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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS
1Institute of Molecular Medicine and State Key Laboratory of Biomembrane Engineering, Peking University, Beijing, 2Department of Physiology, Third Military Medical University, Chongqing, 3College of Life Sciences, Peking University, Beijing, 4Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital of Fudan University, Shanghai, and 5Department of Physiology, Zhejiang University School of Medicine, Hangzhou, China; and 6Departments of Physiology and Pharmacology, West Virginia University, Morgantown, West Virginia
Submitted 1 December 2005 ; accepted in final form 9 October 2006
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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0.5% of current produced by the HCN2 channel or If. The associated increase in Ca2+ influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which If current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca2+ chelator), preactivation of If channels significantly reduced the action potential duration, and the effect was blocked by another selective If channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of If channels had no effects on action potential duration. Our data extend our previous discovery of Ca2+ influx in Ih channels in neurons to If channels in cardiac myocytes. calcium ion flux; hyperpolarization-activated, cyclic nucleotide-gated/cardiac time- and volume-dependent cation current channels
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (4), cyclic nucleotide-gated (CNG) channels (8), and voltage-dependent Ca2+ channels (30). Ca2+ influx through these channels contributes to transmitter release, axon guidance, or muscle contraction (3, 4, 30, 32). In neurons the time- and voltage-dependent inward cation current, Ih, is generated by hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels (10, 14, 23). It has been shown that activation of Ih channels in crayfish neurons facilitates secretion (2). However, only monovalent cations were expected to permeate through the Ih channels. Recently, we demonstrated (28) the presence of a fractional Ca2+ current through Ih channels in dorsal root ganglion (DRG) neurons. We found that Ca2+ influx through Ih channels at negative potentials contributes to activity-evoked secretion in DRG neurons (28).
The cardiac counterpart of Ih, If, shares same molecular components. Among four HCN channel isoforms that have been cloned three, HCN1, HCN2, and HCN4, are present in heart (25). Two isoforms, HCN4 and HCN2, are present in the ventricles (25). Our previous finding of Ca2+ influx through Ih channels raised the possibility of Ca2+ entry through If channels in cardiac myocytes and subsequent contribution to cardiac function at negative membrane potentials.
In this study, we demonstrated that a fractional Ca2+ current is present in currents induced by HCN2 and HCN4 channels, which were ectopically expressed in human embryonic kidney (HEK)293 cells, and in If of rat ventricular myocytes, designated as Pf (If). Preliminary results toward understanding its potential in cardiac function are shown, and future investigation for establishing its physiological role in cardiac pacemaker cells is discussed.
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MATERIALS AND METHODS |
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Cell dissociation.
Adrenal chromaffin cells were isolated from Wistar rats and cultured as previously described (7, 31). Cells were used in experiments after 2–6 days in culture. Single ventricular cardiac myocytes were isolated from adult Sprague-Dawley rats (2–3 mo old; weight 225–300 g; from Shanghai SLAC Laboratory Animal Company, Shanghai and Animal Center of Peking University, Beijing, China) by a previously described Langendorff method (21). Briefly, the heart was removed, placed in Tyrode solution containing (in mM) 137 NaCl, 0.5 MgCl2, 10 glucose, 5.4 KCl, 1.8 CaCl2, and 11.8 HEPES (pH adjusted to 7.4 with NaOH), and squeezed gently to expel the blood. Ventricular myocytes were prepared with a Langendorff perfusion apparatus. Briefly, the hearts were removed and perfused with calcium-free Tyrode containing (in mM) 130 NaCl, 1.2 MgSO4, 5.4 KCl, 1.2 KH2PO4, and 6 HEPES-NaOH (pH adjusted to 7.2 with NaOH) with collagenase (Liberase Blendzyme 4, 0.1 mg/ml, Roche Molecular Biochemicals) for 9 min. After the collagenase was washed out with calcium-free Tyrode, single cells were dissociated by mincing the ventricle and shaking the tissue in Kraftbrühe (KB) solution containing (in mM) 83 KCl, 30 K2HPO4, 5 MgSO4, 5 Na-pyruvate, 5 Na-
-hydroxybutyrate, 5 creatine, 20 taurine, 10 glucose, 0.5 EGTA, 5 HEPES-KOH, and 5 ATP-Na2 (pH adjusted to 7.2 with KOH). Cells were washed and resuspended in KB solution.
The animal protocols in this study were reviewed and approved by the Animal Research Advisory Committees in the Shanghai Institutes of Biological Sciences and Peking University.
Whole cell patch-clamp recordings. Ionic currents were studied in the whole cell patch-clamp configuration with an EPC-9 amplifier (HEKA Elektronik). The membrane was held at –40 mV unless otherwise stated. A RCP-2B perfusion system was used for switching external solutions. The system has a fast exchange time (100 ms) controlled electronically among seven channels (Inbio, Wuhan, China; Ref. 29).
Experiments on chromaffin and HEK293 cells were conducted at room temperature (22–24°C). Ventricular myocytes were studied at 32–35°C.
Solutions used in experiments are defined in Table 1. Pipettes with resistances of 2–5 M
were used for all three types of cells.
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DMEM and fetal bovine serum were purchased from GIBCO/Invitrogen. Fura-2 salt was from Molecular Probes. All other chemicals were from Sigma.
Theory and measurement of fractional Ca2+ currents. Intracellular Ca2+ concentration ([Ca2+]i) was measured by dual-wavelength ratiometric fluorometry. The fura-2 was excited with light alternating between 340 and 380 nm with a monochromator-based system (TILL Photonics), and the resulting fluorescence signals were measured with a cooled charge-coupled device. Relative changes in [Ca2+]i were calculated from the ratio of fluorescence at 340 nm (F340) to fluorescence at 380 nm (F380), which were sampled at 1 Hz. The image data were transferred and analyzed by Igor software (WaveMetrix) (28).
Fractional Ca2+ current Pf is defined as the percentage of Ca2+ current in the total current passing through the cation channels [HCN current (IHCN) in this case]. According to the original definition (28, 32)
 | (1) |
The change of Fd, 
Fd, is the "modified Ca2+-sensitive fura-2 signal" immediately before (Fd') and after (Fd") a voltage pulse-induced Ca2+ influx (30). Under the condition that all entering calcium ions are bound by fura-2, Fd is a measure of Ca2+ influx (30). Fd is determined by the difference of fluorescence signals at 340 and 380 nm.
 | (2) |
 | (3) |
fmax is determined by measuring Ca2+ influx through voltage-gated Ca2+ channels in chromaffin cells under the condition that intracellular fura-2 is sufficiently high (>0.4 mM; Ref. 32).
Under physiological conditions, only calcium ions contribute to the Ca2+ channels (30), or Pf = 100%. From Eq. 1 we have
 | (4) |
To record the time course of fura-2 dialysis, we used the Ca2+-independent fluorescence signal F360 (32), which can be calculated from F340 and F380.
 | (5) |
is the "isocoefficient". According to Eq. 5, can be determined by any experimental recording that shows rapid changes in Ca2+ concentration. In our setup, = 0.35. Since F360 is Ca2+ independent, it can be used as an indicator of the intracellular fura-2 concentration ([fura]i). After the whole cell recording configuration was established, fura-2 was dialyzed into the cell. Dialysis was accompanied by a proportional F360 increase. Once F360 reached a steady-state level, we assumed that [fura]i was equal to the fura-2 concentration in the pipette (see 
Fig. 2 and Ref. 32).
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Data were analyzed with IGOR Pro3.12 software (Wavemetrics, Lake Oswego, OR). Unless otherwise stated, data are presented as means ± SD. Statistical significance was tested with Student's t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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Fd; see Ref. 30). Similar results were observed in five nontransfected cells and five HCN2-transfected cells. In addition, we discovered the requirement of extracellular Ca2+ for hyperpolarization-induced Ca2+ flux. In response to the same pulse shown in Fig. 1B, no Ca2+ flux could be detected in the absence of extracellular Ca2+ (arrow 2, Fig. 1A). However, the Ca2+ flux appeared in the presence of 2 mM Ca2+ (arrows 1 and 3, Fig. 1A). These data support the hypothesis that the extracellular Ca2+ and open HCN channels are required to induce Ca2+ flux. Fractional Ca2+ current through HCN2 and HCN4 channels in HEK293 cells. If calcium ions indeed pass through the HCN channels, the changes in fura-2 Ca2+ signals should be directly associated with the time- and voltage-dependent properties of HCN channels. To test this hypothesis, IHCN2 was elicited by a step to –120 mV for 3, 10, and 20 s (1–3, respectively, in protocol shown in Fig. 2B, inset) from a holding potential of –40 mV. Measurement of Ca2+ fluorescence (Fig. 2, A and C) showed a rise in [Ca2+]i (arrow 1 in Fig. 2, A and C), and this rise was increased with longer pulse durations of 10 (arrow 2) and 20 (arrow 3) s. These data demonstrate a correlation of increasing Ca2+ influx with the prolonged (time dependent) activation of HCN2 channels.
Gating of HCN channels is also voltage dependent. A hyperpolarizing step to –70 mV for 3 s did not activate HCN4 channels (Fig. 3B, middle) and induced no Ca2+ signal (Fig. 3A; Fig. 3B, top), whereas a step to –120 mV for 3 s activated the channels (Fig. 3B, middle) and simultaneously induced Ca2+ influx (Fig. 3A; Fig. 3B, top). The pulse protocol is shown in Fig. 3B, bottom. In Fig. 3A, the peaks between –120 mV-3 s and –70 mV-3 s marks correspond to hyperpolarizing steps to –120, –110, –100, –90, and –80 mV. Decreasing amplitudes of these peaks at various potentials suggest a voltage-dependent change in Ca2+ influx, which simultaneously accompanies the voltage-dependent activation of the channels. Figure 3C elaborates the relationship between normalized Fd and total ion inflow for HCN4 channels at tested pulses. The superimposed traces indicate a correlation between the HCN4 currents and the Ca2+ influx through HCN4 channels.
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Fd2, Fig. 4B, top). The total ion influx charge was calculated from the time integral of ICa trace (Fig. 4B, middle, shaded region). In Fig. 4A we show that in a HEK293 cell expressing HCN2 channels a hyperpolarizing step to –120 mV for 10 s (Fig. 4A, inset) activated the HCN2 current (Fig. 4A, middle) and simultaneously induced an increase in Fd (Fd1, Fig. 4A, top). The time integral of ion flux through HCN2 channels was calculated from the current trace (Fig. 4A, middle, shaded area). The relationship between total ion influx and the corresponding increase in Fd (Fd) obtained with different durations of stimulation (Fig. 4C) was best fitted by a linear equation, indicating a correlation of the increased Fd with the increased ion flux through voltage-dependent calcium channels (Fig. 4B) and HCN2 channels (Fig. 4A). Using Eq. 1, we determined Pf for HCN2 to be 0.47 ± 0.02% (n = 6; Fig. 4D).
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Ca2+ influx through If channels in spontaneously hypertensive rat ventricular myocytes. Although we have demonstrated the Ca2+ flux through HCN2 and HCN4 channels in HEK293 cells and If channels in rat ventricular myocytes, we thought the evidence supporting calcium permeation through If channels would be stronger if we could find the altered change in Ca2+ flux at membrane hyperpolarization in an animal model in which If is naturally altered. In spontaneously hypertensive rat (SHR) ventricle, If current density is significantly increased (5). Figure 6 shows a typical example in that in response to a 10-s pulse to –150 mV from the holding potential of –70 mV, both If (Fig. 6A, top) and Ca2+ influx (Fig. 6A, bottom) through If channels are significantly larger in SHR myocytes than in normal rat myocytes. The averaged If current density at –150 mV is increased by 55% in SHR compared with the control (Fig. 6B). The increase in If is associated with an increase in Ca2+ influx (69%; Fig. 6C).
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On membrane depolarization, L-type Ca2+ channels are activated, allowing Ca2+ to enter the cell, which provides a major inward current contributing to the plateau phase of the action potential in ventricular myocytes (3). It is well documented that Ca2+ entry through L-type Ca2+ channels causes channel inactivation (11, 12). On the other hand, we wondered whether if calcium ions can enter the cell through If channels on hyperpolarization they should also be able to inhibit the subsequent gating of L-type Ca2+ channels, which produces less inward current and, in turn, would shorten the action potential duration.
To investigate the effects of preactivating If on action potential duration, we compared the action potential duration measured at 15% of amplitude with and without preactivating If (Fig. 7A). A hyperpolarizing pulse to –120 mV for 5 s was applied to open If channels before initiation of an action potential (Fig. 7B). Compared to the control (without a preceding hyperpolarizing pulse that opens If channels), the action potential duration was shortened by 32 ± 7% (n = 5, P < 0.05) and returned to control when the hyperpolarizing pulse was removed (Fig. 7, A and C). In addition, when intracellular Ca2+ was buffered by addition of 10 mM EGTA to the pipette solution, the effect of preactivation of If on the shortening of action potential duration was eliminated (99 ± 3%, n = 8; Fig. 7C). This indicates that Ca2+ influx through If channels was functionally involved in the shortening of action potential duration. This conclusion was further supported by two additional experiments. In the first experiment, when the cell was stimulated by a 50-ms depolarization from –40 to 0 mV to activate L-type Ca2+ channels (leading to a Ca2+ influx similar to that of 1-nA If for 5 s), the action potential duration was shortened to a similar degree (27 ± 4%, n = 4, P < 0.05; Fig. 7C). In the second experiment, ZD-7288 (30 µM), which is a specific antagonist of If channels (28), was able to eliminate the shortening effect of If activation on action potential duration (91 ± 5%, n = 3; Fig. 7C). Taken together, all these data support the conclusion that Ca2+ influx through If channels can contribute to the action potential duration.
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DISCUSSION |
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To strengthen the link between the Ca2+ flux and If channel activation at very negative potentials, we need cardiac cells that natively express either higher or no If channels. We chose SHR ventricular myocytes, in which If channel expression is significantly higher compared with the normal rat ventricle (5). Using SHR cells allows us to compare the Ca2+ flux under two native conditions in the same species. The results shown in Fig. 6 provide additional evidence supporting our hypothesis that Ca2+ indeed permeates If channels, although at –150 mV we cannot exclude the possible contribution of other ionic mechanisms such as Na+/Ca2+ exchanger current and Ca2+ release from sarcoplasmic reticulum. The higher percent increase in Ca2+ influx (69%) than in If current (55%) may also reflect the possibility of involving another ionic mechanism. Nonetheless, these data point to a potential role of the fractional Ca2+ influx through If channels during diastole in pathophysiological ventricles where If channel expression is significantly increased (6, 9, 13).
It is well understood that the plateau phase of a ventricular action potential is maintained by a fine balance of outward and inward currents. The major time-dependent inward current that determines the duration of the plateau phase is the L-type Ca2+ current, generated by Ca2+ influx through L-type channels on membrane depolarization. The contribution of this Ca2+ inward current to the action potential duration is limited by the inaction of L-type Ca2+ channels partially caused by Ca2+ influx (3). Within every heartbeat, the amount of calcium that enters the cell during depolarization will have to get out of the cell when the membrane repolarizes to the resting potential via the Na+/Ca2+ exchanger and calcium pump (3). That means that, at negative membrane potentials, what we have learned is the mechanisms that extrude intracellular calcium to set the heart at relax (diastolic) stage ready for the next action potential. The Ca2+ influx through If channels that are open at negative potentials raised the possibility that calcium can still "leak" into the cell at resting or diastolic stage.
Compared with the fractional Ca2+ currents of other cation channels, such as nAChRs (2.5%; Ref. 32), glutamate channels (10% for NMDA; Ref. 17), AMPA/kainate receptors (0.5–5%; Refs. 4, 24), CNG channels (10–80%; Ref. 8), and voltage-dependent calcium channels (100%; Ref. 30), the Pf of HCN/If channels is relatively small (0.47% in HCN2 and 0.6% in If channels). However, given the nature of local calcium signaling, this small Ca2+ flux through If channels may be sufficient to increase the local calcium concentration near the intracellular side of L-type Ca2+ channels, effectively accelerating ICa inactivation, which, in turn, shortens the action potential duration.
Although we have demonstrated the permeation of Ca2+ through If channels in rat ventricular myocytes, more experiments are needed to illustrate the physiological role of fractional calcium current via If channels in cardiac myocytes. Such evidence can only be achieved in a pacing tissue in which If is activated in the physiological voltage range. For example, If appears around –50 mV in cardiac pacemaker sinoatrial (25) and atrial (18) myocytes and –70 mV in neonatal rat ventricular cells (21). In addition, recent studies have shown that under dynamic conditions the activation of HCN1 and HCN2 channels can be dramatically shifted to rather positive voltages (1, 16). Under those conditions, many mechanistic aspects and physiological implications of Ca2+ permeation in If channels could be tested. This can finally establish the physiological relevance of fractional Ca2+ current through HCN.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* X. Yu and X.-W. Chen contributed equally to this work. 
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