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(Circulation Research. 2003;92:950.)
© 2003 American Heart Association, Inc.

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Cardiac Submembrane [Na⁺] Transients Sensed by Na⁺-Ca²⁺ Exchange Current

Christopher R. Weber, Kenneth S. Ginsburg, Donald M. Bers

From the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Donald M. Bers, PhD, Department of Physiology, Loyola University Chicago, 2160 South First Ave, Maywood, IL 60153. E-mail dbers{at}lumc.edu

Abstract

Na⁺ influx via I_Na during cardiac action potentials can raise bulk [Na⁺]_i by 10 to 15 µmol/L. However, larger rises in submembrane [Na⁺] ([Na⁺]_sm) local to Na⁺-Ca²⁺ exchangers (NCX) could enhance Ca²⁺ influx via NCX (and Ca²⁺-induced Ca²⁺ release). We tested whether I_Na could increase [Na⁺]_sm, using NCX current (I_NCX) as a biosensor in rabbit ventricular myocytes (with [Ca²⁺]_i buffered, [Na⁺]_i=10 mmol/L, and other currents blocked). We measured I_NCX as early as 5 ms after I_Na. Prior I_Na activation did not affect I_NCX at physiological membrane potentials (E_m=-100 to +50 mV), but for E_m >+50 mV (where I_NCX is especially sensitive to [Na⁺]_i), I_NCX shifted outward. At 5 ms and +100 mV, I_Na shifted I_NCX outward by 0.23 A/F (corresponding to [Na⁺]_sm=0.24 mmol/L). The effect of I_Na dissipated with a time constant of 15 ms. Thus, the impact of I_Na on NCX is almost undetectable at physiological E_m and short lived. This suggests that I_Na effects on excitation-contraction coupling (via outward I_NCX) are minimal and limited to early during the action potential. However, local [Na⁺]_sm during I_Na may be 60 times higher than bulk [Na⁺]_i.

Key Words: Na⁺ current • excitation-contraction coupling

Cardiac Na⁺-Ca²⁺ exchange (NCX) is reversible with a 3:1 stoichiometry¹ (but see References ² and ³). Net charge flows in the direction of Na⁺ transport, generating current (I_NCX) with an electrochemical reversal potential (E_NCX=3E_Na-2E_Ca). NCX current (I_NCX) direction and amplitude depend on internal and external [Ca²⁺] and [Na⁺] and membrane potential (E_m).⁴ However, these change dynamically during the action potential (AP), and bulk cytosolic [Ca²⁺]_i and [Na⁺]_i may differ from the submembrane [Ca²⁺] and [Na⁺] sensed by the NCX ([Ca²⁺]_sm and [Na⁺]_sm).

During the AP with excitation-contraction coupling intact, [Ca²⁺]_sm rises earlier and is 3 times higher than bulk [Ca²⁺]_i, limiting Ca²⁺ influx via outward I_NCX early in the AP.⁵ But how I_Na alters [Na⁺]_sm during the AP is unknown, making its influence on I_NCX during the AP unclear.

Na⁺ current (I_Na) can evoke sarcoplasmic reticulum (SR) Ca²⁺ release even with Ca²⁺ current (I_Ca) blocked.⁶ Although Na⁺ influx via I_Na is only expected to raise global [Na⁺]_i by 10 to 15 µmol/L (0.1%), locally elevated [Na⁺]_sm could shift NCX, promoting enough Ca²⁺ influx via outward I_NCX to trigger Ca²⁺ release. Others have obtained similar data,⁷ although these findings and conclusions are controversial.^8,9

Acute Na⁺/K⁺-ATPase blockade can enhance SR Ca²⁺ release¹⁰ and retard [Ca²⁺]_i decline¹¹ (without altering bulk [Na⁺]_i). It was inferred that [Na⁺]_sm was elevated for >100 ms and that Na⁺/K⁺-ATPase and NCX colocalize. However, I_Na activation did not activate Na⁺/K⁺-ATPase current in guinea pig myocytes, even as early as 10 ms.¹²

In this study we measured whether I_Na alters [Na⁺]_sm sensed by I_NCX in rabbit ventricular myocytes. Under voltage clamp, with heavy [Ca²⁺] buffering and I_Ca blockade (to limit [Ca²⁺]_sm changes), we recorded I_NCX immediately after I_Na. Within 5 ms of I_Na, the measured I_NCX was only altered by I_Na at E_m 50 mV. We infer that [Na⁺]_sm may rise by 0.25 mmol/L during I_Na but dissipates rapidly. Thus, I_Na may augment Ca²⁺ entry via NCX, but the effect appears small.

Materials and Methods

Rabbit myocytes were isolated, and current was recorded using whole-cell ruptured patch clamp, as previously described (at 36°C).⁵ Patch electrodes (1 to 3 M) contained (in mmol/L) CsCl 40, Cs-glutamate 80, MgCl₂ 0.92, HEPES 10, NaCl 10, MgATP 5, LiGTP 0.3, Cs₄-BAPTA 5, and Br₂-BAPTA 1, with pH set to 7.2 using CsOH (free [Ca²⁺]=100 nmol/L). Cells were pretreated with 0.1 µmol/L thapsigargin to block SR Ca²⁺ transport. Bath solution contained (in mmol/L) NaCl 140, CsCl 4, CaCl₂ 2, glucose 10, HEPES 5, and MgCl₂ 1, pH 7.4, and included 30 µmol/L niflumate, 20 µmol/L nifedipine, and 4 µmol/L N-acetylstrophanthidin to block Ca²⁺-activated Cl current, I_Ca, and Na⁺/K⁺-ATPase. I_NCX was taken as current blocked by 10 mmol/L Ni. Holding E_m (E_hold) was -53, the predicted E_NCX. Access resistance was 3.4±0.37 M, with =635±62 µs and C_m=191±18 pF (n=7). I_Na was activated for 5 ms at -30 mV (just long enough to ensure full I_Na decay). To optimize I_Na control, series R compensation (>70%) with capacitance transient cancellation was applied.

We used our previously characterized equation^5,13 to relate I_NCX to [Na⁺]_o, [Na⁺]_i, [Ca²⁺]_o, [Ca²⁺]_i, and E_m to infer the [Na⁺]_i and V_max in these cells (8.8±0.4 mmol/L, 35.5±6.2 A/F). This also allowed calculation of expected shifts of I_NCX for a given [Na⁺]_i. Data reported are ±SEM.

Results

In the control protocol (Figure 1A), I_Na was unavailable (E_hold=-53), so no I_Na occurred on step to E_m=-30 mV for 5 ms. The I_NCX traces characterize the E_m dependence of I_NCX (without I_Na). With a prepulse to E_m=-120 mV (Figure 1B; allowing I_Na recovery), the step to -30 mV activated large I_Na. Difference currents (I_NCX, Figure 1C) show that I_Na had little effect on I_NCX.

View larger version (28K): [in this window] [in a new window]   Figure 1. INCX with or without prior INa activation. Em protocol (top) used to measure INCX (bottom) without (A) or with (B) INa. C, Difference (A and B) currents, starting from the step to -30 mV.

Because there was little difference, even at E_m=+50 mV, we went beyond physiological E_m, up to +100 mV (where I_NCX is especially [Na⁺]_i sensitive).¹³ Figure 2A shows average I_NCX versus E_m during the 5- to 10-ms period after the -30 mV pulse. Figure 2B shows average I_Na-induced I_NCX (from Figure 2A). The I_NCX for the 5- to 10-ms time window was unaltered for E_m <+50 mV, and the same was true at all E_m for the 95- to 100-ms time window. At E_m 50 mV, there was a transient effect of I_Na to increase outward I_NCX. Dotted lines show shifts in I_NCX expected for a [Na⁺]_i of 0.23 and 0.5 mmol/L. The I_Na-dependent [Na⁺]_sm required to explain the I_NCX is 0.22 to 0.23 mmol/L (at 80 to 100 mV). Because [Na⁺]_sm converges to 0.23 mmol/L at the most positive E_m (where I_NCX is most measurable), we accept this value. The slight inward I_NCX at more negative E_m is not an expected effect of I_Na on I_NCX, and its basis is not obvious.

View larger version (25K): [in this window] [in a new window]   Figure 2. INCX versus Em. A, INCX (Ni2+-sensitive) without INa (blue) and after INa (red). B, Average INCX at 5 to 10 ms was INa-dependent at Em >+50 mV but decayed by 95 to 100 ms. Dotted lines are expected INCX for indicated [Na+]sm (using our equation5 and Vmax obtained from fit of average I-V relationship from all cells, panel A, blue).5,13 C, Average INCX (versus 95 to 100 ms) induced by INa at +50 (n=7) and +100 mV (n=4). Monoexponential declines and calculated [Na+]sm are indicated. Data are average from 7 myocytes and 5 hearts.

Figure 2C shows the transient outward I_NCX component induced by I_Na at +50 and +100 mV (because the increased [Na⁺]_sm decayed by 95 to 100 ms). These difference currents were fit to single exponentials (+100 mV: =16.3±4.6 ms, n=4; +50 mV: =14.7±6.2 ms, n=7). Mean [Na⁺]_i corresponding to peak I_NCX are indicated, suggesting a transient [Na⁺]_sm elevation of 0.24 mmol/L, which decayed with a time constant of 15 ms. Extrapolating back to the time of peak I_Na indicates a mean [Na⁺]_sm of 0.44 mmol/L for +100 mV. The transient I_NCX was inhibited by 30 µmol/L tetrodotoxin, confirming I_Na as the source of [Na⁺]_sm.

Discussion

Here we show that I_NCX is sensitive to local changes in [Na⁺]_sm induced by I_Na and inferred the local [Na⁺]_sm change. At E_m 50 mV, I_NCX transiently shifted outward, consistent with 0.25 mmol/L increase in [Na⁺]_sm within 5 ms of I_Na. The elevated [Na⁺]_sm decayed with 15 ms and was undetectable by 50 ms after I_Na (Figure 2C).

Currents as Sensors of Submembrane Ion Concentrations
Ion currents have been used to sense [Ca²⁺]_sm near Ca²⁺-activated chloride channels,¹⁴ Ca²⁺ channels,¹⁵ and NCX.^5,16 Our experiments were similar to those of Silverman et al,¹² who were unable to detect [Na⁺]_sm changes near Na⁺/K⁺-ATPase by measuring Na⁺ pump currents 10 ms after I_Na. However, at E_m to 50 mV, we also failed to detect I_Na-dependent [Na⁺]_sm elevation. Thus, I_Na-induced perturbations in Na⁺/K⁺-ATPase or I_NCX may be near the detection threshold.

Sources of Error
Our average peak I_Na (10.3±2.9 nA) was smaller than expected in intact myocytes (50 nA).⁴ We probably did not achieve full I_Na availability, but greater I_Na would have complicated voltage clamping, and longer time at E_m=-120 mV could alter [Ca²⁺]_sm. Thus, physiological peak [Na⁺]_sm could be 1 to 2 mmol/L. Offsetting factors where we may overestimate [Na⁺]_sm unphysiologically are that I_Na driving force is greater at E_m=-30 mV than during an AP and Na⁺/K⁺-ATPase was blocked.^10,11

Na⁺ Fuzzy Space: Size, Colocalization, and Excitation-Contraction Coupling
Although [Na⁺]_sm may rise 0.25 mmol/L 5 ms after I_Na, NCX molecules nearer Na⁺ channels would sense peak [Na⁺]_sm earlier and higher. Nevertheless, let us consider a discrete submembrane volume. With our protocol, average integrated I_Na raises global [Na⁺]_i by 3.9 µmol/L cytosol (using 6.4 pF/pL cytosol).⁴ Thus, [Na⁺]_sm is 60 times the expected global [Na⁺]_i. Inversely, this implies a submembrane volume of 1.6% of cytosolic volume or 1% of total cell volume, which exceeds dyadic cleft volume (0.1%).⁴ This agrees with ultrastructural data indicating that neither NCX nor Na⁺ channels are at dyadic clefts (but are in T-tubules).¹⁷ Moreover, NCX and Na⁺ channels may not colocalize¹⁷ yet interact in a diffuse subsarcolemmal volume (ie, 0.5 µm² surface/µm³ cellx0.02 µm deep=1%).⁴

In contrast, Ca²⁺ channels do colocalize with ryanodine receptors at junctional clefts.¹⁷ This gives I_Ca an advantage (versus outward I_NCX) in activating SR Ca²⁺ release (by 4-fold¹⁸). In rabbit ventricle, increasing [Na⁺]_i enhances I_Ca trigger efficacy and contraction at E_m >+40 mV.¹⁹

The small [Na⁺]_sm detected here could work synergistically with I_Ca-dependent [Ca²⁺]_sm elevation in activating I_NCX, but high [Ca²⁺]_sm would also shift I_NCX inward (extruding Ca²⁺).^4,5 The true impact of the I_Na-dependent [Na⁺]_sm reported here on excitation-contraction coupling is complicated. Small [Na⁺]_sm could have disproportionate effects on SR Ca²⁺ release.^6,7,10,19

The effects of I_Na on I_NCX are barely detectable at the most positive physiological E_m values (+50 mV), but I_Na may transiently elevate [Na⁺]_sm by 0.25 to 1 mmol/L (for 15 to 30 ms). Thus, I_Na may slightly enhance Ca²⁺ entry via NCX at very positive E_m early in the AP but has little effect at lower E_m or later in the AP.

Acknowledgments

This work was supported by NIH-HL30077 (to D.M.B.) and American Heart Association predoctoral fellowship 0010180Z (to C.R.W.).

Received February 13, 2003; revision received March 14, 2003; accepted April 3, 2003.

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This Article Abstract Full Text (PDF) All Versions of this Article: 92/9/950    most recent 01.RES.0000071747.61468.7Fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weber, C. R. Articles by Bers, D. M. Search for Related Content PubMed PubMed Citation Articles by Weber, C. R. Articles by Bers, D. M. Related Collections Calcium cycling/excitation-contraction coupling Ion channels/membrane transport