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 I_Na 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.

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 (E_rev), 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 E_rev 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 E_rev.

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. E_rev 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⁺, E_rev 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 (h1) or rat brain (r1) 1 subunits. Confirmation that r1 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 E_rev 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.

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 I_Na 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

REFERENCES AND NOTES

1. A. L. Hodgkin and A. F. Huxley, J. Physiol. 117, 500 (1952) .
2. L. J. Mullins, J. Gen. Physiol. 42, 817 (1959) [Abstract/Free Full Text] .
3. C. L. Armstrong in Membrane Transport, D. C. Tosteson, Ed., (Waverly, Baltimore, MD, 1989), pp. 261-273; B. Sakmann and E. Neher, Single-Channel Recording, (Plenum Press, New York, ed. 2, 1995). A notable exception is the apparent alteration of monovalent cation selectivity in certain K⁺ channels during slow inactivation, as observed by L. Kiss, J. LoTurco, and S. J. Korn [Biophys. J. 76, 253 (1999)].
4. B. Hille, Ionic Channels of Excitable Membranes (Sinauer, Sunderland, ed. 2, 1992).
5. L. F. Santana, A. M. Gómez, W. J. Lederer, Science 279, 1027 (1998) [Abstract/Free Full Text] .
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11. Na⁺ channel  subunit cDNA was subcloned into a mammalian expression vector (GWIH) and co-transfected into CHO-K1 cells with GPF (pGreen Lantern-1, Gibco-BRL). The extracellular recording solution for the Ca²⁺ flux experiments was comprised of (in mM): 125 CsCl, 1 MgCl₂, 5 dextrose, 10 Hepes, pH 7.3 with CsOH, and 10 NaCl or 5 CaCl₂. Cells were dialyzed with an intracellular (pipette) solution containing (in mM): 135 CsCl, 10 TEACl, 10 Hepes, 5 EGTA, 4 MgATP, 0.33 MgCl₂, pH 7.3 with CsOH.
12. J. S. Yang, P. B. Bennett, N. Makita, A. L. George, R. L. Barchi, Neuron 11, 915 (1993) [CrossRef] [ISI] [Medline] ; S. A. Cohen and L. K. Levitt, Circ. Research 73, 735 (1993) [Abstract/Free Full Text].
13. The only accessory subunit expressed in cardiac muscle is 1 (14).
14. E. Marban, T. Yamagishi, G. F. Tomaselli, J. Physiol. 508.3, 647 (1998) .
15. Human and rat Na⁺ channel 1 subunit cDNA were subcloned into a mammalian expression vector (pGFPIrs) for bicistronic expression of the 1 subunit and GFP reporter [ L. L. Isom, et al., Science 256, 839 (1992) [Abstract/Free Full Text] ]; N. Makita, K. Sloan-Brown, D. O. Weghuis, H. H. Ropers, A. L. George, Genomics 23, 628 (1994) [CrossRef] [ISI] [Medline] . D. C. Johns, H. B. Nuss, E. Marban, J. Biol. Chem. 272, 31598 (1997) [Abstract/Free Full Text] . Coexpression of  and 1 Na⁺ channel subunits was accomplished by cotransfecting equal masses of each plasmid into CHO-K1 cells, which provided a molar excess of 1 subunit cDNA due to the differences in length of coding sequences. The control extracellular recording solution was comprised of (in mM): 125 CsCl, 20 NaCl, 2 CaCl₂, 1 MgCl₂, 5 dextrose, 10 HEPES, pH 7.3 with CsOH (). Dibutyryl-cAMP (50 µM) was present in all solutions except the initial control solution (, , and  in the figures). MgCl₂ was increased to 3 mM when CaCl₂ was removed from the bath () CsCl was increased to 145 mM to maintain the osmolarity of the 0 Na⁺ recording solution (). Cells were dialyzed with an intracellular (pipette) solution containing (in mM): 125 CsCl, 20 NaCl, 10 HEPES, 5 EGTA, 4 MgATP, 0.33 MgCl₂, pH 7.3 with CsOH.
16. Reversal potentials were determined by performing a linear regression of the ascending (linear) portion of each individual I-V and averaging the individual values of E_rev for each solution. The experimentally determined E_rev values equal: 1.3 ± 1.0 mV, n = 5 in 20 Na⁺, 2 Ca²⁺ (); 0.1 ± 0.6 mV, n = 5 in 20 Na⁺, 2 Ca²⁺, 50 µM db-cAMP (); 0.6 ± 0.5 mV, n = 5 in 20 Na⁺, 0 Ca²⁺, 50 µM db-cAMP (); 37.8 ± 1.6 mV, n = 4 in 0 Na⁺, 2 Ca²⁺, 50 µM db-cAMP (). Junction potential offsets for the external recording solutions measured with respect to the internal pipette solution were consistently between +0.8 to +1.2 mV. The calculated junction potentials for all four solutions at 22°C equals +0.7 mV with respect to the pipette solution (AxoScope, Axon Instruments, city, state). When the experimentally determined E_rev values (Fig. 2) are corrected for the measured junction potential offsets, the results equal: +0.1 mV for 20 Na⁺, 2 Ca²⁺ (); 1.3 mV for 20 Na⁺, 2 Ca²⁺, 50 µM db-cAMP (); 1.6 mV for 20 Na⁺, 0 Ca²⁺, 50 µM db-cAMP (); and 37.0 mV for 0 Na⁺, 2 Ca²⁺, 50 µM db-cAMP ().
17. M. F. Sheets, B. E. Scanley, D. A. Hanck, J. C. Makielski, H. A. Fozzard, Biophys. J. 52, 13 (1987) [ISI] [Medline] .
18. A predicted shift in the reversal potential of 5.1 mV was calculated with the use of an explicit solution of E_rev for a channel conducting both divalent and monvalent ions using P_Na = 1 and P_Ca = 1.25. The equation was derived from the Goldman-Hodgkin-Katz constant field equation by D. L. Campbell, W. R. Giles, J. R. Hume, D. Noble, and E. F. Shibata [J. Physiol. 403, 267 (1988)].
19. r1 cDNA was cotransfected with the rat skeletal muscle µ1  subunit cDNA in HEK cells. The midpoint of steady-state inactivation measured per J. R. Balser, H. B. Nuss, D. Romashko, E. Marbán, and G. F. Tomaselli [J. Gen. Physiol. 107, 643 (1996)] shifted from 66.9 ± 1.3 mV (n = 3) to 59.8 ± 0.2 mV (N = 3, p < 0.01), in agreement with S. N. Wright, S.-Y. Wang, Y.-F. Xiao, and G. K. Wang [Biophys. J. 76, 233 (1999)]. Current density also increased with 1 coexpression consistent with H. B. Nuss, N. Chiamvimonvat, M. T. Pérez-García, G. F. Tomaselli, and E. Marbán [J. Gen. Physiol. 106, 1171 (1995)]; and T. Yamagishi, G. F. Tomaselli, and E. Marbán, unpublished data.
20. J. S. Cruz, L. F. Santana, W. J. Lederer, Biophys. J. 76, A196 (1999) .
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22. E. Perez-Reves, W. Yuan, X. Wei, D. M. Bers, FEBS Lett. 342, 119 (1994) [CrossRef] [ISI] [Medline] ; K. W. Roche, R. J. O'Brien, A. L. Mammen, J. Bernhardt, R. L. Huganir, Neuron 16, 1179 (1996) [CrossRef] [ISI] [Medline] .
23. S. Berjukow, et al., Br. J. Pharmacol. 118, 748 (1996) [ISI] [Medline] .
24. A. E. Lacerda, E. Perez-Reyes, X. Wei, A. Castellano, A. M. Brown, Biophys. J. 66, 1833 (1994) [ISI] [Medline] .
25. G. Zhu, Y. Zhang, H. Xu, C. Jiang, J. Neurosci. Methods 81, 73 (1998) [CrossRef] [ISI] [Medline] ; M. D. Ashen, B. O'Rourke, K. A. Kluge, D. C. Johns, G. F. Tomaselli, Am. J. Physiol. 268, H506 (1995) [Abstract/Free Full Text] .
26. P. H. Lalik, D. S. Krafte, W. A. Volberg, R. B. Ciccarelli, Am. J. Physiol. 264, C803 (1993) [Abstract/Free Full Text] ; R. Skryma, N. Prevarskaya, P. Vacher, B. Dufy, ibid. 267, C544 (1994) [Abstract/Free Full Text].
27. D. S. Krafte, et al., J. Mol. Cell. Cardiol. 27, 823 (1995) [CrossRef] [ISI] [Medline] . Krafte et al. found that TTX blocked hH1 Na⁺ channels stably expressed in CHO-K1 cells with an IC50 of 2.5 µM.
28. 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.

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 (I_Ca(TTX)), also seen in rat ventricular myocytes (3). I_Ca(TTX) is generated by Na⁺ channels that are functionally distinct from those generating the classical cardiac Na⁺ current (I_Na). The question arises, then, of whether the induced Ca²⁺ current observed by Santana et al. might be identified with I_Ca(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 I_Ca(TTX) channels, and I_Ca(TTX) recorded in the absence of these agents arises from some basal pool of modified classical Na⁺ channels.

I_Ca(TTX) channels are unlikely to arise from PKA-dependent conversion of classical Na⁺ channels, because a substantial increase in I_Ca(TTX) produced by ISO was not accompanied by a reduction in I_Na, as would be required. Moreover, we find all of the TTX-blockable Ca²⁺ current seen in the presence of ISO flows through I_Ca(TTX) and not through classical Na⁺ channels. The enhanced TTX-blockable Ca²⁺ current seen in ISO must be attributed to an increase in I_Ca(TTX) and not to the induction of Ca²⁺ permeability in classical Na⁺ channels.

Because the inactivation kinetics of I_Ca(TTX) are slower than those of I_Na (2), the conversion of any significant fraction of classical Na⁺ channels into I_Ca(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 I_Na and I_Ca(TTX) or with an I_Ca(TTX) component that remains small relative to I_Na, but not with the simple conversion of I_Na into I_Ca(TTX) channels, because _h is slower for I_Ca(TTX). The mean values of _h for I_Ca(TTX) relative to that for I_Na 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 I_Ca(TTX) channels. Therefore, it is unlikely that I_Ca(TTX) channels are just phosphorylated I_Na channels. Moreover, nearly all of the increase in current must be of I_Na, because I_Na at these potentials is considerably larger than I_Ca(TTX) (2).

ISO also increased I_Ca(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 I_Ca(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, I_Ca(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 I_Ca(TTX). They apparently did not attempt to identify which channel type generated the TTX-blockable Ca²⁺ current.

ISO substantially increases the classical I_Na 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

1. L. F. Santana, A. M. Gómez, W. J. Lederer, Science 279, 1027 (1998) .
2. R. Aggarwal, S. R. Shorofsky, L. Goldman, C. W. Balke, J. Physiol. 505, 353 (1997) [Abstract/Free Full Text].
3. 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.
4. 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.
5. 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].

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 (P_Ca) could increase relative to Na⁺ permeability (P_Na) so that the permeability ratio (P_Ca/P_Na) 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 I_Na 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 I_Na in heart cells under conditions that made it possible to measure this current. Low [Na⁺] at cool temperatures kept I_Na 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 I_Na reversal potential, E_rev, of 9 mV to 10 mV, when slip-mode conductance was activated. This shift in E_rev suggested that an increase of P_Ca/P_Na 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 I_Na 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. I_Na 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 E_rev would be expected when PKA is activated. However under all conditions tested, E_rev remained at the Na⁺ equilibrium potential (E_Na), which was 0 mV under control conditions and 0.63 mV in Na⁺- dbcAMP (10). A small increase in I_Na magnitude following the addition of dbcAMP is consistent with published results (9), as is the block of I_Na by TTX (10 µM).

Under the ionic conditions of these experiments, an increase in P_Ca/P_Na would have led to inward current at E_Na and a positive shift in E_rev (10). Because neither was observed, we deduced that there was no significant change in P_Ca/P_Na (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 P_Ca/P_Na 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.

Table 1. Modulation of PCa/PNa. Control PKA activation Comment Conditions ENa (mV) Erev (mV) PCa/PNa ENa (mV) Erev (mV) PCa/PNa 1  : Nai=Nao=10 mM; Cao=2 mM; Cai=0; Monovalent replacement: Cs+ 0 0  0 0 +9 1.2 (2 Ca2+) Ref. (6). 2   in HEK293 cells; Nai=20 mM; Na0=20 mM (control); Nao=20.5 mM (dbcAMP); Cao=2 mM; Cai=100 nM; Monovalent replacement: Cs+ 0  0.23  0 0.63 0.82 0.04 Fig. 1A. 3   in HEK293 cells; Nai=Nao=20 mM (control); Nao=20.5 mM (dbcAMP); Cao=2 mM; Cai=100 nM; Monovalent replacement: Cs+ 0 0.18 0.04 0.63 4.00 0.79 Fig. 1B. 4   in HEK293 cells; Nai=Nao=20 mM (control); Nao=20.5 mM (dbcAMP); Cao=2 mM; Cai=100 nM; Monovalent replacement: Cs+ 0  0.02  0 0.63  0.05  0 Fig. 1C. 5   in HEK293 cells. ; Nai=Nao=10 mM; Cao=5 mM; Cai=0 nM; Monovalent replacement: Cs+; 10 mM TEA 0 0.94 0.04 1.2 7.18 0.32 Fig. 2B 6   in HEK293 cells. Nai=10 mM; Na0=0.5 mM (dbcAMP); Cao=5 mM; Cai=0 nM; Monovalent replacement: Cs+; 10 mM TEA  79 n.d. n.d.  76.2  37  0.11 Fig. 2B 7   in HEK293 cells. Nai=20 mM (control); Nao=20.5 mM (dbcAMP); Cao=2 mM; Cai=100 nM; Monovalent replacement: Cs+; 10 mM TEA 0 0.01  0 0.63 5.0 1.1 Fig. 2G 8   in HEK293 cells; Nai=Nao=20 mM (control); Nao=20.5 mM (dbcAMP); Cao=2 mM; Cai=100 nM; Monovalent replacement: NMG+. 0 0.53 0.11 0.63 6.35 1.48 Fig. 3D n.d., experiment not done.

We measured the current-voltage (IV) relationships for I_Na in HEK293 cells that co-express  and ₁ subunits of the Na⁺ channels under several experimental conditions (Fig. 1B). Under control conditions, E_rev = 0.23 mV, a value statistically indistinguishable from E_Na of 0 mV. After the addition of 500 µM Na⁺-dbcAMP, a significant increase of E_rev was observed (E_rev = 4.00 mV, P < 0.0001, n = 8). This increase in E_rev suggests that P_Ca/P_Na 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 E_rev 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 E_rev following the addition of dbcAMP. In the maintained presence of dbcAMP and with 2 mM [Ca²⁺]_o , TTX significantly reduced I_Na and also produced a significant negative shift in E_rev 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 E_rev back to E_Na. [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 I_Na 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 E_rev 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 E_rev of more than 7 mV, more than was observed in Fig. 1. Unexpectedly, however, P_Ca/P_Na 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 P_Ca/P_Na of 0.95. Thus, although extracellular Ca²⁺ permeates the Na⁺ channel during slip-mode conductance, it also tends to block the conductance.

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 I_Na 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 I_Na, including any component carried by Ca²⁺. This observation was the second unexpected finding in these experiments (Figs. 2A and B). The I_Na, IV plot shows that outward currents began to appear at potentials positive to 37 mV, which suggested that the "reversal potential" for I_Na was 37 mV or more negative. This suggests that under these conditions P_Ca/P_Na 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 P_Ca/P_Na . 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 P_Ca/P_Na during slip-mode conductance should lead to a measured shift in E_rev (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 E_rev. 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 I_Na 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 I_Na (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 I_Na 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 I_na (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, I_Na had a reversal potential at E_Na before slip-mode was activated by PKA (Fig. 2G). After slip-mode had been activated, E_rev moved towards E_Ca by 5.0 mV, which was consistent with an increase in P_Ca/P_Na from 0 to 1.1, a change comparable to the increase in P_Ca/P_Na seen in rat heart cells (P_Ca/P_Na = 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 I_Na better and more reliably than the other subunit combinations we tested. Following PKA activation by dbcAMP, there was a decrease in the peak I_Na 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 (P_NMG/P_Na < 0.03); there is no significant change in E_rev (Fig. 3A). Under control conditions P_Cs/P_Na 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 I_Na 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 I_Na 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 I_Na was reduced by 58.2 ± 16% (p < 0.05, n = 4) following the application of dbcAMP. In these experiments E_Na, the expected E_rev for I_Na, = 59 mV if there is no permeation by K⁺ through Na⁺ channels. We found that under control conditions E_rev = 36.75 ± 0.78 mV (n = 4) indicating P_K/P_Na was about 0.25 under these ionic conditions. Following PKA activation E_rev shifted to 27.73 ± 4.31 mV (p < 0.05, n = 4), which indicated that P_K/P_Na 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.

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⁺, E_rev 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 P_Ca/P_Na = 1.48. (inset in Fig. 3D). Although a large increase in P_Ca/P_Na was observed, there was a reduction of peak I_Na produced by dbcAMP, similar to that seen in Fig. 2G. These results provide additional evidence that specific ionic conditions influence I_Na and PKA-induced changes in P_Ca/P_Na of cardiac Na⁺ channels. In contrast, when Ba²⁺ was used to replace Ca²⁺ in the extracellular solution, no change in E_rev was observed following activation of PKA. E_rev shifts slightly from 0.83 ± 0.27 mV to 0.65 ± 0.4 mV (p = n.s., n = 6). This result suggested that P_Ba/P_Na 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 I_Ca 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 I_Na. 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 E_rev 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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7. 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 h1 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 37^o 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 I_Na (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] ; L. F. Santana, A. M. Gómez, W. J. Lederer, Science 279, 1027 (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 I_Na (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 I_Na 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.
8. I_Na attributed to the  subunit alone did, however, appear to be slowed kinetically compared to the native I_Na . But it is still not clear exactly what the ₁ subunit does. For example, although slow inactivation of I_Na 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, 1171 (1995) [Abstract/Free Full Text] ], rapid inactivation of I_Na 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 I_Na following the co-expression of the ₁ subunit to the  subunit in Xenopus oocytes [ A. Toib, V. Lyakhov, S. Marom, J. Neurosci. 18, 1893 (1998) [Abstract/Free Full Text] ; 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 I_Na 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 I_Na [ 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).
9. I_Na 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, J. Physiol. 498, 309 (1997) [Abstract/Free Full Text]] or when intact rabbit heart cells were exposed to isoproterenol [ J. J. Matsuda, H. Lee, E. F. Shibata, Circ. Res. 70, 199 (1992) ] but I_Na was found to decrease in neonatal rat heart cells [ B. Schubert, A. M. J. VanDongen, G. E. Kirsch, A. M. Brown, Science 245, 516 (1989) [Abstract/Free Full Text] ; B. Schubert, A. M. VanDongen, G. E. Kirsch, A. M. Brown, Am. J. Physiol. 258, H977 (1990) [Abstract/Free Full Text] ].
10. To calculate relative permeability of ions through channels, reversal potential (E_rev) (i.e. the zero-current potential) measurement are made under different ionic conditions. Our experiments presented here use E_rev to calculate the relative permeability of Ca²⁺ (P_Ca) to Na⁺ (P_Na) 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, J. Physiol. 403, 267 (1988) [Abstract/Free Full Text]]; this solution is equivalent to that of C. A. Lewis [ibid. 286, 407 (1979)]. The reversal potential (E_rev) for a channel with two ions that are permeant must lie at or between the equilibrium potentials for the two ions (here E_Na and E_Ca). Since we were examining possible changes in P_Ca/P_Na for the Na⁺ channel, an accurate assessment of measured potential was needed relative to E_Na. We opted to use one of two methods to correct for small (<3 mV) systematic errors in the measured potential compared to E_Na: 1. The zero-current potential for I_Na 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 E_Na 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. P_Cs/P_Na and P_NMG/P_Na are close to zero; 2. P_Cs/P_Na and P_NMG/P_Na do not change following application of PKA activators such as dibutyryl cAMP (dbcAMP) (see Fig. 3).
11. A. Grant, V. S. Chauhan, R. Chandra, C. F. Starmer, Biophys. J. 76, A80 (1999) .
12. 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.
13. R. M. Shah, S. R. Levinson, R. A. Maue, Soc. Neurosci. Abstr. (abstr. 525.4) (1998), p. 1323.
14. T. Zimmer, M. Steinbis, T. Böhle. K. Benndorf. Biophys. J. 76, A193 (1999).
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16. 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.
17. R. Horn and A. Marty, J. Gen. Physiol. 92, 145 (1988) .
18. J. R. Berlin, M. A. Wozniak, M. B. Cannell, R. J. Bloch, W. J. Lederer, Cell Calcium 11, 371 (1990) [CrossRef] [ISI] [Medline] .
19. 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).
20. 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.