Technical Comments

Whether "Slip-Mode Conductance" Occurs

Science  30 Apr 1999:
Vol. 284, Issue 5415, pp. 711
DOI: 10.1126/science.284.5415.711a

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.

Figure 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) α 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 α 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 (▴).

These experiments relied on expression of the α subunit alone. Such a test was motivated by the fact that, in rat myocardium, Na+channels consist only of α subunits (12), making it unlikely that β subunits somehow contributed to the original observations of slip mode conductance in rat heart cells. Nevertheless, to exclude the possibility that β subunit coexpression is required, we performed further experiments in CHO cells that coexpress the α and β1 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 inFig. 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 α + β1 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 (hβ1) or rat brain (rβ1) β1 subunits. Confirmation that rβ1 in fact expresses functional subunits was derived from parallel experiments in which the same β1 cDNA increased current amplitude and shifted inactivation when coexpressed with Na channel α 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.

Figure 2

Membrane currents recorded in CHO-K1 cells cotransfected with the human cardiac Na+ channel (hH1) α subunit and β1 subunit cDNA (2, hβ1 and rat brain β1 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 (hβ1) and rat brain (rβ1) β1 subunits. Two (of five) experiments were performed on cell cotransfected with hH1+GFP-Ir-hβ1; three experiments were performed on cells cotransfected with hH1+rβ1+GFP.

Figure 2

Increased PCa/PNa during slip-mode conductance of the cardiac Na+ channel. INa in HEK293 cells expressing both α and β1 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 (α, β1 and β2) 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 α 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 α 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 Δ[Ca2+]iper 100 depolarizing pulses and peak INa. Solid line is given by Δ[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 (τ = 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 β1 and β2 subunits. HEK293 cells were transfected with hH1α, β1 and β2 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 β-mercaptoethanol in panel (i) and were nonreduced in panel (ii) to detect covalent association of α and β2. Panel (i). Protein immunoblots were probed with antipeptide antibodies to β2 (lanes 1 and 2) or β1 (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 β2. Lane 1: rat brain membranes, Lane 2: HEK293 cells. Positions of molecular weight markers are indicated in kDa. Molecular weights for β1, β2 and α -β2 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).

Figure 3

Permeation by other ions during slip-mode conductance in HEK293 cells expressing α and β1 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 Erevwas observed (control: 55.4 ± 2.1 mV; dbcAMP: 56.7 ± 1.5 mV, n = 7, p=n.s.). A PNMG/PNaratio < 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/PNaof 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 INawas 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/PNaincreased 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 INain 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.

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 β 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 + β1 + 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 α subunit alone nor α + β1 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.

REFERENCES AND NOTES

Whether "Slip-Mode Conductance" Occurs

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. Santanaet 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 (τh; single exponential fit; Fig. 1B, upper curve). The mean ratio of τh,ISO to τh,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 τh is slower for ICa(TTX). The mean values of τh 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).

Figure 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+ 0on 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 (τh; obtained as a best- fit single exponential) in the presence to that in the absence of 1 μM ISO (τh,ISOh,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.

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 τh,ISO to τh,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).

REFERENCES AND NOTES

Whether "Slip-Mode Conductance" Occurs

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 INaand 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 α 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, β1 and β2. Because of the importance of the α subunit, we examined it in HEK293 cells first. INa occurred when only the α subunit of the human isoform of the cardiac Na+channel (hH1α) was expressed in HEK293 cells (Fig. 1A). If slip-mode conductance of the Na+ channel could be produced when only the α subunit was expressed, then a shift of Erev would be expected when PKA is activated. However under all conditions tested, Erevremained 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).

Figure 1

Expression of human heart Na+ channels in HEK293 cells. (A) α subunit only. Membrane current (right) from a single HEK293 cell transfected with the hH1 α 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 Erevwas 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 α and β1 subunits. INa was measured in HEK293 cells expressing both α and β1 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 Erevfrom 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 ΔErevindicates an increase from about 0.04 (control) to 0.79 (dbcAMP). PCa/PNa was calculated from Campbellet 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 α and β1 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) β1only. Averaged INa IV relationships (n = 8) for HEK293 cells expressing only β1 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+).

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 α subunit was expressed (see also Table 1). Because intact heart cells express α and both β subunits (12) and recent evidence suggests that both α and β1 are associated with each other in heart (13) and in heterologous expression systems (14), we examined HEK293 cells that co-express α and β1 subunits.

Table 21

Modulation of PCa/PNa.

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We measured the current-voltage (IV) relationships for INain HEK293 cells that co-express α and β1 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 α and β1 subunits are important for proper Na+ channel function and provides a functional reason role for the β1 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 β1 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 α subunit alone is not enough (Fig. 1) to support this change in selectivity. Instead, it was found that the co-expression of an α Na+ channel subunit along with a β subunit was required for us to observe slip-mode conductance of the Na+ channel in an heterologous expression system. The α 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 α subunit is altered to produce slip-mode conductance. Also we do not know what the β1 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 α and β1 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.

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 α and β, 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 hH1α and β1 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 Balkeet 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: hH1α, the universal β1 subunit, and a β2 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 α and β2 were co-expressed (16). Our overall experience with cardiac Na+ channels suggests that the triple transfection (α, β1, β2) 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 α 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 α, β1 and β2 subunits but not if they expressed the α 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 Δ[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+]ifell towards the prestimulation level (τ = 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 ECaby 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 β1 and β2 subunits were expressed in these HEK293 cells following the triple transfection. β2associated with the α subunit following the triple transfection [Fig. 2H (ii)] (19). The β1 dissociates from α under these preparative conditions because, unlike β2, 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 α and β1subunits. 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.

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. Erevshifts 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 α subunit (hH1α) was expressed along with either the β1 subunit or the β2 subunit, or both. All three combinations of α subunit plus β subunits could produce slip-mode conductance. However, neither α nor β1 nor β2 subunit alone was seen to produce PKA-dependent Ca2+ permeation of the Na+channel. We speculate that it is the α 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 β1 or β2 subunits (or both subunits). We surmise that the β subunits enable the α 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 α subunit function, or help the α subunit associate with other important proteins. Co-transfection of α and one of the β 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 β subunits, with no previously identified function in heart, are necessary to support this conductance mode of the α 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+.

  • * Contributed equally.

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