1 Department of Physiology, University of Maryland School of Medicine, and 2 Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Maryland 21201; and 3 Laboratorio de Membranas Excitáveis, Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte-MG, Brazil
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ABSTRACT |
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The TTX-sensitive Ca2+ current [ICa(TTX)] observed in cardiac myocytes under Na+-free conditions was investigated using patch-clamp and Ca2+-imaging methods. Cs+ and Ca2+ were found to contribute to ICa(TTX), but TEA+ and N-methyl-D-glucamine (NMDG+) did not. HEK-293 cells transfected with cardiac Na+ channels exhibited a current that resembled ICa(TTX) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na+ channel itself gives rise to ICa(TTX). Furthermore, repeated activation of ICa(TTX) led to a 60% increase in intracellular Ca2+ concentration, confirming Ca2+ entry through this current. Ba2+ permeation of ICa(TTX), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na+ channels under our experimental conditions. The report of block of ICa(TTX) in guinea pig heart by mibefradil (10 µM) was supported in transfected HEK-293 cells, but Na+ current was also blocked (half-block at 0.45 µM). We conclude that ICa(TTX) reflects current through cardiac Na+ channels in Na+-free (or "null") conditions. We suggest that the current be renamed INa(null) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between INa(null) and Ca2+ flux through slip-mode conductance of cardiac Na+ channels is discussed in the context of ion channel biophysics and "permeation plasticity."
tetrodotoxin; excitation-contraction coupling; sodium channel; calcium channel; ventricular myocyte
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INTRODUCTION |
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IMPERFECT SELECTIVITY IS A property of ion channels, including cardiac Na+ channels (13). Alterations in selectivity characteristics have been observed in diverse ion channels in many cell types and have been recognized as a means by which ion channels can be modulated (15), a feature we call "permeation plasticity." In heart cells, the tetrodotoxin (TTX)-sensitive Na+ channel has been shown to become permeable to Ca2+ after protein kinase A (PKA)-dependent phosphorylation by a process called "slip-mode conductance" (28). A second TTX-sensitive Ca2+ current also has been identified in heart cells (6, 19) and attributed to a novel protein and not to the cardiac Na+ channel (1, 3, 12, 24). The novel conductance pathway was named ICa(TTX) to recognize its distinctive identity (1).
A number of features of ICa(TTX) support the hypothesis that this unusual current is due to a protein that is distinct from the TTX-sensitive cardiac Na+ channel. Ca2+ permeation is an important distinguishing feature because Ca2+ has been reported to block Na+ channels (25). In addition, the inactivation kinetics of ICa(TTX) are slower than those of Na+ current (INa) (1, 3), and, like Ca2+ current (ICa), it is permeable to both Ca2+ and Ba2+ (19, 24). Recently, ICa(TTX) has been linked to the T-type Ca2+ channel because both ICa(TTX) and T-type Ca2+ current are blocked by 10 µM mibefradil. (12, 24). Together, this array of findings has been interpreted to suggest that ICa(TTX) reflects current through a novel TTX-sensitive Ca2+ channel protein.
Here we investigate the molecular identity of ICa(TTX). While the data noted above support the notion that a novel protein may underlie ICa(TTX), a single feature of ICa(TTX) raises doubt. The remarkable specificity of TTX for the Na+ channel (13) suggested to us that ICa(TTX) may reflect the permeation properties of the cardiac Na+ channel itself in Na+-free conditions. This TTX-blockable Ca2+ permeation of Na+ channels is reminiscent of slip-mode conductance because it, too, is blocked by TTX (7, 28). To rigorously test the hypothesis that ICa(TTX) in heart cells is due to Ca2+ flux through Na+ channels, we examined HEK-293 cells expressing cardiac Na+ channels.
Preliminary reports have been presented to the Biophysical Society (10, 29).
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METHODS |
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Rat Cardiac Myocytes: Cell Isolation and Preparation
Adult rat heart cells were prepared by standard methods (28). Briefly, rats of either sex weighing between 200 and 300 g were killed by lethal intraperitoneal injection of pentobarbital sodium (100 mg/kg). The hearts were rapidly removed and perfused via the Langendorff method with Ca2+-free modified Tyrode solution (see Solutions) until the blood was washed out. Hearts were then perfused with Tyrode solution containing 50 µM CaCl2 along with 1.4 mg/ml collagenase (type 2; Worthington, Lakewood, NJ) and 0.04 mg/ml protease (type XIV; Sigma, St. Louis, MO) until they were soft (~5 min). The hearts were removed from the perfusion apparatus, minced into ~1-mm chunks, and stirred for 4 min in Tyrode solution containing 50 µM CaCl2, 0.7 mg/ml collagenase, and 0.02 mg/ml protease. Cells were filtered through a 200-µm mesh to remove tissue chunks, and extracellular Ca2+ concentration was raised to 0.5 mM over 10 min through three centrifuge cycles. Cells were stored in DMEM until they were used (within 8 h). Experiments were performed at room temperature (22-24°C).HEK-293 Cells: Culture and Transfection
HEK-293 cells obtained from American Type Culture Collection (Manassas, VA) were cultured by standard methods. The Lipofectamine 2000 reagent (Life Technologies, Rockville, MD) was used to transfect cells at 60-95% confluence with the cardiacSolutions
Solution compositions are noted below. Isolated myocytes and HEK-293 cells were initially superfused with modified Tyrode solution containing (in mM) 140 NaCl, 5 KCl, 5 HEPES, 1 NaH2PO4, 1 MgCl2, 2 CaCl2, and 10 glucose (pH 7.4). After the whole cell recording configuration was established, Ca2+-free modified Tyrode was washed in for 2 min, and then cells were superfused with either "Cs+ Tyrode" solution containing (in mM) 150 CsCl, 0 NaCl, 10 HEPES, 1 MgCl2, 2 CaCl2, and 10 glucose (pH 7.4 with CsOH) or "TEA+ (tetraethylammonium) Tyrode," which was identical to Cs+ Tyrode except that TEA-Cl replaced CsCl and TEA-OH was used to adjust the pH to 7.4. For experiments with transfected HEK-293 cells, extracellular solutions contained 8 mM CaCl2 but were otherwise identical to those noted above. At least 2 min elapsed after each solution change before recordings were made. Two basic pipette filling solutions were used: the first contained (in mM) 150 CsCl, 5 EGTA, 10 HEPES, and 4 Mg-ATP (pH 7.2 with CsOH); the second was identical except that tetramethylammonium-Cl was used as a CsCl replacement and pH was adjusted to 7.2 with TEA-OH. In experiments with myocytes a slightly modified Cs+ filling solution was used, in which 20 mM TEA+ was added to the pipette solution, replacing 20 mM Cs+. Citrate-free TTX (Calbiochem) was used in these experiments. Other modifications to these solutions that were made during some experiments are noted in the text and figure legends.Electrophysiology
Cells were voltage clamped in the whole cell mode with 200A and 200B patch-clamp amplifiers from Axon Instruments (Foster City, CA). Data acquisition was performed with pCLAMP (versions 6.01 and 7; Axon Instruments), and pCLAMP, Origin (version 6.0; Microcal, Northampton, MA), and IDL (Research Systems, Boulder, CO) were used for data analysis. Patch-clamp pipettes were pulled to an initial resistance of 1.5-2.0 MFor intracellular Ca2+ concentration
([Ca2+]i) imaging experiments in HEK-293
cells, a perforated-patch method was used, following the method
previously described (7). Amphotericin B (250 µg/ml) was
added to the Cs+ pipette solution. Electrical access that
enabled adequate voltage control (less than ~5 M) was obtained
20-30 min after the gigaseal was formed.
Ca2+ Imaging in HEK-293 Cells
In those HEK-293 cells used for [Ca2+]i imaging, fluo 3-AM was used to load the cells with fluo 3, following the method previously described (7). [Ca2+]i imaging was done using an MRC600 confocal microscope (Bio-Rad). Images were analyzed and processed using COMOS (Bio-Rad), IDL5.2 (Research Systems), and CorelDraw (Corel) software. ![]() |
RESULTS |
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Two groups (1, 3, 19) observed a TTX-blockable, voltage-gated Ca2+ current in cardiac myocytes and named it ICa(TTX) to suggest that this current was due to a novel Ca2+ channel protein. The initial discovery of ICa(TTX) in 1995 was almost as surprising as the more recent discovery of a second TTX-blockable Ca2+ flux in heart in 1998 (7, 28). The second conductance, however, was shown to reflect Ca2+ permeation through cardiac Na+ channels after PKA activation in normal extracellular Na+ in a process called slip-mode conductance. The similar TTX sensitivity of ICa(TTX) (1, 3, 6, 19, 32) and slip-mode conductance of the cardiac Na+ channel (28) suggested to us that the two processes might be related. However, important differences that favor the novel protein hypothesis have been reported, including differences in Ba2+ permeation and "activation" requirements. Because no group has yet made a molecular identification of ICa(TTX), we sought to determine the identity of the protein responsible for ICa(TTX).
Characteristics of ICa(TTX)
Rat heart cells.
Using Na+-free extracellular solutions, we examined
ICa(TTX) as shown in Fig.
1. With Cs+ as the
Na+ replacement, Fig. 1A shows sample recordings
in 2 mM Ca2+ (control) and after addition of the specific
Na+ channel blocker TTX (10 µM ). Virtually all of the
voltage-gated membrane currents seen on step-depolarizations from 100
mV are blocked by TTX. A typical current-voltage (I-V) plot
of the TTX-sensitive component of the current
ICa(TTX) is shown in Fig. 1C (filled circles). This membrane current is similar to that reported earlier (1, 3, 6, 12, 19, 24). Because Cs+ is not
thought to carry inward current through Ca2+ channels in
the presence of Ca2+ but has been shown to permeate
Na+ channels, albeit poorly (7, 18), the
Cs+ permeability of ICa(TTX) may
provide a clue to its molecular identity. We therefore repeated the
experiment shown in Fig. 1A with a Na+
replacement cation that is known not to permeate cardiac
Na+ channels (TEA+). Figure 1B shows
sample recordings made under control conditions when TEA+
rather than Cs+ was used as the Na+ replacement
and after the addition of 10 µM TTX, as well as the TTX-sensitive
current. The time course of individual ICa(TTX) recordings in TEA+ is similar to that seen in
Cs+; however, the magnitude of
ICa(TTX) is much less. Figure 1C
presents the voltage dependence of ICa(TTX) in
Cs+ (filled circles) and TEA+ (open circles)
and shows that the peak ICa(TTX) is more than eight times larger when measured in the presence of Cs+
than in the presence of TEA+.
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Heterologous Expression of Cardiac Na+ Channels in HEK-293 Cells
To assess whether ICa(TTX) is due to Ca2+ flux through a novel TTX-sensitive cardiac Ca2+ channel or through the well-known cardiac Na+ channel, we examined Na+ channels expressed in HEK-293 cells. If ICa(TTX) arises from the former, then we should have observed no ICa(TTX) in these cells. Figure 3A shows sample recordings of INa (left) and the resulting I-V plot (right) obtained in HEK-293 cells expressing cardiac Na+ channels and recorded in external solution containing 140 mM Na+ and 8 mM Ca2+. Figure 3, B-D, shows ICa(TTX) in these cells. In the absence of Na+ channel expression in the HEK-293 cells, neither INa nor ICa(TTX) is observed (data not shown). To simplify the interpretation of experiments on ICa(TTX) performed in Na+-free conditions, we eliminated Cs+ from the internal and external solutions because it appears to permeate ICa(TTX), as noted above. Figure 3B shows sample recordings of ICa(TTX) (left) and the I-V plot of ICa(TTX) (right) recorded with 140 mM TEA+ and 8 mM Ca2+ in the bath solution. The blockade of ICa(TTX) by TTX (10 µM) is also shown. Figure 3, C and D, which displays pooled data, shows that ICa(TTX) is present whether TEA+ (Fig. 3C) or N-methyl-D-glucamine (NMDG+) (Fig. 3D) is used to replace Na+ and that ICa(TTX) is abolished when Mg2+ replaces extracellular Ca2+. We thus note that the following four major features of ICa(TTX) recorded in cardiac myocytes are also seen in HEK-293 cells that express cardiac Na+ channels: 1) ICa(TTX) is activated at negative potentials, 2) ICa(TTX) is blocked by 10 µM TTX, 3) ICa(TTX) disappears when Ca2+ is replaced by Mg2+, and 4) ICa(TTX) magnitude is greater when Cs+ (rather than TEA+ or NMDG+) is the Na+ replacement cation (data not shown; see also Fig. 6). From these experiments, we deduce that ICa(TTX) can be seen in HEK-293 cells expressing cardiac Na+ channels and that both Ca2+ and Cs+ can carry charge through ICa(TTX) but that TEA+, NMDG+, and Mg2+ cannot. As expected for ICa(TTX), TTX can block the inward membrane current.
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We next sought to examine other characteristics of
ICa(TTX) observed in previous studies. Because
the measured Ba2+ current through
ICa(TTX) was an important element in the
suggestion that ICa(TTX) represented membrane
current through a novel Ca2+ channel, we attempted to
determine whether Ba2+ could carry charge through
ICa(TTX) in cardiac ventricular myocytes or in
HEK-293 cells that express cardiac Na+ channels. Figure
4A shows sample recordings of
membrane current when either Ca2+ (top left) or
Ba2+ (bottom left) is present and the average
I-V plot for such data from heart cells (right).
Figure 4B shows sample recordings of membrane current and
average I-V plots from HEK-293 cells expressing cardiac
Na+ channels from experiments similar to those shown in
Fig. 4A. These data show that
ICa(TTX) is reduced to zero when
Ba2+ replaces Ca2+. We conclude from these
experiments that Ba2+ cannot permeate
ICa(TTX). These data therefore cast doubt on the
novel channel hypothesis.
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A surprising result reported by others, however, at first glance
supports the hypothesis that ICa(TTX) arises
from a novel Ca2+ channel protein. This is the observation
that ICa(TTX) is blocked by mibefradil, a known
Ca2+ channel blocker (12). However, for the
novel protein hypothesis to be supported by this finding, mibefradil
must be shown to be a relatively pure blocker of Ca2+
channels, and INa should not be affected
significantly by mibefradil. We therefore examined the actions of
mibefradil in our heterologous expression system. Figure
5A shows samples of
INa measured on depolarization from 100 to
30 mV at different concentrations of mibefradil. The I-V
relationships at different mibefradil concentrations are shown in Fig.
5B, and the dose-response curve is shown in Fig.
5C. Half-block was achieved at 0.45 µM mibefradil. Figure 5D shows that, similar to INa,
ICa(TTX) observed in HEK-293 cells expressing
cardiac Na+ channels is fully blocked by 10 µM
mibefradil. Importantly, this is the concentration of mibefradil used
to block ICa(TTX) in the experiments of Heubach
et al. (12). Thus we conclude that all of the effects of
mibefradil on ICa(TTX) can be explained by the actions of mibefradil on cardiac Na+ channels.
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The absence of outward membrane current when
ICa(TTX) is measured in heart cells could be
explained by many mechanisms. Clearly, if a novel Ca2+
channel were responsible for ICa(TTX), then the
absence of significant amounts of charge carrier (i.e.,
Ca2+) in the intracellular compartment could account for
this observation (1, 3, 19), as is the case for
ICa measured in heart cells. If, however, the
cardiac Na+ channel was responsible for
ICa(TTX) as suggested by our results, then
outward current should be readily observed when a permeant charge
carrier is present. To test this hypothesis, we measured ICa(TTX) with equal concentrations of
Cs+ (150 mM) inside and outside the cell and with 8 mM
extracellular Ca2+. Intracellular TEA+ was
removed because it can block outward current through the Na+ channel. Figure 6 shows
the inward current carried by both Cs+ and Ca2+
and the outward current carried by Cs+. The presence of an
outward current is important, as is the value of the reversal potential
of ICa(TTX) . The reversal potential for
Cs+ is 0 mV, and under these conditions the reversal
potential (Erev) of
ICa(TTX) is 8 mV. If only Cs+ were
to permeate the Na+ channel, then
Erev for ICa(TTX) would
be 0 mV. The observed Erev of +8 mV suggests
that the ratio of Ca2+ to Cs+ permeability
(PCa/PCs) is significant
and has a value of 4.1 (5, 20).
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Ca2+ Influx via ICa(TTX)
The measured permeability of Ca2+ through ICa(TTX) (deduced from Fig. 6 and supported by Figs. 1-4) should be confirmed by measuring the accumulation of intracellular Ca2+ under conditions in which ICa(TTX) is observed. Using HEK-293 cells to express the cardiac Na+ channel proteins, we examined how changes in [Ca2+]i were related to ICa(TTX). Figure 7A shows sample images of two cells. The top pair of images (Fig. 7A, i) shows a control cell that was transfected but did not express INa or ICa(TTX) to any significant level; the bottom pair of images (Fig. 7A, ii) shows a cell expressing 4.5 nA INa and significant levels of ICa(TTX) (26.2 pA). The images in Fig. 7A, left, show that [Ca2+]i is at control levels in the absence of stimulation; the images in Fig. 7A, right, show how [Ca2+]i changed in response to a series of 200 depolarizations from
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Inactivation of ICa(TTX)
The kinetics of ICa(TTX) inactivation were used by Aggarwal et al. (1) and Balke et al. (3) to distinguish ICa(TTX) from INa. For that reason we compared the inactivation kinetics of ICa(TTX) to those of INa in both rat ventricular myocytes and HEK-293 cells expressing the cardiac Na+ channel. Figure 8 shows sample inward currents obtained in myocytes on step depolarizations from
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DISCUSSION |
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By examining TTX-sensitive current in Na+-free conditions in rat heart cells, we have been able to confirm the existence of a small but significant Ca2+-dependent current. Furthermore, by showing that an identical inward current appears in HEK-293 cells only when the cardiac Na+ channel is expressed, we have been able to identify the Na+ channel as the protein responsible for this Ca2+ flux. While Ca2+ contributes to this current, called ICa(TTX) up to now, Cs+ is also able to permeate. Together, our findings suggest four reasons that the membrane current called ICa(TTX) should not be called a Ca2+ current. First, it is the Na+ channel that is responsible for the current. Second, at least one other ion (i.e., Cs+) readily permeates the channel under diverse experimental conditions. Third, Ba2+ does not contribute to ICa(TTX), while it permeates established Ca2+ channels as well as or better than Ca2+ itself. Fourth, the current as such arises under special conditions, namely, Na+-free ("null") conditions. We therefore prefer the term INa(null) because it properly reflects these four important factors. While the need for Na+-free conditions suggests that INa(null) may not be physiologically important, the existence of INa(null) and its properties help to broaden our understanding of the cardiac Na+ channel and its biophysical properties.
Implications of INa(null)
Ca2+ influx via INa(null) and Ca2+ influx via slip-mode conductance. The measurement of Ca2+ influx through cardiac Na+ channels is unexpected under any condition because Ca2+ has been reported to block Na+ channels (8, 25, 27). Nevertheless, a strong case has been made for such Ca2+ influx in two very distinct modes of behavior of the cardiac Na+ channel: 1) in the absence of extracellular Na+, Ca2+ permeates the channel via INa(null), and 2) in the presence of extracellular Na+ and after activation of PKA, Ca2+ permeates the channel via slip-mode conductance. The finding that Ca2+ can permeate Na+ channels in these two very different circumstances does suggest that Ca2+ permeability is a property of the cardiac Na+ channel, even if it takes special circumstances to recruit that property. It is hard to directly compare the amount of Ca2+ that enters under two very different conditions, but it is clear that much more Ca2+ can permeate via slip-mode conductance than via INa(null), as assessed by the measured increases in [Ca2+]i. When slip-mode conductance was activated, 100 depolarizations were necessary to produce an increase in [Ca2+]i in HEK-293 cells of 250 nM (7), whereas here [Ca2+]i only increased by 60 nM after 200 depolarizations to activate INa(null). Work to date indicates that there is little, if any, Ca2+ permeation via INa (i.e., when extracellular Na+ is present and slip-mode conductance has not been activated). For the "Na+ channel null current" INa(null) to exist, Na+-free conditions appear to be needed. Interestingly, slip-mode conductance occurs in normal Na+ (7, 28) but requires activation (e.g., PKA) and is blocked by very low Na+ (i.e., 0.5 mM). Thus the Na+-free mode of Ca2+ permeation appears to be INa(null), while Ca2+ permeation in normal extracellular Na+ depends on slip-mode conductance. In the absence of those two conditions, there is essentially no Ca2+ permeation via the cardiac Na+ channel.
Mechanisms of Ca2+ permeation through
the Na+ channel to produce
INa(null).
While slip-mode conductance requires the heterotrimeric cardiac
Na+ channel (,
1 and
2) or
the
-subunit and at least one of the two
-subunits (see Refs.
7 and 28), INa(null) can be
observed whenever the
-subunit is expressed and Na+-free
conditions are established. In contrast, slip-mode conductance appears
to need Na+ in the extracellular solution along with PKA
activation. INa(null) is readily observed in the
absence of explicitly activated PKA, as shown in Figs. 1-8. Given
the earlier reports (21, 22, 26) on changes in
INa after PKA activation, however, clarification of the actions of PKA activation on INa(null)
seemed warranted.
Role of PKA activation in INa(null).
Experiments examining PKA activation and
INa(null) in HEK-293 cells expressing the
cardiac Na+ channel were carried out, and the results are
shown in Fig. 9. With PKA inhibitory
peptide (PKI) in the pipette (Fig. 9A), a robust
INa(null) was observed, and forskolin did not
alter the magnitude of INa(null). We conclude
from this experiment that PKA activation is not necessary for
INa(null) to be seen and that no independent
action of forskolin (an activator of PKA) materially affects
INa(null). In addition, activation of PKA by
forskolin in the absence of PKI in the pipette failed to alter the
magnitude of INa(null) (Fig. 9B).
This finding is surprising in light of the results of Balke et al.
(3), who showed that isoproterenol increased the magnitude
of INa(null) in heart cells. Experiments in
myocytes demonstrated the integrity of our reagents, because forskolin
altered the magnitude and voltage dependence of the L-type
Ca2+ current only when PKI was absent from the pipette
(data not shown). We then hypothesized that PKA activation affects
INa(null) in a manner that depends strongly on
the holding potential, as suggested for INa by
Ono et al. (26). Representative results displayed in Fig.
9C show that forskolin increases the magnitude of
INa(null) when the holding potential is 120
mV. Additionally, in cardiac myocytes, isoproterenol can affect
INa via G protein signaling that is independent
of PKA activation (21), and this may account for some of
the observations of Balke et al. (3) in heart cells.
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Permeation Plasticity
Role of permeant ions in channel behavior and permeation plasticity. Permeant ions have been known for some time to have significant effects on channel permeation (13). For example, the L-type Ca2+ channel can readily conduct Na+ and other monovalent cations in the absence of Ca2+ (2), and the relative permeabilities of this channel to Ca2+ and Ba2+ depend on the ionic concentrations (anomalous mole fraction effect) (11). Similar behaviors are observed in certain K+ channels (17) . That permeation depends on the concentration and identity of the permeant ion is thus a feature broadly seen. What has only been appreciated more recently is that the ionic milieu can also have effects on channel gating, such as the decrease in Na+ channel open probability observed upon removal of extracellular Na+ (34, 35) and the slowing of C-type inactivation by K+ occupancy of the pore in Shaker K+ channels (4, 16). INa(null) displays aspects of both phenomena: the removal of intracellular and extracellular Na+ changes the permeability properties as well as the inactivation kinetics of the Na+ channel.
In summary, the primary conclusion from this study and other recent reports is that channel selectivity and kinetics are not rigid features of the assembled Na+ channel proteins but are remarkably dynamic, flexible, and subtle. There are now many examples of channels that change selectivity, including the cardiac Na+ channel (current study and Refs. 7 and 28), the Shaker K+ channel (30, 31, 36), and ligand-gated channels (14, 15). Channel properties can be influenced by phosphorylation or by interaction with permeant ions, even at sites apparently distant from the "selectivity" filter or channel gates. These features may depend critically on protein-protein interactions (e.g., among channel subunits or cytoskeleton proteins or among extracellular matrix proteins or other proteins). Selectivity can be dynamically modified by both physiological interventions (e.g., PKA-dependent phosphorylation in slip-mode conductance) and more severe interventions [e.g., Na+ removal for INa(null) investigations]. ![]() |
ACKNOWLEDGEMENTS |
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We thank C. A. Frederick and N. Agarwal for laboratory support and E. Moczydlowski, A. Goldin, L. Isom, A. George, M. Cahalan, L. F. Santana, and H. Hartmann for reagents, clones, and advice.
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FOOTNOTES |
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* S. Guatimosim, E. A. Sobie, and J. dos Santos Cruz contributed equally to this work.
This work was supported by a grant from the National Heart, Lung, and Blood Institute, by DRIF and Medical Biotechnology Center special accounts funding from the University of Maryland, Baltimore, by the University of Maryland Biotechnology Institute, and by the National Institutes of Health Muscle Training Program at the University of Maryland, Baltimore.
Address for reprint requests and other correspondence: W. J. Lederer, Medical Biotechnology Center, Univ. of Maryland Biotechnology Institute, 725 W. Lombard St., Baltimore, MD 21201 (E-mail: lederer{at}umbi.umd.edu).
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.
Received 19 September 2000; accepted in final form 18 December 2001.
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