for Molecular Medicine, University of Cologne (CMMC), Germany

* Corresponding author. Tel: +49 221 478 32396; fax: +49 221 478 32397. E-mail address: uta.hoppe{at}uni-koeln.de

Received 25 June 2007; revised 7 January 2008; accepted 28 January 2008

Time for primary review: 27 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The hyperpolarization-activated cyclic nucleotide-gated (HCN) current If/IHCN is generally thought to be carried by Na+ and K+ under physiological conditions. Recently, Ca2+ influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca2+ permeation through If/IHCN is still lacking.

Methods and results: To possibly provide direct evidence of Ca2+ influx through IHCN/If, we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human If, since Ca2+-mediated If/IHCN currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca2+ inward currents upon hyperpolarization with a single-channel amplitude of –0.87 ± 0.06 pA, a low open probability of 3.02 ± 0.48% (at –110 mV, n = 6, Ca2+ 2 mmol/L), and a Ca2+ conductance of 8.9 ± 1.2 pS. IHCN2-Ca2+ was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific If inhibitor ivabradine, clearly confirming that Ca2+ influx indeed was conducted by HCN2 channels. Changing [Na+] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca2+ caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na+/Ca2+ conductance, whereas the K+ conductance of HCN2 increased significantly in the absence of external Ca2+ with increasing K+ concentrations. The mixed K+–Ca2+ conductance, however, was unaffected by the external K+ concentration. Notably, we could also record hyperpolarization-activated Ca2+ permeation of single native If channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca2+ conductance of human If, 9.19 ± 0.34 pS; amplitude, –0.81 ± 0.01 pA; open probability, 1.05 ± 0.61% at –90 mV).

Conclusion: We directly show Ca2+ permeability of native rat and, more importantly, human If at physiological extracellular Ca2+ concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased If density and HCN expression.

KEYWORDS HCN channels; Calcium; Pacemaker current; Electrophysiology; Ion channels; Single-channel


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
The hyperpolarization-activated cation inward current, termed If in cardiac tissue, plays a key role in the initiation and modulation of rhythmic activity in cardiac pacemaker cells and neonatal cardiocytes.1–3 If is generated by voltage-dependent hyperpolarization-activated cyclic nucleotide-gated (HCN) channels.4,5 In heart failure, myocardial hypertrophy and atrial fibrillation If current densities and/or mRNA levels of its molecular correlate HCN are increased compared with controls.6–9 Therefore, it has been postulated that If might contribute to arrhythmogenesis in disease states.6–9

Four HCN gene family members have been cloned (HCN1–4) with varying message levels in different cardiac regions.4,5,10–13 All four HCN subunits share an identical pore region, indicating that their ion selectivity should be similar. If and IHCN are generally thought to be carried by sodium and potassium under physiological conditions.4,6,14 However, recently calcium influx through HCN channels has indirectly been postulated.15,16 Since calcium is the most important second messenger contributing to numerous cellular processes, i.e. transcription of various proteins, muscle contractility, cellular secretion and neurotransmitter release, calcium conductance of If might have profound physiological and pathophysiological implications, particularly in diseased states with increased current densities. However, direct functional evidence of calcium permeation through HCN channels is still lacking.

Precise understanding of the molecular structure and function of the pacemaker channel is critical to any future therapeutic modulation of this current in myocardium and neurons. Since calcium influx through HCN channels or native If may not readily be demonstrated in whole-cell recordings, we provide single-channel analysis of IHCN/If cation permeability. Our recordings directly show permeation of calcium through IHCN and human native If under physiological external calcium concentrations.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and conforms to the Declaration of Helsinki.

2.1 Plasmid construction and transient transfections
The expression plasmids pAdCGI-HCN2 encoding the full-length sequences of mHCN2 has been described.1,17,18 Twenty four hours prior to transfection, CHO-K1 cells (ATCC CCL 61, American Type Culture Collection, Manassas, VA, USA) were seeded at a density of 2.0 x 105 per 35 mm.19 Cells were transfected with 0.5 µg/well plasmid DNA using Lipofectamine Plus (Life Technologies, Gaithersburg, MD, USA) as directed by the manufacturer.20 After 4 h, transfection media were replaced with normal growth media.

2.2 Myocyte isolation
Right atrial appendage specimens were obtained from patients (two men, 71.5 ± 5.5 years; two women, 70 ± 14 years) undergoing coronary bypass surgery. A standard isolation method was used to prepare atrial myocytes as described previously.18,21 The local Ethics Committee approved experimental use of tissue samples. Neonatal cardiomyocytes of Sprague–Dawley (1–2 days old) rats were enzymatically dissociated as previously reported.1,22

2.3 Electrophysiology
Single-channel experiments were carried out using standard microelectrode patch–clamp techniques with an Axopatch 200B amplifier and Digidata 1200 interface (Axon instruments, Foster City, CA, USA) at room temperature (21–23°C) while sampling at 10 kHz and filtering at 2 kHz (–3 dB, four-pole Bessel), if not otherwise indicated.18,23 After seal formation, capacitive currents of the membrane (range 15–105 pF) were compensated to a minimum using the capacitance neutralization circuit implemented in the Axopatch 200B amplifier. Single HCN channels were recorded in the inside-out and cell-attached configuration, as indicated, at 2000-fold amplification.

For inside-out recordings, both the pipette and bath solution were composed of (mmol/L): KCl 160, MgCl2 1, HEPES 10, EGTA 1; pH 7.4 with KOH. For calcium inside-out recordings, both the pipette and bath solution contained (mmol/L): Ca-glutamate 2 or 20 and TEA-bromide 136 or 100, respectively (as indicated); pH 7.4 with TEA-OH.

For cell-attached recordings, the pipette solution contained (mmol/L): K-glutamate 1, 4, 7, 10 or Na-glutamate 10, 100, and/or Ca-glutamate 2, 20 and HEPES 10. In some experiments, the divalent cation concentration was maintained constant to 4 mmol/L containing (mmol/L): Ca-glutamate 2 and Mg-glutamate 2 vs. Mg-glutamate 4 in the presence of Na-glutamate 128 and K-glutamate 4. A total molarity of 140 mmol/L was always maintained by adding TEA-bromide (pH 7.4 with TEA-OH). The bath-solution was composed of (mmol/L): K-glutamate 140, Mg-glutamate 1, Ca-glutamate 0.1, and HEPES 10. For cell-attached recordings, a high external potassium concentration in the bath solution is absolutely necessary to achieve a resting membrane potential of zero outside the patch.18,23

For If recordings of cardiomyocytes, nifedipine 10 µmol/L (Sigma, Taufkirchen, Germany), efonidipine 10 µmol/L (Nissan Chemical Industry, Japan), KB-R79435 10 µmol/L (Calbiochem, Darmstadt, Germany), 6-carboxyeosin 10 µmol/L (Sigma), and SKF-96365 10 µmol/L (Sigma) were added to block L-type calcium channels, T-type calcium channels, the sodium calcium exchanger, the plasma-membrane calcium ATPase, and store-operated calcium channels, respectively.24–26

In some experiments, 8-bromo-adenosine 3',5'-cyclic monophosphate (8Br-cAMP, Sigma) or ivabradine (stock solution 10 mmol/L; in double-destilled H2O; kindly provided by the Institut de Recherches Servier, Suresnes, France) was added to the bath solution, as indicated.

Single channels were depolarized or hyperpolarized (as indicated) continuously for a total duration of 3 s, from a holding potential of –35 mV. In experiments evaluating the effect of ivabradine, cells were repeatedly hyperpolarized (–90/–110 or –130 mV, as indicated; 150 ms) and depolarized (10 mV; 150 ms) for a total duration of 3.0 s. A xenon arc lamp was used to view EGFP at 488/530 nm (excitation/emission).

2.4 Data analysis
Single-channel measurements and analysis were performed using custom software as previously reported.18,27 Linear leak and capacity currents were digitally subtracted using the average currents of non-active sweeps. Closed-time and first-latency analyses were carried out only in one-channel patches. The open probability (defined as the relative occupancy of the open state during active sweeps) and the availability (fraction of sweeps containing at least one channel opening) were calculated from single-channel and multi-channel patches with a maximum of three active channels in one patch. Single-channel amplitudes were determined by direct measurements of fully resolved openings (conductance-calculation) or as the maximum of Gaussian fits on amplitude histograms. n, the number of the channels in the patch, was defined as the maximum current amplitude observed, divided by the unitary current. Peak current was corrected by division through n. The availability was corrected by the square root method: (1 – availabilitycorrected) is the nth root of (1 – availabilityuncorrected). The corrected open probability was calculated on the basis of the corrected number of active sweeps, i.e. total open time divided by (n x availabilitycorrected x number of test pulses x pulse length).

Pooled data are presented as mean ± SEM. Comparisons between groups were performed with one-way ANOVA. Significant ANOVAs were followed by post hoc tests applying the Bonferroni correction for multiple comparisons. Probability values of P < 0.05 were deemed significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
Since available data about HCN single-channel properties vary depending on the cell-type and recording technique,18,28–31 we first provided further proof that the channel openings in our recordings indeed are produced by HCN channels. Although single-channel recording of one individual channel does not result in kinetics comparable to macroscopic currents,18 we performed additional inside-out experiments of multi-channel patches, which allow in contrast to ‘pure’ one-channel recording cooperative HCN channel gating of single-channels.31 As clearly demonstrated in Figure 1A, original traces of these multi-channel patches from heterologously expressed HCN2 channels exhibited a time- and voltage-dependent kinetic similar to macroscopic IHCN/If current. HCN2 single-channel conductance of these multi-channel patches (24.0 ± 4.62 pS, n = 3) was comparable to our previous cell-attached recordings (considering the symmetrical K+-solution in inside-out recordings).18 Although we previously showed characteristic stimulation of single HCN2 channels by forskolin in the cell-attached configuration,18 we now extended these observations by direct application of cAMP to the inner side of the membrane in single-channel inside-out recordings. Expectedly, sympathetic stimulation with cAMP (1 mmol/L) significantly increased HCN2 open probability from 24.7 ± 11.1% to 51.0 ± 14.1% (n = 4, P < 0.05) without altering single-channel amplitude (Figure 1B and C). Moreover, we demonstrated a significant reduction of HCN2 open probability by the specific If inhibitor ivabradine (50 µM) (25.8 ± 7.07% vs. 6.08 ± 3.45%, n = 4, P < 0.05) (Figure 1B and C). These data clearly prove that our recordings were obtained from HCN channels.


Figure 1
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  		Figure 1 Electrophysiological and pharmacological properties of IHCN/If on single-channel level from inside-out recordings in CHO cells. (A) Representative HCN2 single-channel currents (sampling frequency: 5 kHz, corner frequency: 1 kHz, bath- = pipette-solution (in mmol/L: 160 KCl, 1 MgCl2, 10 HEPES, 0.5 EGTA, pH 7.4 with KOH) of inside-out recordings from one multi-channel (n ~ 50 channels) patches at different test potentials (–90 to –130 mV). (B) Pharmacological characteristics of HCN2 single-channels (test potential: –90 mV, holding potential: –35 mV). Ivabradine (50 µM) blocks HCN2 single-channel current during repetitive activation/deactivation steps (–90 mV, 150 ms/+10 mV, 600 ms). The observations that ivabradine significantly reduced the open probability (25.8 ± 7.07% vs. 6.08 ± 3.45%, n = 4, P < 0.05), the mean open time (1.03 ± 0.12 ms vs. 0.61 ± 0.16 ms, n = 4, P < 0.05), and the availability (75.9 ± 10.1% vs. 25.3 ± 6.98%, n = 4, P < 0.05) suggests an open-channel blockade by a fast and a slow gating mechanism. cAMP induced an increase of the channel activity (for data, see text). The data were sampled at 10 kHz and filtered at 2 kHz. (C) Effect of ivabradine (Iva) and cAMP on single-channel activity. Open probability (Po) decreased after ivabradine (50 µM) and increased after cAMP (1 mmol/L) application, respectively. Data recorded as in (B).

 
3.1 Direct evidence of calcium conductance of IHCN
Recently, indirect evidence suggested calcium permeability of If channels.15,16 However, macroscopic If currents in whole-cell technique exclusively carried by calcium may not be recorded, presumably due to small current size. Thus, to provide direct evidence of calcium influx through HCN channels, we performed single-channel recordings in the inside-out technique with calcium at a physiological concentration (2 mmol/L) being the only possible charge carrier. Original current traces indeed demonstrated HCN2 calcium inward currents upon hyperpolarization with a current amplitude of –0.87 ± 0.06 pA and an open probability of 3.02 ± 0.48% (at –110 mV, n = 6) (Figure 2A, Table 1). Current amplitude increased at more negative potentials. Slope conductance calculated by linear regression of individual recordings exhibited a calcium conductance of 8.90 ± 1.20 pS for 2 mmol/L Ca2+. Increase in the Ca2+ concentration to 20 mmol/L resulted in a reduction of Ca2+ conductance to 5.78 ± 1.08 pS (Figure 2B). To further confirm that calcium was indeed permeating HCN channels, we tested the effect of direct stimulation by cAMP and inhibition by the specific blocker ivabradine in the presence of physiological Ca2+ concentration (2 mmol/L). The ‘HCN2-calcium channel’ was significantly activated by the addition of cAMP with an increase in the open probability (3.02 ± 0.48%, no cAMP vs. 8.66 ± 2.28%, with cAMP 10 µmol/L, at –110 mV, n = 4; P = 0.027), and a shorter mean first latency, whereas current amplitude remained unchanged (Figure 2C, Table 1). The mean closed time was significantly reduced in the presence of cAMP (4.21 ± 0.85 ms vs. 2.37 ± 0.93 ms; P = 0.049). Moreover, the open probability of IHCN2-Ca2+ was significantly suppressed by ivabradine (10 µmol/L) following repetitive hyperpolarization/depolarization steps (3.67 ± 1.08% before ivabradine vs. 1.16 ± 0.90% with ivabradine, at –110 mV, n = 4; P = 0.033) (Figure 2D). These observations clearly confirmed that calcium influx indeed was conducted by HCN2 channels.


Figure 2
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  		Figure 2 IHCN2 is permeable for calcium. (A) HCN2 single-channel traces recorded in inside-out mode with Ca2+ (2 mmol/L) as the only charge carrier in the pipette and bath solution in continuous pulse mode (3 s) at –110 mV from a holding potential of –35 mV. (B) HCN2 single-channel amplitude (i) calculated from inside-out recordings as a function of test potentials. Slope conductances for Ca2+ 2 and 20 mmol/L, respectively, were determined by linear regression in individual experiments. Single-channel activity of IHCN2-Ca2+ (2 mmol/L) was significantly increased by cAMP (C; 10 µmol/L) and decreased by ivabradine (D; 10 µmol/L). For data, see text.

 


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  		Table 1 HCN2 single-channel properties recorded with Ca2+ being the only charge carrier at physiological external (2 mmol/L) Ca2+ concentrations

 
3.2 Calcium permeates native rat and human If
Given that HCN2 is the dominant mRNA transcript in atrial and ventricular myocardium,5,11 native If should also conduct calcium. Therefore, we performed additional single-channel recordings of If in cardiomyocytes using the inside-out configuration with calcium as only possible charge carrier in the presence of blockers for all known cardiac calcium conduction pores.24–26 Indeed, original recordings and mean data obtained from neonatal rat ventriculocytes clearly demonstrated calcium influx at hyperpolarized voltages, where other voltage-dependent calcium channels are inactivated (Figure 3, Table 2). Single-channel currents were detectable at physiological membrane potentials with a current amplitude of –0.79 ± 0.01 pA and an open probability of 3.53 ± 1.59% (at –90 mV; n = 6). Slope conductance calculated by linear regression of individual recordings exhibited a calcium conductance similar to heterologously expressed HCN2, i.e. 9.63 ± 0.24 pS.


Figure 3
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  		Figure 3 Single-channel recordings of native If-Ca2+ in neonatal rat ventricular myocyte (inside-out). (A) The top represents 20 consecutive sweeps and the bottom trace depicts ensemble average current from 180 traces (holding potential: –35 mV, test potential: –90 mV). Scale bars indicate 50 ms and 1 pA (unitary current traces) or 1 s and 20 fA (ensemble averages), respectively. (B) Patches were depolarized from –35 mV (holding potential) to various test potentials (TPs), as indicated. Two traces are shown for each indicated TP. (C) Single-channel amplitude of Ca2+-conducting ventricular If channels as a function of test potential. Values of single-channel amplitude were measured using fully resolved openings as indicated in (B). Slope conductances were calculated based on individual data (n = 3). See Table 2 for average values determined by linear regression.

 


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  		Table 2 Native If single-channel properties of neonatal rat ventriculocytes and human atrial myocytes recorded with Ca2+ being the only charge carrier at physiological external (2 mmol/L) Ca2+ concentrations

 
Given evidence for increase in If currents in human atrial fibrillation and the pathophysiological contribution of altered calcium handling to the perpetuation of this arrhythmia,9,32,33 it was of particular interest whether If in human atrial myocardium also conducts calcium. Therefore, we performed additional inside-out recordings of single human If channels from atrial myocytes. Notably, while blocking all known calcium channels/transporters, we could also record hyperpolarization-activated calcium permeation of native If in human atrial myocytes (Figure 4, Table 2). We obtained clear calcium inward currents at the physiological resting membrane potential, i.e. –70 mV (Figure 4B). Current amplitude increased at more negative voltages. Human If-Ca2+ exhibited a low open probability of 1.05 ± 0.61% with a similar single-channel amplitude of –0.81 ± 0.01 pA compared with neonatal rat cardiomyocytes and heterologously expressed HCN2 (at –90 mV). Linear regression revealed a single-channel calcium conductance of 9.19 ± 0.34 pS. This indicated that native rat If and, more importantly, human If conducts calcium at physiological external Ca2+ concentrations and physiological membrane potentials.


Figure 4
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  		Figure 4 Single-channel recordings of native If-Ca2+ in human atrial myocyte (inside-out). (A) The top represents 20 consecutive sweeps and the bottom trace depicts ensemble average current from 180 traces (holding potential: –35 mV, test potential: –90 mV). (B) Patches were depolarized from –35 mV (holding potential) to various test potentials (TPs), as indicated. Two traces are shown for each indicated TP. (C) Single-channel amplitude of Ca2+-conducting atrial If channels as a function of test potential. Values of single-channel amplitude were measured using fully resolved openings as indicated in (B). Slope conductances were calculated based on individual data (n = 4). See Table 2 for average values determined by linear regression.

 
3.3 HCN2 conducts calcium in the presence of Na+ or K+
The influence of both main conducting cations on the HCN-pore and on gating properties of the IHCN-Ca2+-current is still unknown. Therefore, we further analysed the permeability of Ca2+ in the presence of Na+ or K+ under physiological and non-physiological conditions using the cell-attached configuration, which is the best method for analysing ion channels in physiological environment.

Consistent with inside-out recordings, increasing external Ca2+-concentration from 2 to 20 mmol/L on cell-attached level decreased HCN conductance (gCa2mM 7.90 ± 0.89 pS, gCa20mM 4.59 ± 0.73 pS, n = 4, P = 0.04; Figure 5A) and resulted in a positive shift of the reversal potential (Vrev-Ca2mM 15.3 ± 4.23 mV, Vrev-Ca20mM 50.4 ± 6.20 mV, n = 4, P = 0.008; Figure 5A), indicating a change of the surface potential in the positive direction. In comparison to the pure HCN2-Ca2+ conductance, the HCN2-Na+-Ca2+ conductance was lower (6.43 ± 0.39 pS, Na+ 100 mmol/L, Ca2+ 2 mmol/L; n = 4, P = ns), whereas HCN2-K+-Ca2+ conductance was significantly higher (10.9 ± 0.48 pS, K+ 4 mmol/L, Ca2+ 2 mmol/L; n = 3, P < 0.05).


Figure 5
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  		Figure 5 HCN2 single-channel characteristics for different ion concentrations (cell-attached). (A/B) Single-channel amplitudes (i) of HCN2 channel as a function of the test potential. Holding potential was –35 mV throughout. Slope conductances were calculated based on individual data (n = 3–6 for each concentration). (A) Increasing Ca2+ from 2 to 20 mmol/L decreased the HCN2 conductance and shifted the reversal potential in positive direction. (B) Changing the Na+-concentration (10 vs. 100 mmol/L) in the presence (dotted lines) or absence of 2 mmol/L Ca2+ caused a simple shift of the reversal potential along the voltage axis. (C) Selectivity (measured by single-channel conductance ratios, gx/gy) and permeability ratios (Px/Py, calculated by using the modulated Goldman–Hodgkin–Katz equation) of HCN2 single-channel (*P < 0.05, one-way ANOVA). For equation and data see text.

 
Changing the Na+-concentration (10 vs. 100 mmol/L) in the presence or absence of 2 mol/L Ca2+ caused a simple shift of the reversal potential along the voltage axis, without significantly affecting Na+/Ca2+ conductance [5.74 ± 0.59 pS (10 mmol/L Na+) vs. 5.84 ± 0.39 pS (10 mmol/L Na+ plus Ca2+); 6.21 ± 0.45 pS (100 mmol/L Na+) vs. 6.43 ± 0.39 pS (100 mmol/L Na+ plus Ca2+); n = 4–6, P = ns; Figure 5B]. In comparison to HCN2-Na+ conductance, the K+ conductance of HCN2 increased significantly in the absence of extracellular Ca2+ with increasing K+ concentrations (1 mmol/L K+ 10.0 ± 1.15 pS, 4 mmol/L K+ 12.8 ± 1.0 pS, 7 mmol/L K+ 15.2 ± 0.54 pS, 10 mmol/L K+ 21.0 ± 0.57 pS, n = 3–6, P < 0.05/one-way ANOVA; Figure 6A/B), whereas the mixed K+–Ca2+ conductance was unaffected by the external K+ concentration (1 mmol/L K+ 9.23 ± 0.94 pS, 4 mmol/L K+ 10.9 ± 0.48 pS, 7 mmol/L K+ 10.8 ± 0.45 pS, 10 mmol/L K+ 11.5 ± 0.29 pS, n = 3–6, P= ns, one-way ANOVA; Figure 6A/C). In addition, the open-probability (Po), the main parameter of fast and slow gating of HCN2, was significantly decreased in the presence of Ca2+ (Figure 6D). Moreover, under physiological K+-conditions (4 mmol/L), Ca2+ induced a shortening of the mean open time (1.09 ± 0.13 ms vs. 0.67 ± 0.07 ms, n = 3–5, P < 0.05), supporting the hypothesis that Ca2+ directly modulates IHCN gating.


Figure 6
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  		Figure 6 HCN2 single-channel characteristics at different K+ concentrations (cell-attached). (A) Single-channel behavior of HCN2 in the absence (left) and presence (right) of 2 mmol/L Ca2+ on cell-attached level. Patches were depolarized from –35 mV (holding potential) to various test potentials (TPs), as indicated. Two traces are shown for each TP (mV). (B and C) Single-channel amplitudes of HCN2 channel as a function of the test potential. Slope conductances were calculated based on individual data (n = 3–6) without Ca2+ (B) or with 2 mmol/L Ca2+ (C) for 1 mmol/L K+ (K1), 4 mmol/L K+ (K4), 7 mmol/L K+ (K7), and 10 mmol/L K+ (K10). For data, see text. (D) The fast and slow gating of HCN2 (TP: –110 mV) given by the ‘mixed’ parameter Po (open probability within active traces) at different K+ concentrations was unaffected. Adding Ca2+ in the pipette solution resulted in a significant decrease of Po vs. no Ca2+ (n = 3–6).

 
To analyse the ‘ion selectivity’ and the ‘ion permeability’ of IHCN2 under physiological conditions (in mmol/L: K+ 4, Na+ 100, Ca2+ 2), we used the single-channel conductance ratio (gx/gy) and the permeability ratio (Px/Py), respectively. The ion selectivity sequence for HCN2 was gK/gCa (1.44 ± 0.12) > gNa/gCa (0.80 ± 0.04) > gNa/gK (0.49 ± 0.04) (P < 0.05 for n = 4–5, one-way ANOVA). The permeability rates were calculated using Ohm's law and Goldman's equation, which allow the conversion of the single-channel permeability (Px): g x (E – Erev) = P x z2 (E x F2/RT) {[X]i – [X]o exp–zEF/RT/(1 – exp–zEF/RT)}, where g is the single-channel conductance, E is the membrane potential, Erev the reversal potential, Px is the ion permeability, and z, F, R, T have their usual meanings. It should be noted that the permeability ratios calculated from single-channel current cannot be compared with those from whole-cell configuration (Pwhole-cell = Psingle-channel x number of channels x probability under zero-current conditions). At physiological ion concentrations, we received from our single-channel permeability rates (in cm3/s: PK 4.79 ± 1.81 x 10–13, PNa 2.31 ± 0.01 x 10–14, and PCa 2.94 ± 0.02 x 10–13), the permeability ratios: PK/PCa (1.79 ± 0.33) > PNa/PCa (0.08 ± 0.01) > PNa/PK (0.07 ± 0.01) (P < 0.05 for n = 4, one-way ANOVA). The single-channel selectivity sequences agreed well with the permeability sequences (Figure 5C).

To exclude a shift of the reversal potential (Vrev) only due to a non-specific screening effect of Ca2+, we performed additional experiments to evaluate Ca2+ permeation under physiological conditions, i.e. in the presence of Na+ 128 mmol/L and K+ 4 mmol/L with constant concentrations of divalent cations (4 mmol/L) by adjusting Mg2+. Adding Ca2+ (2 mmol/L) in the prescence of Mg2+ induced (i) a significant depolarizing shift of Vrev (with Ca2+ –6.50 ± 0.29 mV, n = 8 vs. –13.2 ± 0.44 mV without Ca2+, n = 6, P < 0.05), and (ii) a reduction of the single-channel conductance (gMg2mM-Ca2mM 10.9 ± 0.59 pS vs. gMg4mM 17.2 ± 1.19 pS, n = 6, P < 0.05; Figure 7), confirming that Ca2+ is a true conducting ion of the HCN2 channel pore.


Figure 7
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  		Figure 7 HCN2 conducts Ca2+ under physiological conditions (cell-attached). (A) True shift of the reversal potential in the presence of Na+, K+, and a constant concentration of divalent cations. Single-channel amplitudes of HCN2 channel as a function of the test potential. Slope conductances were calculated based on individual data; Na+–K+–Mg2+ (mmol/L): 128 Na-glutamate, 4 K-glutamate, 4 Mg-glutamate (n = 6); Na+–K+–Mg2+–Ca2+ (mmol/L): 128 Na-glutamate, 4 K-glutamate, 2 Mg-glutamate, 2 Ca-glutamate (n = 8). For data, see text. (B) Single-channel behavior of HCN2 in absence (left) and presence (right) of 2 mmol/L Ca2+ on cell-attached level. Patches were depolarized from –35 mV (holding potential) to various test potentials (TPs), as indicated. Two traces are shown for each TP (mV).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
There has been a lot of controversy about the functional role of If in working myocardium under physiological conditions and in diseased states, mainly because If activates at more negative voltages in working myocardium than in pacemaker tissue.6,7,21,34 Near the resting membrane potential, If/IHCN channels are believed to conduct predominantly Na+ into and less K+ out of the cell, generating a net inward current. Recently, indirect evidence indicated that HCN channels additionally permit calcium influx at negative voltages, which might have a functional significance for If modulating cardiac calcium handling and neural secretion.15,16 In the present study for the first time, we directly demonstrate calcium permeation through IHCN2 and native If at physiological external calcium concentrations and physiological membrane potentials.

Since available data about single-channel properties vary depending on the cell type analysed and the recording technique,18,28–31 we further confirmed that channel openings in our experiments indeed were produced by HCN channels. Single traces and averages obtained from one individual HCN channel in our previous report and the present study, expectedly, did not result in kinetics similar to macroscopic IHCN/If,18 as ‘pure’ single-channel recordings do not allow cooperative channel gating which underlies the typical time-dependent activation of IHCN/If.31 Consistent with others, recordings from multi-channel patches in the present study, however, exhibited characteristic time- and voltage dependence comparable to macroscopic currents.1,4,28,29,31 In these inside-out recordings, we obtained a single-channel conductance in the same range as in our previous cell-attached experiments considering the different ion concentrations.18 Moreover, we could demonstrate facilitation of single-channel activity following cAMP application to the inner side of the membrane confirming direct sympathetic stimulation of HCN channels.28,30,31,35 Furthermore, we proved HCN single-channel identity by blockade with the specific inhibitor ivabradine.36

Although If/IHCN generally is being considered a monovalent cation current, recently Zhou and coworkers15,16 indirectly postulated fractional Ca2+ influx through HCN channels. Using fura-2 Ca2+ imaging, these authors demonstrated intracellular Ca2+ elevation induced by hyperpolarization, which could be blocked by the If inhibitors Cs+ and ZD7288. If activation was accompanied by elevated secretion in dorsal ganglion neurons and action potential prolongation in rat cardiomyocytes.15,16 Although these data may have profound physiological and pathophysiological implications, experiments thus far do not provide any direct evidence that HCN channels indeed permit calcium conductance. Therefore, to prove this hypothesis, we performed single-channel recordings in the inside-out configuration with calcium being the only possible charge carrier. Permeation of any other ion except for calcium was excluded since all other anions and cations present were non-permeable macromolecules. Though HCN2 channel activity was considerably lower than in the presence of monovalent cations,18 we indeed could record clear HCN2 channel openings mediated by calcium at hyperpolarized potentials. The small single-channel amplitude mediated by calcium and the very low open probability compared with K+/Na+ current properties are consistent with the notion that whole-cell IHCN/If calcium currents may not readily be recorded. Perfusion of the intracellular side of the patch with cAMP significantly reduced the mean closed time and increased the open probability without affecting single-channel amplitude, in agreement with cAMP-induced facilitation of HCN channel opening.29 Moreover, blockade of Ca2+ influx by the selective inhibitor ivabradine further confirmed that calcium in our single-channel recordings indeed was conducted by HCN channels.

To exclude that Ca2+ permeation through HCN channels in the absence of any other permeating ions might represent an artefact, we performed additional experiments under more physiological conditions, which also demonstrated Ca2+ permeation in the presence of physiological K+ and Na+ concentrations in the cell-attached configuration. Expectedly, under cell-attached conditions increasing Ca2+ concentrations resulted in a shift of the reversal potential, consistent with Ca2+ screening of fixed membrane charges. Beside this well-known divalent-induced shift by the Gouy–Chapman theory, we demonstrated a true shift of the HCN reversal potential by Ca2+ in the presence of a constant divalent external cation concentration, confirming Ca2+ permeation of HCN channels. Moreover, our results suggest that external Ca2+ interferes with monovalent ion permeation through the HCN2 channel possibly by binding within the pore (multi-ion process). Figure 6A–C demonstrates that in the presence of external Ca2+, the driving force for cation influx increased, whereas the single-channel amplitude decreased. This indicates that when Ca2+ ions are forced into the HCN2-channel pore, monovalent cations—especially K+—cannot permeate quickly so that net K+–Ca2+ current is reduced. Thus, Ca2+ seems to ‘stabilize’ IHCN/If by making it more independent of alterations of external K+ concentrations.

In atria and ventricle, HCN2 is the dominant HCN isoform based on northern blotting and RNAse protection.5,11 Therefore, we hypothesized that native If would also admit calcium. Indeed, we could demonstrate Ca2+ influx through native neonatal ventricular and human atrial If at negative potentials, which inactivate other voltage-dependent calcium channels, and in the presence of blockers for all known cardiac calcium conduction pores.24–26 Notably, Ca2+ permeability of native If was observed at physiological extracellular calcium concentrations at the physiological resting membrane potential. Thus, despite the small amount, calcium influx through If/IHCN might have functional significance, as even minor changes in [Ca2+]i have been demonstrated sufficient to affect transcription of various proteins.37,38 This might have particular implications in diseased states with increased If density and HCN expression like atrial fibrillation and heart failure, in which alterations of calcium handling play a profound role in the progress of the disease.6–9


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ho 2146/3-1), the Köln Fortune Program, and the Marga und Walter Boll-Stiftung. N.Z. received a fellowship of the Deutsche Herzstiftung.


   Acknowledgements
 
We thank N. Henn and I. Berg for skilful technical assistance.

Conflict of interest: none declared.


   Notes
 
{dagger} These authors contributed equally to this work. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

  1. Er F, Larbig R, Ludwig A, Biel M, Hofmann F, Beuckelmann DJ, et al. Dominant-negative suppression of HCN channels markedly reduces the native pacemaker current I(f) and undermines spontaneous beating of neonatal cardiomyocytes. Circulation (2003) 107:485–489.[Abstract/Free Full Text]
  2. Stieber J, Herrmann S, Feil S, Loster J, Feil R, Biel M, et al. The hyperpolarization-activated channel HCN4 is required for the generation of pacemaker action potentials in the embryonic heart. Proc Natl Acad Sci USA (2003) 100:15235–15240.[Abstract/Free Full Text]
  3. Ludwig A, Budde T, Stieber J, Moosmang S, Wahl C, Holthoff K, et al. Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2. EMBO J (2003) 22:216–224.[CrossRef][Web of Science][Medline]
  4. Ludwig A, Zong X, Jeglitsch M, Hofmann F, Biel M. A family of hyperpolarization-activated mammalian cation channels. Nature (1998) 393:587–591.[CrossRef][Medline]
  5. Santoro B, Liu DT, Yao H, Bartsch D, Kandel ER, Siegelbaum SA, et al. Identification of a gene encoding a hyperpolarization-activated pacemaker channel of brain. Cell (1998) 93:717–729.[CrossRef][Web of Science][Medline]
  6. Hoppe UC, Jansen E, Südkamp M, Beuckelmann DJ. A hyperpolarization-activated inward current (If) in ventricular myocytes from normal and failing human hearts. Circulation (1998) 97:55–65.[Abstract/Free Full Text]
  7. Cerbai E, Pino R, Porciatti F, Sani G, Toscano M, Maccherini M, et al. Characterization of the hyperpolarization-activated current, If, in ventricular myocytes from human failing heart. Circulation (1997) 95:568–571.[Abstract/Free Full Text]
  8. Cerbai E, Sartiani L, DePaoli P, Pino R, Maccherini M, Bizzarri F, et al. The properties of the pacemaker current I(F)in human ventricular myocytes are modulated by cardiac disease. J Mol Cell Cardiol (2001) 33:441–448.[CrossRef][Web of Science][Medline]
  9. Lai LP, Su MJ, Lin JL, Tsai CH, Lin FY, Chen YS, et al. Measurement of funny current (I(f)) channel mRNA in human atrial tissue: correlation with left atrial filling pressure and atrial fibrillation. J Cardiovasc Electrophysiol (1999) 10:947–953.[Web of Science][Medline]
  10. Shi W, Wymore R, Yu H, Wu J, Wymore RT, Pan Z, et al. Distribution and prevalence of hyperpolarization-activated cation channel (HCN) mRNA expression in cardiac tissues. Circ Res (1999) 85:e1–e6.[Abstract/Free Full Text]
  11. Ludwig A, Zong X, Stieber J, Hullin R, Hofmann F, Biel M. Two pacemaker channels from human heart with profoundly different activation kinetics. EMBO J (1999) 18:2323–2329.[CrossRef][Web of Science][Medline]
  12. Moosmang S, Stieber J, Zong X, Biel M, Hofmann F, Ludwig A. Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissues. Eur J Biochem (2001) 268:1646–1652.[Web of Science][Medline]
  13. Xiao J, Nguyen TV, Ngui K, Strijbos PJ, Selmer IS, Neylon CB, et al. Molecular and functional analysis of hyperpolarisation-activated nucleotide-gated (HCN) channels in the enteric nervous system. Neuroscience (2004) 129:603–614.[CrossRef][Web of Science][Medline]
  14. DiFrancesco D, Ferroni A, Mazzanti M, Tromba C. Properties of the hyperpolarizing-activated current (if) in cells isolated from the rabbit sino-atrial node. J Physiol Lond (1986) 377:61–88.[Abstract/Free Full Text]
  15. Yu X, Duan KL, Shang CF, Yu HG, Zhou Z. Calcium influx through hyperpolarization-activated cation channels (Ih channels) contributes to activity-evoked neuronal secretion. Proc Natl Acad Sci USA (2004) 101:1051–1056.[Abstract/Free Full Text]
  16. Yu X, Chen XW, Zhou P, Yao L, Liu T, Zhang B, et al. Calcium influx through If channels in rat ventricular myocytes. Am J Physiol Cell Physiol (2007) 292:C1147–C1155.[Abstract/Free Full Text]
  17. Hoppe UC, Marbán E, Johns DC. Distinct gene-specific mechanisms of arrhythmia revealed by cardiac gene transfer of two long QT disease genes, HERG and KCNE1. Proc Natl Acad Sci USA (2001) 98:5335–5340.[Abstract/Free Full Text]
  18. Michels G, Er F, Khan IF, Südkamp M, Herzig S, Hoppe UC. Single-channel properties support a potential contribution of HCN channels and If to cardiac arrhythmias. Circulation (2005) 111:399–404.[Abstract/Free Full Text]
  19. Lam JS, Huang H, Levitz SM. Effect of differential N-linked and O-linked mannosylation on recognition of fungal antigens by dendritic cells. PLoS ONE (2007) 2:e1009.[CrossRef]
  20. Fowler MA, Sidiropoulou K, Ozkan ED, Phillips CW, Cooper DC. Corticolimbic expression of TRPC4 and TRPC5 channels in the rodent brain. PLoS ONE (2007) 2:e573.[CrossRef]
  21. Hoppe UC, Beuckelmann DJ. Characterization of the Hyperpolarization-activated Inward Current (If) in Isolated Human Atrial Myocytes. Cardiovasc Res (1998) 38:788–801.[Abstract/Free Full Text]
  22. Brenner JS, Dolmetsch RE. TrpC3 regulates hypertrophy-associated gene expression without affecting myocyte beating or cell size. PLoS ONE (2007) 2:e802.[CrossRef]
  23. Hullin R, Matthes J, von Vietinghoff S, Bodi I, Rubio M, D'Souza K, et al. Increased expression of the auxiliary beta2-subunit of ventricular L-type Ca2+ channels leads to single-channel activity characteristic of heart failure. PLoS ONE (2007) 2:e292.[CrossRef]
  24. Lee TS, Kaku T, Takebayashi S, Uchino T, Miyamoto S, Hadama T, et al. Actions of mibefradil, efonidipine and nifedipine block of recombinant T- and L-type Ca channels with distinct inhibitory mechanisms. Pharmacology (2006) 78:11–20.[CrossRef][Medline]
  25. Watanabe Y, Koide Y, Kimura J. Topics on the Na+/Ca2+ exchanger: pharmacological characterization of Na+/Ca2+ exchanger inhibitors. J Pharmacol Sci (2006) 102:7–16.[CrossRef][Medline]
  26. Nakayama H, Wilkin BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. Faseb J (2006) 20:1660–1670.[Abstract/Free Full Text]
  27. Michels G, Er F, Eicks M, Herzig S, Hoppe UC. Long-term and immediate effect of testosterone on single T-type calcium channel in neonatal rat cardiomyocytes. Endocrinology (2006) 147:5160–5169.[CrossRef][Web of Science][Medline]
  28. DiFrancesco D. Characterization of single pacemaker channels in cardiac sino-atrial node cells. Nature (1986) 324:470–473.[CrossRef][Medline]
  29. DiFrancesco D, Mangoni M. Modulation of single hyperpolarization-activated channels (i(f)) by cAMP in the rabbit sino-atrial node. J Physiol Lond (1994) 474:473–482.[Abstract/Free Full Text]
  30. Simeone TA, Rho JM, Baram TZ. Single channel properties of hyperpolarization-activated cation currents in acutely dissociated rat hippocampal neurones. J Physiol (2005) 568:371–380.[Abstract/Free Full Text]
  31. Dekker JP, Yellen G. Cooperative gating between single HCN pacemaker channels. J Gen Physiol (2006) 128:561–567.[Abstract/Free Full Text]
  32. Hove-Madsen L, Llach A, Bayes-Genis A, Roura S, Rodriguez Fonte E, Aris A, et al. Atrial fibrillation is associated with increased spontaneous calcium release from the sarcoplasmic reticulum in human atrial myocytes. Circulation (2004) 110:1358–1363.[Abstract/Free Full Text]
  33. Gassanov N, Brandt MC, Michels G, Linder M, Er F, Hoppe UC. Angiotensin II-induced changes of calcium sparks and ionic currents in human atrial myocytes: potential role for early remodeling in atrial fibrillation. Cell Calcium (2006) 39:175–186.[CrossRef][Web of Science][Medline]
  34. Yu H, Chang F, Cohen IS. Pacemaker current exists in ventricular myocytes. Circ Res (1993) 72:232–236.[Abstract/Free Full Text]
  35. Kole MH, Hallermann S, Stuart GJ. Single Ih channels in pyramidal neuron dendrites: properties, distribution, and impact on action potential output. J Neurosci (2006) 26:1677–1687.[Abstract/Free Full Text]
  36. Bucchi A, Baruscotti M, DiFrancesco D. Current-dependent block of rabbit sino-atrial node I(f) channels by ivabradine. J Gen Physiol (2002) 120:1–13.[Abstract/Free Full Text]
  37. Benitah JP, Perrier E, Gomez AM, Vassort G. Effects of aldosterone on transient outward K+ current density in rat ventricular myocytes. J Physiol (2001) 537:151–160.[Abstract/Free Full Text]
  38. Cartin L, Lounsbury KM, Nelson MT. Coupling of Ca(2+) to CREB activation and gene expression in intact cerebral arteries from mouse: roles of ryanodine receptors and voltage-dependent Ca(2+) channels. Circ Res (2000) 86:760–767.[Abstract/Free Full Text]

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