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Science 30 April 1999: Vol. 284. no. 5415, p. 711 DOI: 10.1126/science.284.5415.711a
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Technical Comments
Whether "Slip-Mode Conductance" Occurs
Excitable cells rely on selective ionic conductances for
electrical signaling. The work of Hodgkin and Huxley (1) propelled the modern dissection of the mechanisms of selectivity. Their
formulation postulated two independent sets of ionic conductances, Na+ and K+, whose relative permeabilities
changed during the course of excitation. Mullins subsequently proposed
that Na+ and K+ traverse the membrane through a
single set of pores that changed from being Na+-selective
to K+-selective (2). That view is no longer held
as tenable. Several lines of experimental evidence, notably
single-channel recordings, established convincingly that the membrane
contains distinct sets of pores, each with its own distinctive
selectivity properties (3, 4). Nevertheless, the
prevailing view that ion channel selectivity is preserved during normal
electrical activity was recently challenged in a report by L. F. Santana et al. (5). Voltage clamp
experiments in rat ventricular cells led to the proposal that
voltage-dependent Na+ channels change their selectivity in
response to cyclic AMP-dependent phosphorylation.
Unexpectedly, such channels conduct Ca2+ well; similar
"slip-mode conductance" responses were seen during exposure to
cardiac glycosides. The extensive evidence against flexible
selectivity, as well as major technical concerns over that report
(5), prompted us to question whether or not this
idea has a genuine biological basis. Ventricular myocytes are large
cells in which voltage control is notoriously difficult to achieve
(6); the large number of Ca2+-sensitive
ionic currents (7) further complicates the interpretation of the results. To test for the existence of slip-mode conductance, we expressed human hH1 (8) or rat rH1
(9) cardiac Na+ channels heterologously in
Chinese hamster ovary (CHO) cells. These small cells are readily
voltage-clamped, have few endogenous channels, and support cyclic
AMP-mediated responses (10).
We performed whole-cell patch clamp recordings of membrane current
(11). In 20 mM external Na+, a family of typical
Na+ currents was elicited (Fig.
1A) by depolarizing voltage pulses from
100 mV to potentials from 80 mV to +60 mV. As shown in the I-V
relation (panel B), the currents reach a maximum amplitude at 20 mV
and demonstrate a reversal potential near +60 mV. Such currents
increased in amplitude in response to 1 µM isoproterenol or 10 µM
dibutyryl cyclic AMP (32 ± 5%, n = 9; panel C,
), confirming the well-established response to
phosphorylation (10). However, no ionic current
was measurable when the external solution was switched to 0 Na+/10 mM Ca2+ (1 ± 2% of basal current,
n = 7; panel C, ). That Na+ channels
were still present and functional was verified by restoring Na+ to the external solution (panel C, ). Figure 1D
shows the time course of the phosphorylation-mediated
increase in peak INa and flux of Na+ ( )
versus Ca2+ ( ) through the modified rH1 Na
channels. Similarly, there was no measureable Ca2+
flux through phosphorylated hH1 channels in 0 Na+/5 Ca2+ solution (Fig. 1E). Results were
comparable with either the human or rat channels, excluding a possible
species-specific response.
Fig. 1.
Testing for Ca2+ flux through
phosphorylated Na+ channels. (A)
Membrane currents recorded in CHO-K1 cells transfected with the rat
cardiac Na+ channel (rH1) subunit cDNA (3).
(B) I-V relation peaks at 20 mV and reverses near +60 mV.
(C) Basal Na+ currents ( ) recorded with the
use of 20 mM external Na+ as the permeant ion are increased
in magnitude when phosphorylated ( ) during exposure to
dibutyryl cyclic AMP (10 µM). Complete exchange of the extracellular
solution from 20 mM Na+/0 Ca2+ to 0 Na+/10 mM Ca2+ abolishes all inward currents
( ). Na+ current is restored on washout of
Ca2+ and return of Na+ (20 mM) to the external
solution ( ). (D) Peak inward current at 20 mV plotted
over the course of the above experiment illustrates the time course for
the phosphorylation-mediated increase in peak
Na+ current ( ) and the complete exchange of
Ca2+ for Na+ in the recording solution. There
was no measurable inward current in the absence of external
Na+ with 10 mM Ca2+ as the putative charge
carrier ( ). Washout of Ca2+ with the 20 mM
Na+/0 Ca2+ solution fully restored the current.
Solution changes were repeated with similar results. (E)
Human heart Na+ channel subunit expressed in CHO-K1
cells (2) also provided no evidence for Ca2+
flux in the absence of external Na+. Exposure to
isoproterenol (1 µM) produced the
phosphorylation-mediated increase in peak current ( ).
Replacement of external Na+ (10 mM) with Ca2+
(5 mM) did not produce inward current ( ). Washout of
Ca2+ with Na+ (10 mM) confirmed that there was
no loss of functional channels during this manipulation ( ).
[View Larger Versions of these Images (13 + 21 + 14K GIF file)]
These experiments relied on expression of the subunit alone. Such a
test was motivated by the fact that, in rat myocardium, Na+
channels consist only of subunits (12), making it unlikely that subunits somehow contributed to the original
observations of slip mode conductance in rat heart cells. Nevertheless,
to exclude the possibility that subunit coexpression is required, we performed further experiments in CHO cells that coexpress the and 1 subunits (13, 14). In these experiments we
quantified both major manifestations of ion selectivity: reversal potential (Erev), and ion flux. These two reflections of
selectivity are complementary. The reversal potential in solutions of
mixed ion composition yields relative ion permeabilities
(4), but Erev measurements can be
problematic methodologically because small changes, resulting from
junction potential drift for example, are difficult to exclude. For
Na+ currents, the very nature of the measurements
necessitates that small inward and outward currents be quantified
reliably, which is difficult to do in large cells (for example, cardiac
myocytes) with a variety of ionic conductances. In principle, the use
of TTX subtraction may help in distinguishing among various currents, but it does not prevent problems resulting from cumulative junction potential drift or endogenous TTX-sensitive Na+ currents.
The alternative approach, that of measuring ion flux directly (as in
Fig. 1), has various advantages. First, it is model-independent: if net
current is carried by a given ion, then that ion must be permeant.
Secondly, net flux is the parameter of physiological importance. In the
case of slip mode, Ca2+ flux through Na+
channels is postulated to suffice to trigger excitation-contraction coupling (5). If that is the case, a sizable Ca+
current should be measurable through Na+ channels: there is
no reason to rely solely on shifts of Erev.
We took membrane current recordings from a representative CHO cell that
coexpressed + 1 subunits (Fig.
2A). The Na+ equilibrium
potential was set to 0 mV by including 20 mM [Na+] in
both the internal and external solutions (15). Membrane current was first recorded at baseline in the absence of
cyclic AMP, but with 2 mM external [Ca2+]. The currents
reversed at 0 mV ( ). The addition of 50 µM dibutyryl cAMP
increased both inward and outward Na current, as expected from a simple
increase in Na channel open probability. Erev did not
change despite the continuing presence of Ca2+ in the
external solution (16) ( ). Subsequent removal of
external Ca2+ increased the amplitude of the Na currents at
negative potentials; this effect is expected from the known
voltage-dependent block of Na channels by external Ca2+
(17), but is in the opposite direction to the change that would have been expected had the channels been permeable by
Ca2+. Once again, the current reversed at 0 mV ( ). The
single experiment shown in Fig. 2, A to C, was representative of five
cells, whose mean current-voltage relations are shown (B). On removal of Ca2+ in the continued presence of external
Na+, Erev did not change
(16). This stability differs from the shift of 5.1 mV (18), which would have been seen had the relative
Ca2+/Na+ permeabilities equaled 1.25, as stated
in the report (5). Inward currents disappeared (Fig.
2B) when the cells were bathed with an external solution containing 2 mM Ca2+ but no Na+ ( ). This observation
further confirms the absence of an appreciable calcium conductance
through Na channels. Results were indistinguishable between hH1
channels coassembled with human heart (h 1) or rat brain
(r 1) 1 subunits. Confirmation that r 1 in fact expresses functional subunits was derived from parallel experiments in which the
same 1 cDNA increased current amplitude and shifted inactivation when coexpressed with Na channel subunits (19). Because no Erev shift was observed in mixed
Na+/Ca2+ solutions, there is no basis for the
notion that "slip mode" requires the modulatory presence of
external Na+ ions.
Fig. 2.
Membrane currents recorded in CHO-K1 cells
cotransfected with the human cardiac Na+ channel (hH1) subunit and 1 subunit cDNA (2, h 1 and rat brain 1 refs).
(A) Basal Na currents ( ) recorded in 20 mM
Na+ + 2 mM Ca2+ on step depolarizations to
30, 0, and +30 mV reverse at 0 mV with 20 mM internal
Na+. Inward and outward currents were increased in
magnitude when the channels were phosphorylated ( )
during exposure to dibutyryl cAMP (50 µM) without any change in the
reversal potential. After washout of Ca2+ from the external
solution, the currents still reversed at 0 mV ( ). Complete exchange
of the extracellular solution from 20 mM Na+/0
Ca2+ to 0 Na+/2 mM Ca2+ abolished
all inward currents (currents not shown). (B)
Current-voltage relationships did not show a rightward shift of
Erev with modification by db-cAMP ( ) or a leftward shift
of Erev on removal of external Ca2+
(16, ). Only outward Na+ currents were
recorded in the 0 Na+/2 mM Ca2+ external
solution as internal Na+ was fixed at 20 mM ( ). Results
were indistinguishable between hH1 channels coassembled with human
heart (h 1) and rat brain (r 1) 1 subunits. Two (of five)
experiments were performed on cell cotransfected with hH1+GFP-Ir-h 1;
three experiments were performed on cells cotransfected with
hH1+r 1+GFP.
[View Larger Version of this Image (20K GIF file)]
Our results contradict findings in rat ventricular myocytes
(5) and in HEK cells (20). We have used
CHO cells, which are known to support cyclic AMP-mediated responses and which contain few endogenous conductances. The first reports of
cyclic AMP-dependent upregulation of Na currents were from expression
studies in CHO cells (10). HEK cells have the virtue of
being readily transfected (21), but they do not consistently
support cyclic AMP-dependent responses; several lines of evidence
indicate that protein kinase activity is so high in the basal condition
that kinase inhibitors must be added to reveal directionally
appropriate responses (22). HEK cells also contain a variety
of endogenous conductances that may interfere with the quantification
of Na+ current reversal potentials. These include an
endogenous TTX-sensitive voltage-dependent Na+ channel
(21) and a dihydropyridine-sensitive Ca+ channel
(23). The Ca+ channel literature raises the
caution that endogenous channels may interact with exogenously
expressed subunits in an unanticipated manner, clouding the
interpretation of multisubunit expression studies
(24). HEK cells also possess a variety of endogenous K+ and Cl conductances
(25), which may complicate attempts to measure small
differences in Na+ current reversal potential.
We have not observed Na+ currents in nontransfected CHO
cells, but such cells have occasionally been reported to express small endogenous TTX-sensitive Na+ currents (26,
27). To verify that our findings reflect the behavior of
TTX-resistant cardiac Na+ channels, we measured
INa in hH1 + 1 + GFP transfected CHO cells under drug-free conditions and in the presence of low- and high-dose TTX (n = 7). The lower concentration of 100 nM would be
expected to block >95% of TTX-sensitive channels, but only suppressed
the current by 10.7 ± 1.4% (n = 7). In contrast,
a higher concentration appropriate to inhibit cardiac channels (5 µM
TTX) blocked 62.1 ± 2.9% (n = 7) of the current
in agreement with Krafte et al. (27). Our results
indicate that at least 90% of current in transfected cells arises from
the TTX-resistant hH1 (cardiac) Na+ current. This
conclusion is further bolstered by the observation that nongreen cells
in the same dishes (n = 2) and CHO cells transfected with GFP alone (n = 4) had no observable
Na+ currents under identical recording conditions.
In summary, our experiments show that cardiac Na+ channels
are up-regulated by cyclic AMP, but that such up-regulation is not accompanied by changes in their Na+/Ca2+
selectivity. Na+ channels are not measurably permeant to
Ca2+ either in the basal state or after cyclic
AMP-dependent stimulation. Our experiments were designed to
investigate the molecular basis of the reported "slip mode";
neither the cardiac subunit alone nor + 1 subunits suffice
to confer such a phenomenon. Furthermore, cyclic AMP-dependent
upregulation occurs without an associated change in selectivity, which
implies that "slip mode" does not reflect a direct consequence of
phosphorylation of either subunit. For these reasons, we
conclude that the reports of "slip-mode conductance" represent a
technical artifact, possibly arising from suboptimal voltage control.
H. Bradley Nuss
Eduardo Marbán
Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA E-mail: marban{at}jhmi.edu
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Supported by National Institutes of Health grant R01-HL-52768
(EM) and an American Heart Association Scientist Development Grant
(HBN). We thank W. Catterall for providing the rat cardiac Na channel
subunit cDNA.
22 June 1998; revised 19 January 1999; accepted 18 February
1999
Santana et al. (1) observed a
tetrodotoxin (TTX)-blockable Ca2+ current in rat
ventricular myocytes after several pharmacological treatments [cyclic
AMP, isoproterenol (ISO), or the cardiotonic steroids, ouabain and
digoxin]. This TTX-blockable Ca2+ current was attributed
to a Ca2+ permeability induced in classical cardiac
Na+ channels that normally express little or none. Santana
et al. described such treated Na+ channels as
"promiscuous" (1). In the absence of any of these
agents, we previously described (2) a TTX-blockable
Ca2+ current (ICa(TTX)), also seen in rat
ventricular myocytes (3). ICa(TTX) is generated
by Na+ channels that are functionally distinct from those
generating the classical cardiac Na+ current
(INa). The question arises, then, of whether the induced Ca2+ current observed by Santana et al. might be
identified with ICa(TTX). Possibly, protein kinase A
(PKA)-dependent phosphorylation of classical Na+ channels
(or whatever the nature of the modification produced by cardiotonic
steroids) converts them to ICa(TTX) channels, and ICa(TTX) recorded in the absence of these agents arises
from some basal pool of modified classical Na+ channels.
ICa(TTX) channels are unlikely to arise from PKA-dependent
conversion of classical Na+ channels, because a substantial
increase in ICa(TTX) produced by ISO was not accompanied by
a reduction in INa, as would be required. Moreover, we find
all of the TTX-blockable Ca2+ current seen in the presence
of ISO flows through ICa(TTX) and not through classical
Na+ channels. The enhanced TTX-blockable Ca2+
current seen in ISO must be attributed to an increase in
ICa(TTX) and not to the induction of Ca2+
permeability in classical Na+ channels.
Because the inactivation kinetics of ICa(TTX) are slower
than those of INa (2), the conversion of any
significant fraction of classical Na+ channels into
ICa(TTX) channels must necessarily slow the inactivation time course of the TTX-blockable current. In 1 mm external
Na+ (Na+O) plus 0.5 mm external
Ca2+ (Ca2+O), 1 µM ISO
substantially increased the total TTX blockable current (Fig. 1A; mean
increase: 73.2 ± 8.7% SEM; 19 determinations from four cells). This
increase occured without a detectable change in the current time course
(as shown by the scaled and superimposed traces of Fig. 1A, top
row). ISO did not produce a significant change in the inactivation time constant ( h; single
exponential fit; Fig. 1B, upper curve). The mean ratio of
h,ISO to h,control was 0.988 (range:
0.826 to 1.208). These results are consistent with either a roughly
parallel increase in the amplitudes of INa and
ICa(TTX) or with an ICa(TTX) component that
remains small relative to INa, but not with the simple
conversion of INa into ICa(TTX) channels,
because h is slower for ICa(TTX). The mean values of h for ICa(TTX) relative to that
for INa ranged from 2.90-fold slower at 50 mV to
1.70-fold slower at 30 mV (2). However, the inactivation
time course was unchanged by ISO, indicating that there was no
detectable conversion of classical Na+ channels into
ICa(TTX) channels. Therefore, it is unlikely that ICa(TTX) channels are just phosphorylated
INa channels. Moreover, nearly all of the increase in
current must be of INa, because INa at these
potentials is considerably larger than ICa(TTX)
(2).
Fig. 1.
Effects of isoproterenol (ISO) in rat ventricular
myocytes. (A) Whole cell patch clamp current records from
freshly isolated adult cells (3). For all experiments, holding
potential was 100 mV, and currents were isolated with 10 µM TTX.
(Top row) Currents from the same cell recorded in 1 mM
Na+0 plus 0.5 mM Ca2+0
on a step to 35 mV. Left record was obtained in the absence and
center record in the presence of 1 µM ISO. ISO increased the current
amplitude by 75% without a change in time course, as shown in the
right panel, which again presents the record in the absence of ISO,
with that in ISO superimposed. Record in ISO has been scaled down so
that its peak matches that in the absence of ISO. (Bottom row) Currents
form a different cell recorded in 0 Na+0 and 1 mM Ca2+0 on a step to 35 mV, in the absence
(left panel) and presence (center panel) of 1 µM ISO. This cell
expresses the largest ICa(TTX) seen in this series. ISO
increases the current by 82%, again without a detectable change in the
time course as shown by the scaled and superimposed records of the
right panel. For both experimental conditions, the fact that current
amplitudes can be substantially increased without a detectable change
in time course suggests that these data have not been affected to any
appreciable extent by any residual uncompensated series resistance
errors. Scale: 500 pA, 20 ms. (B) Ratio of the time constant
of inactivation ( h; obtained as a best- fit single
exponential) in the presence to that in the absence of 1 µM ISO
( h,ISO/ h,control). Filled squares ( )
indicate means and brackets indicate total range of variation. Currents
with a peak amplitude less than 150 pA were not used for kinetic
analysis. (Top curve) Ratios obtained in 1 mM
Na+0 plus 0.5 mM Ca2+0.
Means of 1 ( 55 mV), 4 ( 50 mV, 45 mV, 40 mV), 3 ( 35 mV), 2 ( 30 mV), and 1 ( 25 mV) determinations. (Bottom curve) Ratios
obtained in 0 Na+0 and 1 mM
Ca2+0. Means of 2 ( 40 mV), 4 ( 35 mV), 5 ( 30 mV), and 2 ( 25 mV) determinations.
[View Larger Version of this Image (17K GIF file)]
ISO also increased ICa(TTX). In 1 mM
Ca2+0 and 0 Na+0, 1 µM ISO increased the (TTX subtracted) current by 83.6 ± 10% SEM.
(13 determinations from five cells), again without a change in current time course (Fig. 1A, scaled and superimposed traces, bottom row; different cell from top row). One cell (Fig. 1A, bottom row) expressed a particularly large amplitude ICa(TTX) component. The mean
ratio of h,ISO to h,control in
Ca2+0 only was 0.913 (range: 0.712 to 1.250;
Fig. 1B, bottom curve). In the absence of ISO, all the TTX-blockable
Ca2+ current is generated by a single population of
channels, ICa(TTX). The activation curve for the
TTX-blockable current is well described by a single Boltzmann function
in Ca2+0 only, but requires the sum of two
Boltzmann functions for a good description in
Na+0 plus Ca2+0
(2). Similarly, the TTX-blockable current inactivates with a
single exponential time course in Ca2+0 only,
but with two exponentials in Na+0 plus
Ca2+0 (2). These data indicate that,
in the presence of ISO, the TTX-blockable Ca2+ current is
still generated by a single population of channels because a nearly
twofold increase in current amplitude is not accompanied by any change
in the inactivation time course. Under our experimental conditions (TTX
subtracted currents; no more than 1 mM Na+0),
the increased current in ISO seen in Na+0 plus
Ca2+0 must represent primarily increased
Na+ current through classical Na+ channels and
not Ca2+ current. The increase in TTX-blockable
Ca2+ current reported by Santana et al.
(1) may simply reflect an increase in ICa(TTX).
They apparently did not attempt to identify which channel type
generated the TTX-blockable Ca2+ current.
ISO substantially increases the classical INa without a
change in its kinetics (Fig. 1A, upper traces). Thus, if ISO induces any appreciable Ca2+ permeability in classical
Na+ channels, this slippage should be evidenced by the
appearance of a faster inactivating component in the TTX-blockable
Ca2+ current, but none was detected (Fig. 1A, lower
traces). Therefore, our experiments are not consistent with a change in
selectivity of classical Na+ channels induced by conditions
that promote channel phosphorylation, but are in agreement with
findings in guinea pig, rabbit, and canine ventricular myocytes, as
well as in rat cardiac Na+ (SkM2) channels expressed
heterologously in frog oocytes (4).
C. William Balke
Larry Goldman
Rajesh Aggarwal
Stephen R. Shorofsky
Departments of Physiology and Medicine, School of Medicine, University of Maryland, Baltimore, Baltimore, MD 21201, USA
REFERENCES AND NOTES
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L. F. Santana,
A. M. Gómez,
W. J. Lederer,
Science
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(1998)
.
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R. Aggarwal,
S. R. Shorofsky,
L. Goldman,
C. W. Balke,
J. Physiol.
505,
353
(1997)
[Abstract/Free Full Text].
-
Isolated rat ventricular myocytes were obtained by a
standard enzymatic dispersion technique described previously [
C. W. Balke and
W. G. Wier,
Proc. Natl. Acad. Sci. U.S.A.
89,
4417
(1992)
[Abstract/Free Full Text]
]. Membrane currents were recorded using the
whole-cell configuration of the patch clamp technique. Patch pipettes
were as described previously (2). Series resistance
compensation was used in all experiments. Currents were filtered at 2 kHz and digitized at 20 kHz. The external solution was composed of 140 mM tetraethylammonium chloride (TEA-Cl), 10 mM glucose, 10 mM Hepes, 10 mM CsCl, 1 mM MgCl2 (pH adjusted to 7.3 with CsOH) and
either 1 mM Na+ plus 0.5 mM Ca2+ or 1 mM
Ca2+ only as noted. La3+ (10 µM) was included
in all external solutions to suppress L-type Ca2+ currents.
When indicated, isoproterenol (1 µM plus 10 µM EDTA to prevent
oxidation) was added to this solution. The electrode filling solution
contained 120 mM glutamic acid, 120 mM CsOH, 10 mM Hepes, 0.33 mM
MgCl2, 20 mM TEA-Cl, 4 mM adenosine 5'-triphosphate (Mg
salt), and 5 mM EGTA (pH adjusted to 7.3 with CsOH). Temperature was 21 to 23°C. The holding potential was
100 mV. Depolarizing pulses
varied between 60 and 25 mV, in increments of 5 mV. Analyses were
performed on records subtracted with the use of 10 µM TTX.
-
K. Ono,
T. Kiyosue,
M. Arita,
Am. J. Physiol.
256,
C1131
(1989)
[Abstract/Free Full Text]
;
J. J. Matsuda,
H. Lee,
E. F. Shibata,
Circ. Res.
70,
199
(1992)
[Abstract/Free Full Text]
;
K. Ono,
H. A. Fozzard,
D.
A. Hanck,
Pflügers Arch.
429,
561
(1995)
[CrossRef] [ISI] [Medline];
W. Schreibmayer,
et al.,
Receptors Channels
2,
339
(1994)
[ISI] [Medline]
. The studies using canine ventricular cells (Ono et
al., 1995) were about the effects of forskolin. However, these
forskolin effects are apparently not mediated through protein kinase A
phosphorylation.
-
Animals used in the present study were maintained in
accordance with the guidelines of the Institutional Animal Care and Use
Committee of the University of Maryland School of Medicine and the
Guide for the Care and Use of Laboratory Animals
[Department of Health and Human Services publication No. (NIH) 85-23, revised 1985].
4 May 1998; accepted 18 February 1999
Response: The TTX-sensitive Na+ channel is
selective for Na+ over other monovalent and divalent
cations (1-5). Recently, however, we reported that
the ion selectivity of cardiac Na+ channels could be
dynamically modulated (6). Following phosphorylation by protein kinase A (PKA), the
Ca2+ permeability of the Na+ channel
(PCa) could increase relative to Na+
permeability (PNa) so that the permeability ratio
(PCa/PNa) was greater than 1. Called
"slip-mode conductance" of the Na+ channel, this
behavior was shown to be functionally important for heart cells when it
was activated. First, it was shown that Ca2+ influx through
Na+ channels alone could trigger Ca2+-induced
Ca2+-release (CICR); activating Ca2+ sparks and
small [Ca2+]i transients. Second, it was
demonstrated that it was possible to evaluate the contributions of
Ca2+ influx through Na+ channels under
near-physiological conditions using action potential "clamp"
experiments. These results demonstrated that INa could activate measurable Ca2+ influx, Ca2+ sparks or
[Ca2+]i transients only when slip-mode
conductance was activated. We also carried out quantitative examination
of INa in heart cells under conditions that made it
possible to measure this current. Low [Na+] at cool
temperatures kept INa to values that could be reliably measured (1 to 4 nA). With the use of this approach, we measured large
and unambiguous positive shifts in the INa reversal
potential, Erev, of 9 mV to 10 mV, when slip-mode
conductance was activated. This shift in Erev suggested
that an increase of PCa/PNa from a very low
value (0.0 to 0.1) to large values (1.2 to 1.4) occurred when slip-mode
conductance was activated. In their comments, neither Nuss and
Marbán nor Balke et al. have repeated these
experiments in heart cells under conditions identical to those of our
report (6). Consequently their comments do not
directly address our report (6) and must be
interpreted in light of the assumptions they have made. Balke et
al. discuss a phenomenon that appears to be unrelated to slip-mode
conductance of the Na+ channel. Nuss and Marbán,
however, did raise a question that we too have been examining for the
past year: Can slip-mode conductance of the cardiac Na+
channel be observed in an heterologous expression system? The experiments presented below, in contrast to those of Nuss and Marbán, show that Ca2+ can permeate Na+
channels in an heterologous expression system and thus provide strong
evidence in support of our original findings and hypotheses (6).
If slip-mode conductance of the cardiac Na+ channel only
relies on appropriate phosphorylation of the cardiac
Na+ channel and the presence of Na+ channels,
then it should be possible to reproduce the changes in INa
and in Ca2+ flux that we have seen in heart using an
heterologous expression system. A key factor in any such experiment is
setting up the conditions in the correct manner. We chose to use HEK293
cells because of the low level of voltage-gated channels expressed in these cells (7). However, we did not know a priori what part of the heterotrimeric Na+ channel was responsible
for slip-mode conductance. Although virtually all features of cardiac
Na+ channels have been attributed to the subunit and
can be observed when this subunit of the Na+ channel is
expressed in an heterologous system (2, 8), there are two
other Na+ channel subunits, 1 and
2. Because of the importance of the subunit, we
examined it in HEK293 cells first. INa occurred when only
the subunit of the human isoform of the cardiac Na+
channel (hH1 ) was expressed in HEK293 cells (Fig.
1A). If slip-mode conductance of the
Na+ channel could be produced when only the subunit was
expressed, then a shift of Erev would be expected when PKA
is activated. However under all conditions tested, Erev
remained at the Na+ equilibrium potential
(ENa), which was 0 mV under control conditions and 0.63 mV
in Na+- dbcAMP (10). A small increase in
INa magnitude following the addition of dbcAMP is
consistent with published results (9), as is the block of
INa by TTX (10 µM).
Fig. 1.
Expression of human heart
Na+ channels in HEK293 cells. (A) subunit only. Membrane current (right) from a single HEK293 cell
transfected with the hH1 subunit are shown as are current-voltage
(IV) plots averaged from 8 cells (left). Control conditions:
[Na+]o = 20 mM;
[Na+]i = 20 mM;
[Ca2+]o =2 mM;
[Ca2+]i = 0 nM;
[Cs+]i = 120 mM;
[Cs+]o = 120 mM;
[Mg2+]o = 1 mM; [Mg-ATP]i = 4 mM. Voltage protocol (top right) indicates that depolarizations from
113 mV to test potentials over the range 83 mV to +17 mV at 5 mV
intervals were imposed. Voltage-dependence of Na+ channel
current (INa) is compared under four conditions: control
(n = 8), following the addition of 500 µM
Na+-dibutyryl cAMP (dbcAMP) with 2 mM Ca2+
(n = 7), following the addition of 500 µM
Na+-dbcAMP (0 Ca2+) and following the addition
of 500 µM Na+-dbcAMP (2 Ca2+ and 10 µM
TTX) (n = 4). Measured mean Erev
was close to ENa for each curve: Erev = 0.23 ± 0.72mV (n = 8) (control), +0.82 ± 0.86 mV (n = 7) (dbcAMP, 2 mM Ca2+),
0.63 ± 0.77 mV (n = 7) (dbcAMP, 0 mM
Ca2+), 2.04 ± 0.16 mV (n = 4)
(dbcAMP, 2 mM Ca2+, TTX). TTX blocks
87.1 ± 0.7% (n = 4, p < 0.05)
of INa. Expected ENa was 0 mV for control and
+0.63 for all others because Na+-dbcAMP was used, thus
adding 0.5 mM Na+ to the extracellular solutions. No
significant difference was observed between the expected
Erev (control) and Erev (dbcAMP with, 2 mM
Ca2+). (B) Co-expression of and
1 subunits. INa was measured in HEK293 cells
expressing both and 1 subunits of the human heart
Na+ channel. Voltage protocol similar to (A), but the
holding potential was 110 mV and the test potential range was between
80 mV and +15 mV. Averaged IV plots (n = 8) are shown
for control conditions (identical to panel A), following the addition
of 500 µM Na+-dbcAMP (2 Ca2+), following the
addition of 500 µM Na+- dbcAMP (0 Ca2+) or
following the addition of 500 µM Na+-dbcAMP (2 Ca2+ and 10 µM TTX). When compared to
control, the addition of dbcAMP led to a significant increase in peak
INa (21 ± 6% (p < 0.05, n = 8), with a significant shift in Erev
from 0.18 ± 0.65 mV to +4.00 ± 0.36 mV
(p < 0.0001, n = 8).
Erev = 0.63 ± 0.76 mV (dbcAMP, 0 Ca2+);
Erev = 0.80 ± 1.25 mV (dbcAMP, 2 Ca2+,
TTX). (*) indicates a significant difference between
dbcAMP average INa and control INa
(p < 0.05). An enlargement of the region of
the reversal potentials shown as an insert.
PCa/PNa for the measured Erev
indicates an increase from about 0.04 (control) to 0.79 (dbcAMP). PCa/PNa was calculated from Campbell
et al.(10). Addition of TTX (10 µM)
reduced INa at 30 mV by 78.8 ± 4.7%
(p < 0.05, n = 8) with a
significant shift of Erev back towards the control
0.59 ± 1.25 mV (p < 0.05, n = 8) when compared to the Erev for INa in
dbcAMP alone. (C) PKA-inhibitory peptide. Averaged
INa IV relationships (n = 5) for HEK293
cells expressing and 1 subunits of the human cardiac
Na+ channel observed when PKA-inhibitory peptide (PKA-I)
was added (100 µM) to the pipette filling solution (2). Control
solutions were otherwise similar to those in A. IV
relationships of INa were obtained under control
conditions, following the addition of 500 µM Na+-dbcAMP
(2 Ca2+) and following the addition of 500 µM
Na+-dbcAMP (2 Ca2+) with 10 µM TTX.
Erev = 0.02 ± 0.44 mV (Control), Erev = 0.05 ± 0.41 mV (dbcAMP, 2 Ca2+), Erev = 0.2 ± 0.84 mV (dbcAMP, 2 Ca2+, TTX).
Thus Erev was similar in all conditions (control, dbcAMP,
dbcAMP + TTX) and close to ENa. (0 mV,
0.63 mV and 0.63 mV respectively). (D) 1
only. Averaged INa IV relationships (n = 8) for
HEK293 cells expressing only 1 subunits only under
control conditions and following exposure to 500 µM
Na+-dbcAMP (left) with INa records from a
single cell (right). No significant measured INa is
observed. (E) Sham transfection. Averaged INa IV
relationships (n = 6) for HEK293 cells exposed to all
aspects of the transfection process but with no added vector (left)
with INa records from a single cell (right). No significant
INa is observed. ENa = 0 under control
conditions (20 mM Na+), ENa = 0.63 mV in dbcAMP
(20.5 Na+, 2 Ca2+), ENa= 0.63 mV in
dbcAMP (20.5 Na+, 0 Ca2+).
[View Larger Version of this Image (38K GIF file)]
Under the ionic conditions of these experiments, an increase in
PCa/PNa would have led to inward current at
ENa and a positive shift in Erev
(10). Because neither was observed, we deduced that
there was no significant change in PCa/PNa
(10), a finding similar to that observed by Grant et
al. (11). How can these findings (Fig. 1A) be
reconciled with the finding that Na+ channels in rat heart
became permeable to Ca2+ following the activation of PKA
(2)? Could our earlier results with rat heart cells
(6) simply be wrong? That conclusion seemed
unlikely, because multiple investigative methods were used to
demonstrate slip-mode conductance of the cardiac Na+
channel in rat cardiac myocytes. We thus examined the possibility that
a missing factor or channel subunit was responsible for the absence of
any PKA-activated increase in PCa/PNa when only
the subunit was expressed (see also Table
1). Because intact heart cells express
and both subunits (12) and recent evidence suggests that both and 1 are associated
with each other in heart (13) and in heterologous expression systems (14), we examined HEK293 cells that
co-express and 1 subunits.
We measured the current-voltage (IV) relationships for INa
in HEK293 cells that co-express and 1 subunits of
the Na+ channels under several experimental conditions
(Fig. 1B). Under control conditions, Erev = 0.23 mV, a
value statistically indistinguishable from ENa of 0 mV.
After the addition of 500 µM Na+-dbcAMP, a significant
increase of Erev was observed (Erev = 4.00 mV,
P < 0.0001, n = 8). This increase in
Erev suggests that PCa/PNa has
increased from close to zero to 0.79 (10), indicating that
Ca2+ was almost as permeable through Na+
channels as was Na+. The altered Erev was
returned to the control level by the removal of extracellular
Ca2+ (replaced by Mg2+), a result that also
supports the conclusion that Ca2+ permeation underlies the
shift of Erev following the addition of dbcAMP. In the
maintained presence of dbcAMP and with 2 mM [Ca2+]o
, TTX significantly reduced INa and also produced a
significant negative shift in Erev back to control
conditions. We thus concluded that first, the observed TTX-sensitivity
confirms the involvement of cardiac Na+ channels in the
measured currents. Second, Na+ channels altered by PKA may
be more sensitive to TTX when in slip-mode conductance than when they
are not. If Na+ channels in slip-mode and those not in
slip-mode were equally sensitive to TTX, then TTX would not have
shifted Erev back to ENa. [This finding is
identical to that observed in intact rat ventricular myocytes
(6).] Third, protein-protein interactions between
the and 1 subunits are important for proper Na+ channel function and provides a functional reason role
for the 1 subunit of the Na+ channel in
heart. PKA-dependent phosphorylation underlies the activation of slip-mode conductance (Fig. 1C), which can be prevented by adding intracellular PKA inhibitory peptide (PKA-I, 100 µM).
Two control experiments (Fig. 1, D and E) indicated that neither the
expression of the 1 subunit alone nor a sham
transfection of HEK293 cells altered the HEK293 cells to produce
INa under control conditions or following exposure to
dbcAMP. We thus conclude that there are no interfering voltage-gated
currents in these cells to confound interpretation.
Ca2+ permeation through the cardiac Na+ channel
is thus confirmed (Fig. 1) in support of our report
(6). The subunit alone is not enough (Fig. 1) to
support this change in selectivity. Instead, it was found that the
co-expression of an Na+ channel subunit along with a
subunit was required for us to observe slip-mode conductance of the
Na+ channel in an heterologous expression system. The subunit is central to our understanding of ion permeation through
Na+ channels because it contains the ion channel, the
selectivity filter, the TTX binding site, and the PKA consensus
phosphorylation sites. However, we have no structural
information on how the subunit is altered to produce slip-mode
conductance. Also we do not know what the 1 subunit does
and whether or not its action requires other factors. Intuition from
nonselective channels, including mutated Na+ channels
(15), suggests that larger changes in
Erev might be observed in bi-ionic conditions (for example,
high Ca2+ outside and high Na+ inside with low
Ca2+ inside and low Na+ outside). One finding
(Fig. 2, A and B) was counterintuitive. HEK293 cells were made to
express and 1 subunits (as was shown in Fig. 1), but
with somewhat reduced intracellular and extracellular [Na+] (10 mM) and increased extracellular
[Ca2+] (5 mM). Slip-mode conductance was readily
activated by dbcAMP, leading to a positive shift of Erev of
more than 7 mV, more than was observed in Fig. 1. Unexpectedly,
however, PCa/PNa was only 0.32. Experiments
(16) similar to those of Fig. 2, A and B, but with extracellular
[Ca2+] at 2 mM led to a larger calculated
PCa/PNa of 0.95. Thus, although extracellular
Ca2+ permeates the Na+ channel during slip-mode
conductance, it also tends to block the conductance.
Fig. 2.
Increased
PCa/PNa during slip-mode conductance of the
cardiac Na+ channel. INa in HEK293 cells
expressing both and 1 subunits of the human cardiac
Na+ channel was measured on depolarization from 112 mV to
the test potential. (A) INa records are shown
for four depolarizations ( 32, 2, +3, and +8 mV) under three
conditions (control in 10 mM Na+, dbcAMP in 10.5 mM
Na+, dbcAMP in 0.5 mM Na+). (B) IV
plot of INa for the same three conditions. In these
experiments [Ca2+]o = 5 mM;
[Ca2+]i = 0 nM;
[Cs+]i = 125 mM;
[Cs+]o = 125 mM; TEA-Cli = 10 mM;
[Mg2+]o = 1 mM; [Mg-ATP]i = 4 mM; [EGTA]i = 5 mM. When comparing control conditions to
dbcAMP, INa at 32 mV increased significantly by 17.5 ± 4.7% (n = 7, p < 0.001).
Erev shifted significantly (p < 0.001) to 7.18 ± 0.73 mV (n = 7) in dbcAMP
(plus 5 mM Ca2+) from 0.94 ± 1.14 mV (n = 7) in control conditions. PCa/PNa changes
from 0.04 under control conditions to 0.32 in dbcAMP. Under the same
conditions, with the maintained superfusion of dbcAMP and 5 mM
[Ca2+]o, extracellular Na+ was
reduced to 0.5 mM. There was no significant INa measured.
The most positive zero-current voltage was 37.1 mV. This is the upper
limit of Erev and it would correspond to a
PCa/PNa of 0.11. The (*) symbol indicates
significant differences between control and dbcAMP (with 5 mM
[Ca2+]o and with 10.5 mM
[Na+]o). ENa = 0 under control conditions, ENa = 1.2 mV in dbcAMP (10.5 Na+), ENa= 76 in dbcAMP (0.5 Na+). Calculations of PCa/PNa make
use of Campbell et al.(10). (C)
Increased [Ca2+]i resulting from
Ca2+ flux through Na+ channels. Top: Triply
transfected ( , 1 and 2) had spatially
averaged [Ca2+]i following 100 depolarizations from 100 mV to 30 mV for 20 ms (at 200 Hz) of 101 nM (control), 325 nM (dbcAMP); 103 nM (recontrol). Middle: Triply
transfected cells in the presence of TTX (10 µM) had
spatially averaged [Ca2+]i following 100 depolarizations of 106 nM (control), 105 nM (dbcAMP), 108 nM
(recontrol). Bottom: when only the subunit was transfected,
INa was measured but no increase in
[Ca2+]i was observed following 100 depolarizations (109 nM (control); 110 nM (dbcAMP), 104 nm
(recontrol)). [Ca2+]i measurements used cells
exposed for 30 min to 1.5 µM fluo-3AM. Resting
[Ca2+]i was assumed to be 100 nM in 2 mM
extracellular Ca2+ and 20 mM extracellular Na+.
Recontrol levels were measured at steady-state in
zero [Ca2+]o. Scale bar:
10 µm. (D) Relationship between the number of
depolarizations (protocol and cells as in C) and increase in
[Ca2+]i in 500 µM dbcAMP (n = 6) (solid line) and in 500 µM dbcAMP and 10 µM TTX
(n = 3)(black dashed line). HEK293 cells transfected
with only the subunit of the Na+ channel, the same
relationship between number of depolarizations and
[Ca2+]i in the presence of 500 µM dbcAMP is
shown by the red dashed line (n = 3). (E)
Relationship between the average [Ca2+]i
per 100 depolarizing pulses and peak INa. Solid line is
given by [Ca2+]i = 41.98 nM + 0.91 * INa, with INa values in pA. Correlation
coefficient (r2) was 0.93, with p < 0.0001 for the null hypothesis that the slope was zero. Dashed red lines mark
the 95% confidence limits. (F) Decay of
[Ca2+]i in 0 [Ca2+]o following an elevation in dbcAMP ( = 36 s, n = 6). (G) Shift of
Erev produced by slip mode conductance in triply
transfected cells. Control: Erev = 0.01 ± 0.27 mV
(n = 6); dbcAMP: Erev = 5.00 ± 0.44 (n = 6). PCa/PNa(control) = 0.0; PCa/PNa (dbcAMP) = 1.1. Peak
INa at 30 mV in dbcAMP is 0.71 ±0.1 (n = 6) compared to control INa peak of 1.00 ± 0.01 (n = 6). (H) Protein immunoblots of
Na+ channel 1 and 2 subunits.
HEK293 cells were transfected with hH1 , 1 and
2 subunits as above. After 3 days, cells were scraped
from the culture dishes, washed with PBS, and solubilized in 50 mM Tris
(pH 8), 10 mM EGTA, 2% SDS. For comparison, membranes containing
neuronal Na+ channels (from whole rat brain) were prepared
as previously described (19) (14). Proteins were reduced
with -mercaptoethanol in panel (i) and were nonreduced in
panel (ii) to detect covalent association of and 2.
Panel (i). Protein immunoblots were probed with antipeptide antibodies
to 2 (lanes 1 and 2) or 1 (lanes 3 and 4). Lanes 1 and 3: HEK293 cells. Lanes 2 and 4: rat brain membranes. Panel (ii).
Protein immunoblot was probed with the anti-peptide antibody to
2. Lane 1: rat brain membranes, Lane 2: HEK293 cells.
Positions of molecular weight markers are indicated in kDa. Molecular
weights for 1, 2 and - 2 are 36, 33, and 290 kDa respectively, but modest
differences in positions of bands (arrows) are not unexpected given
differences in post-translational processing (for example,
glycosylation and phosphorylation).
[View Larger Version of this Image (28K GIF file)]
To further examine the role of extracellular Na+ in
producing slip-mode conductance, we reduced extracellular
[Na+] from 10.5 to 0.5 after slip-mode had been activated
by dbcAMP, and we recorded INa at four select potentials
from one cell (Fig. 2A). Switching to an extracellular Na+
of 0.5 mM with continued exposure to dbcAMP led to the virtual abolition of measured Ca2+ flux through Na+
channels. Fig. 2B shows the three IV plots (n = 7) that
correspond to the conditions noted above. Reduction of extracellular
Na+ to 0.5 mM led to the apparent abolition of inward
INa, including any component carried by Ca2+.
This observation was the second unexpected finding in these experiments
(Figs. 2A and B). The INa, IV plot shows that outward currents began to appear at potentials positive to 37 mV, which suggested that the "reversal potential" for INa was
37 mV or more negative. This suggests that under these
conditions PCa/PNa is 0.11 or less
(10). Taken together, these data (Fig. 1 and Fig. 2, A and
2B) suggest that even when slip-mode conductance is activated by PKA,
as [Na+]o declines, so does
PCa/PNa . These experiments, as noted above, also support a blocking action of high extracellular
[Ca2+]. Dual actions of extracellular Ca2+
are consistent with the hypothesis that two independent processes are
involved, one involving Ca2+ that blocks Na+
channels (as previously established) and another that permits it to
permeate.
We do not find (Fig. 2, A and B)
support for the inward current described by Balke et al.. In
HEK293 cells that co-express and , subunits of the cardiac
Na+ channel, the absence of inward current in 0.5 mM
[Na+]o in 5 mM
[Ca2+]o suggests that the current they
describe is not the result of hH1 and 1 subunits.
Furthermore, our experiments (Fig. 2) rule out their suggestion that
slip-mode conductance of the cardiac Na+ channel is the
result of the same putatively novel channel protein that Balke
et al. state is responsible for the current they observe.
Just as the increase in PCa/PNa during
slip-mode conductance should lead to a measured shift in
Erev (Figs. 1 and Fig. 2, A and B), it should also produce
measurable Ca2+ influx in HEK293 cells. With the use of an
amphotericin perforated patch-clamp method (17) with
cells loaded with the Ca2+-sensitive indicator fluo-3, we
measured [Ca2+]i during Na+
channel activation. This method permitted patch-clamp control while
measuring [Ca2+]i and avoided the loss of
Ca2+ into the pipette which can severely distort
[Ca2+]i measurements (18). Because
this approach uses an entirely different method to investigate
Ca2+ entry through cardiac Na+ channels, it
provided an independent check on the earlier measured changes in
Erev. In particular, this method of measuring
Ca2+ flux through Na+ channels does not depend
on tip-potential measurements. For these experiments, we transfected
the HEK293 cells with all three relevant cardiac Na+
channel subunits: hH1 , the universal 1 subunit, and a
2 subunit cloned from human heart (13). We
did this after finding that slip-mode conductance of cardiac
Na+ channels could also be measured when only and
2 were co-expressed (16). Our overall
experience with cardiac Na+ channels suggests that the
triple transfection ( , 1, 2) provides the most reliable expression of INa and the most robust
slip-mode conductance. We took fluorescence images (Fig. 2C) of HEK293
cells containing the Ca2+ indicator fluo-3 under control
conditions (that is, no PKA activation) following 100 depolarizing
pulses to activate INa (left), and then following 100 depolarizing pulses in the presence of 500 µM dbcAMP (middle), and
following the removal of extracellular Ca2+ (right).
External [Na+] and pipette [Na+]
concentrations were 20 mM and extracellular [Ca2+] was 2 mM (as in Fig. 1). The top set of images in Fig. 2C shows triply
transfected cells, the middle set shows triply transfected cells in the
presence of 10 µM TTX, and the bottom set shows cells transfected
with only the subunit. Supporting our interpretation of Figs. 1B
and 2B, we found (Fig. 2C) that the addition of dbcAMP enabled
Na+ channels in HEK293 cells to become permeable to
Ca2+ if they were composed of , 1 and
2 subunits but not if they expressed the subunit
only. The increase of [Ca2+]i arising from
the flux of Ca2+ through Na+ channels depended
on the number of depolarizing pulses and thus on the number of times
that INa was activated (Fig. 2D) and was blocked by TTX. An
increase of [Ca2+]i from 100 nM to about 325 nM after 100 pulses is consistent with a Na+ current having
a magnitude of 300 pA with an inactivation time constant of 2 ms if
10% of the current is carried by Ca2+ and this
Ca2+ flux enters a 16-µm-diameter cell with a buffering
power of 60. The [Ca2+]i achieved after
100 pulses was proportional to the measured peak Ina (Fig.
2E), a finding also consistent with the hypothesis that
Ca2+ can permeate Na+ channels that exhibit
slip-mode conductance. The elevated [Ca2+]i
fell towards the prestimulation level ( = 36 s) when
extracellular Ca2+ was removed (Fig. 2F). When HEK293 cells
were triply transfected, INa had a reversal potential at
ENa before slip-mode was activated by PKA (Fig. 2G). After
slip-mode had been activated, Erev moved towards ECa
by 5.0 mV, which was consistent with an increase in PCa/PNa from 0 to 1.1, a change comparable to
the increase in PCa/PNa seen in rat heart cells
(PCa/PNa = 1.2) following PKA activation.
Finally, protein immunoblots [Fig. 2H(i)] indicated that both
1 and 2 subunits were expressed in these
HEK293 cells following the triple transfection. 2
associated with the subunit following the triple transfection
[Fig. 2H (ii)] (19). The 1 dissociates from
under these preparative conditions because, unlike
2, it is not attached by disulfide bonds. These results and others (13, 14) provide direct evidence that
the three cardiac Na+ channel subunits are associated to
form the normal cardiac Na+ channel. All three subunits
also contribute functionally to Na+ channel behavior. The
triply transfected HEK293 cells expressed INa better and
more reliably than the other subunit combinations we tested. Following
PKA activation by dbcAMP, there was a decrease in the peak
INa at 30 mV in the triply transfected HEK293 cells, a
finding different to that observed in rat heart cells and in Figs. 1B
and 2B. Additional studies will be needed to better understand this
property of Na+ channels.
A question raised by these findings (Figs. 1 and 2) is whether the
Na+ channel may also become permeable to other cations
under conditions that activate slip-mode conductance. This topic was
investigated in HEK293 cells expressing and 1
subunits. In the absence of extracellular Ca2+,
NMG+ does not readily pass through the Na+
channel before or after activation of slip-mode conductance
(PNMG/PNa < 0.03); there is no significant
change in Erev (Fig. 3A).
Under control conditions PCs/PNa was about 0.10 and, following PKA activation, increased insignificantly to 0.12 (Fig.
3B). Thus, the Na+ channel permitted a very small amount of
Cs+ and almost no NMG+ to permeate; the
permeability of neither cation was significantly modulated by PKA
activation. Consistent with these observations was the insignificant
reduction of peak INa that followed PKA activation in
Cs+ (decreasing 28.5 ± 21.2%, p = n.s., n = 6). In NMG+ the reduction of
24.6 ± 5.9% (p = n.s., n = 6) was also insignificant. In contrast, K+ ions were more
readily conducted by Na+ channels (Fig. 3C) under control
conditions than either Cs+ or NMG+ ions. We
examined, in the absence of Ca2+, the relative changes in
INa when K+ and Na+ were present
and when NMG+ was the impermeant cation (chosen because it
is the least permeant) (Fig. 3A). In the presence of K+,
the maximum INa was reduced by 58.2 ± 16%
(p < 0.05, n = 4) following the
application of dbcAMP. In these experiments ENa, the
expected Erev for INa, = 59 mV if there is no
permeation by K+ through Na+ channels. We found
that under control conditions Erev = 36.75 ± 0.78 mV
(n = 4) indicating PK/PNa was about
0.25 under these ionic conditions. Following PKA activation
Erev shifted to 27.73 ± 4.31 mV (p
< 0.05, n = 4), which indicated that
PK/PNa almost doubled to 0.47. We concluded
that K+ permeation of the cardiac Na+ channel
(in the absence of Ca2+) is also modulated by PKA-dependent
phosphorylation. Nevertheless, compared to the single amino
acid mutations of the selectivity filter (15), the changes
in ion selectivity that we observed were subtle and specific following
PKA-dependent phosphorylation.
Fig. 3.
Permeation by other ions during
slip-mode conductance in HEK293 cells expressing and
1 subunits. N-methyl-d-glucamine (NMG+)
was used in these experiments. (A) Selectivity of the
Na+ channel for NMG+ (compared to
Na+) in the absence of Ca2+ before and after
PKA activation. Solutions had the following ion composition:
[Na+]o = 120 mM;
[Na+]i = 10 mM;
[Ca2+]o = 0 mM;
[Ca2+]i = 0 nM;
[NMG+]i = 120 mM;
[NMG+]o = 10 mM;
[Mg2+]o = 1 mM; [Mg-ATP]i = 4 mM. Depolarizations from a holding potential of 110 mV to a potential
over the range 100 to +80 mV in 5 mV steps were applied. IV
relationships before and after 500 µM Na+-dbcAMP are
shown. A statistically insignificant reduction of INa was
observed following dbcAMP, 24 ± 5.9%, (n = 6. p = n.s.). No significant change in Erev
was observed (control: 55.4 ± 2.1 mV; dbcAMP: 56.7 ± 1.5 mV, n = 7, p=n.s.). A PNMG/PNa
ratio < 0.03 can account for the measured Erev under
both conditions. (B) Selectivity of the Na+
channel for Cs+ (compared to Na+) in the
absence of Ca2+ before and after PKA activation. Ion
composition: [Na+]o = 100 mM;
[Na+]i = 10 mM;
[Ca2+]o = 0 mM;
[Ca2+]i = 0 nM;
[Cs+]i = 60 mM;
[Cs+]o = 27 mM;
[NMG+]i = 75 mM;
[NMG+]o = 18 mM;
[Mg2+]o = 1 mM; [Mg-ATP]i = 4 mM. Depolarizations from a holding potential of 110 to potential over
the range 80 mV to +70 in 10 mV steps were applied. IV relationships
before and after dbcAMP are shown. A statistically insignificant
reduction of INa was observed following dbcAMP, 28.5 ± 21.2% reduction, (n = 4, p = n.s.).
No significant change in Erev was observed.
Erev = 47.2 ± 1.9 mV under control conditions and
45.2 ± 1.8 mV in dbcAMP (n = 4, p = n.s.) corresponding to similar ratios PCs/PNa
of 0.10 (control) and 0.12 (dbcAMP). (C) Selectivity of
Na+ channel for K+ compared to Na+
in the absence of Ca2+ before and after PKA activation. Ion
composition: [Na+]o = 100 mM;
[Na+]i = 10 mM;
[Ca2+]o = 0 mM;
[Ca2+]i = 0 nM;
[K+]i = 60 mM;
[K+]o = 27 mM;
[NMG+]i = 75 mM;
[NMG+]o = 18 mM;[Mg2+]o = 1 mM; [Mg-ATP]i = 4 mM. Depolarizations from a holding potential of 110 mV to a
potential in the range from 80 to +60 mV in 10 mV steps were applied.
IV relationships before and after the addition of 500 µM
Na+-dbcAMP are shown. A large reduction of INa
was observed following dbcAMP (58 ± 16%, n = 4, p < 0.05). A significant negative shift of
Erev was measured from 36.75 ± 0.78 mV (n = 4) under control conditions to 27.73 ± 4.31 mV
(n = 4) following PKA activation (p < 0.05), which indicated that PK/PNa
increased from 0.25 to 0.47. These values for
PK/PNa are larger than those found in other
Na+ channels (1) and are nearly doubled by
dbcAMP. (D) Selectivity for Ca2+ over
Na+ in the presence of NMG+. Depolarizations
from a holding potential of 108 mV to potential in the range 78 to
+17 mV in 5 mV steps were applied. IV relationships for INa
in control conditions and after the application of 500 µM
Na+-dbcAMP for 5 cells are shown. Control conditions
include [Na+]o = 20 mM;
[Na+]i = 20 mM;
[Ca2+]o = 2 mM;
[Ca2+]i = 100 nM;
[K+]i = 0 mM; [K+]o = 0 mM; [NMG+]i = 125 mM;
[NMG+]o = 125 mM;
[Mg2+]o = 0 mM; [Mg-ATP]i = 4 mM. A 16.1 ± 0.8% reduction of INa was observed
following dbcAMP. Erev increased from 0.53 ± 1.5 mV
to 6.35 ±1.76 mV (n = 4, p < 0.05),
which suggests that PCa/PNa increased from 0.11 to 1.48. (E). Selectivity for Ba2+ through
Na+ channels. Depolarizations from a holding potential of
110 mV to a range of potentials from 80 to +15 mV in 5 mV steps
were applied. IV relationships in control conditions and after the
application of 500 µM Na+-dbcAMP are shown for six cells.
Control conditions include [Na+]o = 20 mM;
[Na+]i = 20 mM;
[Ba2+]o = 2 mM;
[Ba2+]i = 100 nM;
[K+]i = 0 mM; [K+]o = 0 mM; [NMG+]i = 125 mM;
[NMG+]o = 125 mM;
[Mg2+]o = 0 mM; [Mg-ATP]i = 4 mM. A statistically insignificant reduction of peak INa was
observed following dbcAMP. No change in Erev was observed;
it remained nearly constant and close to ENa. Control:
0.83 ± 0.27 mV; after dbcAMP: 0.65 ± 0.4 mV,
(n = 6, p=n.s.). PBa/PNa could
not be distinguished from zero.
[View Larger Version of this Image (28K GIF file)]
Because Ca2+ permeation
arises as PKA-activation occurs, and because Ba2+ permeates
all known Ca2+ channels (1), we examined the
extent to which Ba2+ could permeate Na+
channels in slip-mode conductance. Because NMG+ was the
least permeant monovalent cation tested, it was used as the impermeant
monovalent cation during these experiments. We examined
Ca2+ flux through Na+ channels (Fig. 3D) under
conditions similar to those used in the Ba2+ permeation
experiments (below). First, with 2 mM extracellular Ca2+,
Erev shifted from 0.53 ± 1.5 mV (control) to
+6.35 ± 1.76 mV (dbcAMP) (p < 0.05, n = 4), which suggested that following PKA activation
PCa/PNa = 1.48. (inset in Fig. 3D). Although a
large increase in PCa/PNa was observed, there
was a reduction of peak INa produced by dbcAMP, similar to
that seen in Fig. 2G. These results provide additional evidence that
specific ionic conditions influence INa and PKA-induced
changes in PCa/PNa of cardiac Na+
channels. In contrast, when Ba2+ was used to replace
Ca2+ in the extracellular solution, no change in
Erev was observed following activation of PKA. Erev
shifts slightly from 0.83 ± 0.27 mV to 0.65 ± 0.4 mV (p = n.s., n = 6). This
result suggested that PBa/PNa was about zero
whether PKA was activated or not. Although Ba2+ normally
can permeate Ca2+ channels, in other biological processes
Ba2+ is readily distinguished from Ca2+. For
example, although Ca2+ flux through L-type Ca2+
channels augments ICa inactivation, Ba2+ flux
through L-type Ca2+ channels does not. A second example is
found in the "Ca2+-induced Ca2+-release"
phenomenon in heart. Normally Ca2+-release channels in the
sarcoplasmic reticulum cryanodine receptors are rapidly activated by
Ca2+, but not by Ba2+. The absence of
Ba2+ permeation though Na+ channels activated
by PKA adds to the evidence that the permeation process during
slip-mode conduction of the Na+ channel is distinctive and
involves regulation by the permeant ions themselves.
We thus confirm the ability of PKA phosphorylation of cardiac Na+
channels to enable Ca2+ flux as we originally
proposed in our report (6). The experiments
presented here were carried out in an heterologous expression system
using HEK293 cells. These cells do not have the numerous voltage-gated
currents (Fig. 1) seen in heart muscle cells. The human heart
Na+ channel subunit (hH1 ) was expressed along with
either the 1 subunit or the 2 subunit, or
both. All three combinations of subunit plus subunits could
produce slip-mode conductance. However, neither nor
1 nor 2 subunit alone was seen to produce PKA-dependent Ca2+ permeation of the Na+
channel. We speculate that it is the subunit that has the ability to produce slip-mode conductance, but only under specific conditions. One condition that is necessary is the co-expression of either 1 or 2 subunits (or both subunits). We
surmise that the subunits enable the subunit to function
properly by one or more of the following means. They may help it fold
properly in the SL membrane and stabilize important conformations of
the protein, or directly modulate subunit function, or help the subunit associate with other important proteins. Co-transfection of and one of the subunits did not always enable measurable slip-mode
conductance of INa. Why such variability occurred and how
it depended on conditions known to affect subunit assembly (that is
stoichiometry of expression, expression conditions, unidentified
co-factors, the cell line used for expression, timing and conditions of
the transfection, or other factors) remains unknown at this time.
Presumably one or more of these conditions was not met in the
experiments carried out by Nuss and Marbán and "different
conditions" accounted for their negative results. We have directly
addressed each of the concerns expressed in their comment. Also we have
more completely characterized the molecular requirement for slip-mode
conductance of cardiac Na+ channels.
We have identified and characterized important new features of
TTX-sensitive Na+ channels from human heart. Most
significantly, we demonstrate that Ca2+ can permeate
cardiac Na+ channels following PKA activation during
"slip-mode" conductance in an heterologous expression system. This
permeation by Ca2+ of Na+ channels is evidenced
by shifts in Erev and by measured Ca2+ influx
seen as increases in [Ca2+]i. That the subunits, with no previously identified function in heart, are
necessary to support this conductance mode of the subunit was
unexpected. Additional unexpected features of slip-mode conductance of
the cardiac Na+ channel have been identified and include
facilitation by Na+, block and permeation by
Ca2+, and permeation by K+.
Jader dos Santos Cruz*
Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA and Laboratorio de Membranas Excitáveis, Departamento de Bioquimica e Imunologia, Universidade Federal de Minas Gerais, CP 486 ICB-UFMG, Belo Horizonte-MG, Brazil
L. F. Santana*
Institute of Neurobiology, University of Puerto Rico, San Juan, PR 00901 USA
Cecilia A. Frederick
Department of Physiology, University of Maryland School of Medicine
Lori L. Isom
Jyoti Dhar Malhotra
Laura N. Mattei
Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109-0632, USA
R. S. Kass
J. Xia
R-H. An
Department of Pharmacology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
W. J. Lederer
Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201 and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA E-mail: jlederer{at}umaryland.edu * Contributed equally.
Reprints
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Experimental Procedures: General. Patch clamp
experiments were carried out in whole cell mode. HEK293 cells are a
human embryonic kidney cell line used because of the low background of
ion channel expression. The HEK293 cells were transfected with the
subunit of the human sodium channel and/or the 1 subunit
and/or the 2 subunit of the human cardiac sodium
channel. The subunit (also called SCN5A, Genbank accession No.
M77235) was cloned [M. E. Gellens, A. L. George, Jr., L. Chen, M. Chahine, R. Horn et al., Proc. Natl.
Acad. Sci., U.S.A. 89, 554 (1992)] and inserted into pcDNA
(Invitrogen Corporation, Carlsbad CA). The human 1
subunit (also called h 1 or Scn1b, Genbank accession No. U12192) was
cloned [
N. Makita,
P. B. J. Bennett,
A. L. J. George,
J. Biol. Chem.
269,
7571
(1994)
[Abstract/Free Full Text]
]. We
would like to thank Drs. M. Keating, P. Bennett and A. George for the
gifts of the cDNAs. HEK293 cells were transfected using the
"lipofectamine plus" reagent (Life Technology, Rockville, MD) and
the instructions provided were followed. Sub-confluent HEK293 cells
were exposed to Lipofectamine Plus and the Na+ channel
subunits (in pcDNA) for six hours. The cells were incubated overnight
at 37o C in MEM supplemented with 10% fetal bovine serum,
100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine,
then split and plated onto 25 mm No. 1 glass coverslips at low density.
After one to two days, the cells were examined without removing them
from the coverslips. HEK293 cells prepared in this manner without
transfection or transfected with pcDNA containing the 1
or 2 subunit alone, had no measurable INa
(see Fig. 1D). Patch clamp methods: Membrane currents were
measured using the conventional [
R. H. An,
R. Bangalore,
S.
Z. Rosero,
R. S. Kass,
Circ. Res.
79,
103
(1996)
[Abstract/Free Full Text]
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L. F. Santana,
A. M. Gómez,
W. J. Lederer,
Science
279,
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(1998)
] or the perforated patch
configuration [
R. Horn and
A. Marty,
J. Gen. Physiol.
92,
145
(1988)
[Abstract/Free Full Text]
]. Standard patch-clamp pipettes were
prepared from borosilicate filamented glass (WPI, Instruments)
with uncompensated series resistance of between 1.0 and 3.0 M-Ohms.
When the perforated patch method was employed, 250 µg/ml amphotericin
B (Sigma) was included in the pipette filling solution. Using this
concentration of amphotericin B, maximal electrical access to the cell
was achieved within 5 to 10 min after G seal formation. Cells were
examined using a whole cell method using pClamp 6.03 (Axon Instruments)
software and an Axopatch 200A patch clamp amplifier (Axon Instruments).
Cells were not accepted for recording if the initial seal resistance
was <2G or if the peak INa (when
[Na+]o = 135 mM) was <5 nA. Voltage errors
were minimized using series resistance compensation (generally 75 to
80%). The uncompensated capacitance transients along with linear leaks
were removed. Data collection using conventional patch clamp methods
was only initiated at least 5 min after break-in and when
INa had stabilized after intracellular dialysis with
pipette solution. Data was recorded during continuous perfusion of the
extracellular solutions. Analysis was carried out using pClamp 6.02, Sigmaplot v.2 and Origin v.5 software (Microcal, Inc.).
Solutions: Intracellular (pipette) solutions contained (in
mM): 10 HEPES, 4 Mg-ATP, 5 EGTA, and pH 7.4. All extracellular
solutions contained (in mM) 10 glucose, 10 HEPES, pH 7.4. TTX
(10 µM) was used as noted in the Figures. The other constituents and
changes in these solutions are indicated in the Fig. legends.
All experiments were carried out at 22°C.
-
INa attributed to the
subunit alone did,
however, appear to be slowed kinetically compared to the native
INa . But it is still not clear exactly what the
1 subunit does. For example, although slow inactivation
of INa is observed when skeletal muscle subunits were
expressed in Xenopus oocytes [
H. B. Nuss,
N. Chiamvimonvat,
M. T. Perez-Garcia,
G. F. Tomaselli,
E. Marbán,
J. Gen. Physiol.
106,
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[Abstract/Free Full Text]
], rapid inactivation of INa was observed when the
subunits were expressed in HEK293 cells [
C. Ukomadu,
J. Zhou,
F. J. Sigworth,
W. S. Agnew,
Neuron
8,
663
(1992)
]. This raises the question of the mechanism of acceleration of
inactivation of the brain and skeletal muscle INa following
the co-expression of the 1 subunit to the subunit in
Xenopus oocytes [
A. Toib,
V. Lyakhov,
S. Marom,
J. Neurosci.
18,
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[Abstract/Free Full Text]
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H. B. Nuss,
N. Chiamvimonvat,
M. T. Perez-Garcia,
G. F. Tomaselli,
E. Marbán,
J. Gen. Physiol.
106,
1171
(1995)
]. Nevertheless, it is widely suggested that the
1 subunit does appear to significantly influence
INa kinetics when co-expressed with non-cardiac subunits. In heart, however, the question is murky. Marbán and
co-workers have examined the issue and note that there are clear,
albeit much smaller, kinetic effects of 1 on hH1 subunit dependent INa [
H. B. Nuss,
N. Chiamvimonvat,
M. T. Pérez-García,
G. F. Tomaselli,
E. Marbán,
J. Gen. Physiol.
106,
1171
(1995)
]. Nevertheless, their overall-assessment is that "... the
functional role of this subunit in heart is uncertain ..."
Perhaps they were influenced by the absence of demonstrable association
between and 1 in heart [
N. Makita,
P. B. J. Bennett,
A. L. J. George,
J. Biol. Chem.
269,
7571
(1994)
] combined with the small effects observed.
More recent experiments now suggest there is an association between
cardiac and 1 subunits (12-14).
-
INa was reported to increase when the
subunit
was expressed in Xenopus oocytes and activated by cAMP [
B. Frohnwieser,
L. Q. Chen,
W. Schreibmayer,
R. G. Kallen,
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498,
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rabbit heart cells were exposed to isoproterenol [
J. J. Matsuda,
H. Lee,
E. F. Shibata,
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70,
199
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] but INa was found to decrease in neonatal rat
heart cells [
B. Schubert,
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A. M. Brown,
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258,
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].
-
To calculate relative permeability of ions through channels,
reversal potential (Erev) (i.e. the zero-current potential)
measurement are made under different ionic conditions. Our experiments
presented here use Erev to calculate the relative
permeability of Ca2+ (PCa) to Na+
(PNa) through the Na+ channel. We used a
solution of the Nernst-Planck equation with constant field assumption
presented by Campbell et al. in 1988 [
D. L. Campbell,
W. R. Giles,
J. R. Hume,
D. Noble,
E. F. Shibata,
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403,
267
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[Abstract/Free Full Text]]; this
solution is equivalent to that of C. A. Lewis [ibid.
286, 407 (1979)]. The reversal potential
(Erev) for a channel with two ions that are permeant must
lie at or between the equilibrium potentials for the two ions (here
ENa and ECa). Since we were examining possible
changes in PCa/PNa for the Na+
channel, an accurate assessment of measured potential was needed
relative to ENa. We opted to use one of two methods to
correct for small (<3 mV) systematic errors in the measured
potential compared to ENa: 1. The zero-current potential
for INa was measured in Na+-containing
solutions (and known [Na+]o and
[Na+]i) but with 0 mM
[Ca2+]o and 0 mM
[Ca2+]i. It was assumed this potential should
be equal to ENa as calculated by the known Na+
concentrations and all potentials were adjusted by the small measured
error. 2. A steady-state tip potential was measured using the relevant
pipette solution. All potentials were then adjusted by this estimated
error. The adjustments were less than 3 mV and only a single adjustment
was applied to a complete data set. Since the first method is
preferred, it was applied whenever possible (Fig. 1, A and B, and Fig.
3, A, B, C, and E). The second method was applied elsewhere. The first
method assumes that any monovalent cations present in the intracellular
or extracellular solutions other than Na+ do not contribute
significantly to measured potentials. We determined that
Cs+ and NMG+ could be used because they have
two important properties--1. PCs/PNa and
PNMG/PNa are close to zero; 2. PCs/PNa and PNMG/PNa do
not change following application of PKA activators such as dibutyryl
cAMP (dbcAMP) (see Fig. 3).
-
A. Grant,
V. S. Chauhan,
R. Chandra,
C. F. Starmer,
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76,
A80
(1999)
.
-
Two
subunits have been identified in
heart, 1 and 2. There is only one gene
for 1, which is responsible for expression of this
subunit in all tissues. 2 identified in brain [L.
L. Isom, D. S. Ragsdale, K. S. DeJongh, R. E. Westenbroek, B. F. X. Reber, W. A. Catterall.
Cell 83, 433 (1995)] was recently characterized
by L. N. Mattei and L. L. Isom in heart (unpublished). A cDNA
clone for a human fetal heart sodium channel 2 subunit
was obtained by searching the TIGR/ATCC Special Collection of human
cDNA clones for homology to the rat brain 2 subunit.
Clone 149022 (in pBluescript SK ) was obtained and
sequenced with ThermoSequenase (Amersham) using oligonucleotide
primers specific to 2. Analysis of the deduced
amino acid sequence (accession number AF107028) confirmed that this
clone was indeed a 2 subunit that was approximately 93%
identical to that cloned from rat brain. For mammalian cell expression
(e.g., such as the HEK293 cells used here), clone 149022 was subcloned
into the EcoRI and XhoI sites of pcDNA3.1/Zeo(+) (Invitrogen) and
resequenced to confirm the orientation as well as the lack of
mutations. Evidence by western of subunit association is shown in Fig.
2.
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R. M. Shah, S. R. Levinson, R. A. Maue,
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T. Zimmer, M. Steinbis, T. Böhle. K. Benndorf.
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J. S. Cruz, L. F. Santana, C. A. Frederick, R-H. An, J. Xia,
J. D. Malhotra, L. N. Mattei, L. Isom, W. J. Lederer, R. S. Kass, data
not shown.
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R. Horn and
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J. R. Berlin,
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L. L Isom, T. Scheuer, A. B. Brownstein, D. S. Ragsdale, B. J. Murphy, W. A. Catterall. J. Biol.
Chem. 270, 3306 (1995). The antibodies used here were rabbit
polyclonal antibodies generated against multiple-antigenic peptides
(MAP) by Research Genetics (Huntsville, AL). MAPs specific to the
cytoplasmic domains of
1 (LAITSESKENCTGVQVAE) or
2 (KCVRRKKEQKLSTD) were synthesized by the Protein and
Carbohydrate Structure Core facility at the University of Michigan. The
doublet observed for the brain 1 subunit in Fig. 2H(i)
was first noted by E. M. Sutkowski and W. A. Catterall
[J. Biol. Chem. 265, 12393 (1990)].
Additional investigations of - 1 and
- 2 interactions by L. L. Isom and co-workers
supports our findings (work in progress).
-
We would like to thank Xiao-Li Wang for support in preparing
constructs and Keith Dilly, W. H. duBell, T. B. Rogers, and
Laura Martin for comments on the manuscript. This work has been
supported by NIH grants (HL25675, HL36974, HL56810), by NSF EPSCoR
grant (UPR-RP program), by University of Maryland DRIF support funds
and by funds from the University of Maryland Biotechnology Institute
and Medical Biotechnology Center.
27 May 1998; accepted 18 February 1999
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