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(Circulation Research. 1995;77:950.)
© 1995 American Heart Association, Inc.

Articles

Description of a Nonselective Cation Current in Human Atrium

William J. Crumb, Jr, John D. Pigott, Craig W. Clarkson

From the Departments of Pharmacology (W.J.C., C.W.C.), Pediatrics (W.J.C.), and Surgery (J.D.P.), Tulane University School of Medicine, New Orleans, La.

Correspondence to Dr William J. Crumb, Jr, Department of Pediatrics, Division of Cardiology #SL37, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112-2699.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Ion currents were examined in isolated human atrial myocytes by using the whole-cell patch-clamp technique. When currents were recorded with a K⁺-containing pipette solution, depolarizing voltage pulses elicited a rapidly activating outward current that decayed to an apparent steady state. Exposure of cells to 10 mmol/L 4-aminopyridine markedly reduced current amplitude; however, a rapidly activating current that was 30% of the steady state current amplitude remained. When pipette K⁺ was replaced with Cs⁺, a similar rapidly activating current that reversed polarity at 0 mV was recorded. This current was seen in 100% of the cells tested from 17 different hearts (n=142), and its amplitude was 40% of the amplitude of the steady state current recorded in the presence of pipette K⁺. The current amplitude was not significantly different in cells isolated from adult (6.31±1.35 pA/pF, n=8) and pediatric (5.54±1.04 pA/pF, n=9) hearts. Studies designed to determine the charge-carrying species indicated that changes in bath Cl^- concentration had no effect on either the amplitude or the reversal potential of this current, whereas removal of pipette Cs⁺ and bath Na⁺ dramatically reduced this current. In addition, this current was not modulated by either isoproterenol (1 µmol/L, 22°C) or cell swelling. This study provides the first description of a nonselective cation current in human atrial myocytes, which may play an important role in repolarization in human atria.

Key Words: humans • atrial myocytes • cation • Cs⁺ • electrophysiology

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Repolarization in cardiac tissue is due in part to an efflux of K⁺ ions through a variety of K⁺ channels that each display different conductances and kinetic properties.^{1 2 3 4 5} Interestingly, the type of K⁺ currents found in cardiac tissue is species dependent.^{4 5 6} Although much is known about the currents involved in repolarization of the cardiac action potential in a number of different animal species,^{7 8 9} much less is known about the currents involved in repolarization in human myocardium. At present, I_to,^{10 11 12 13 14} I_K1,¹³ a delayed rectifier K⁺ current,¹⁵ and an ultra–rapidly activating K⁺ current^{5 16} have been identified in human atrial myocytes. In the present study, we report the first description of a nonselective cation current in isolated human atrial myocytes that is carried by both Na⁺ and K⁺ under physiological conditions.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human myocytes were isolated from specimens of human right atrial appendage obtained, in accordance with Tulane University School of Medicine Institutional guidelines, during surgery from the hearts of 17 patients undergoing cardiopulmonary bypass. Myocytes were obtained from six neonates (age, 1 to 14 days), six infants (age, 1 to 16 months), and five adult patients (age, 45 to 63 years). All atrial specimens were described as grossly normal at the time of excision, and all patients had normal P waves on electrocardiography. None of the pediatric patients had received Ca²⁺ channel blocking agents or antiarrhythmic agents. Some adult patients had received cardioactive drugs, including Ca²⁺ channel blockers, digitalis, and adrenergic blocking agents. The cell isolation procedure was similar to that described by Fermini et al¹⁴ and is described in detail by Crumb et al.¹³

Isolated human atrial myocytes were superfused with an "external" solution that consisted of (mmol/L) NaCl 137, CsCl 4, MgCl₂ 1, CaCl₂ 1.8, glucose 11, and HEPES 10, adjusted to a pH of 7.4 with NaOH. In some experiments, the Cl^- concentration of the bath solution was reduced by replacing all NaCl with sodium isethionate. Glass pipettes (electrodes) were filled with an "internal" solution that consisted of (mmol/L) CsCl 140, Na₂-ATP 4, MgCl₂ 1, EGTA 5, and HEPES 5, adjusted to a pH of 7.2 with CsOH. In one series of experiments, all pipette Cs⁺ was replaced with TEA. Experiments were performed in the presence of 200 µmol/L Cd²⁺, 1 mmol/L Ba²⁺, and 1 µmol/L tetrodotoxin to block Ca²⁺, inward rectifier K⁺, and Na⁺ channels, respectively. In some experiments, either 100 nmol/L or 10 µmol/L nisoldipine instead of Cd²⁺ was added to the bath solution to block L-type Ca²⁺ channels. In addition, in one series of experiments the effects of Ba²⁺ (1.8 mmol/L) were examined. For experiments performed in the presence of K⁺, cells were perfused with an "external" solution that consisted of (mmol/L) NaCl 137, KCl 4, MgCl₂ 1, CaCl₂ 1.8, glucose 11, and HEPES 10, adjusted to a pH of 7.4 with NaOH. Glass pipettes were filled with an "internal" solution that consisted of (mmol/L) potassium aspartate 120, KCl 20, Na₂-ATP 4, EGTA 5, and HEPES 5, adjusted to a pH of 7.2 with KOH. Experiments were performed in the presence of 200 µmol/L Cd²⁺ and 1 mmol/L Ba²⁺. All experiments were performed at room temperature (22°C to 23°C).

Acceptable atrial myocytes were rod-shaped and lacked any visible blebs on the surface. Currents were measured by using the whole-cell variant of the patch-clamp method.¹⁷ Pipette tip resistance was 1.0 to 2.0 M when the pipettes were filled with the internal solution. Seal resistances were 10 G. Analog capacity compensation and 40% to 60% series resistance compensation was used in all experiments to yield voltage drops across uncompensated series resistance of <3 mV. Mean cell capacitance was 28.91±1.09 pF for neonatal myocytes (n=31) and 64.85±4.68 pF for adult myocytes (n=27). Liquid junction potentials resulting from the substitution of Cl^- were typically 2 to 3 mV and were not adjusted. Currents were not leak-subtracted. An unpaired Student’s t test was used for statistical analysis. Data are presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of 4-AP on Sustained Current
We initially sought to examine the properties of I_sus recorded in isolated human atrial myocytes.¹⁶ This current remaining after the decay of I_to has recently been attributed to a highly 4-AP–sensitive (IC₅₀, 50 µmol/L) Kv1.5-like current.^{5 16} Therefore, we began our study of I_sus by performing experiments to construct a concentration-response curve. Recordings were made in the presence of pipette and bath K⁺ (see "Materials and Methods"). Currents were elicited during depolarizing steps from a holding potential of -40 mV to inactivate the Na⁺ current, and both the L-type Ca²⁺ current and I_K1 were blocked by the addition of 200 µmol/L Cd²⁺ and 1 mmol/L Ba²⁺ to the external solution.^{13 16} As illustrated in Fig 1A, in the absence of drug (control), voltage pulses to +60 mV elicited a rapidly activating current (I_to), which decayed to an apparent steady state over the time course of a 500-millisecond pulse. When this first pulse was rapidly followed by a second depolarizing voltage pulse to +60 mV, only a rapidly activating noninactivating outward current (I_sus) could be elicited, indicating that the interpulse interval (5 milliseconds) was too brief for I_to to recover from inactivation.¹³ After the addition of 4-AP (10 mmol/L) to the bath solution, I_to was virtually abolished while I_sus was reduced by 50%. Fig 1B shows mean concentration-response data for 4-AP inhibition of I_sus. The solid line represents a best fit of a Hill equation. The IC₅₀ for the noninactivating current was 32.6 µmol/L, and the calculated value for the Hill coefficient was 0.9, values very similar to those previously reported for 4-AP block of I_sus (49 µmol/L and 1.45, respectively).¹⁶ The concentration-response relation, however, reached a plateau at 1 mmol/L, and no further reduction in the amplitude of I_sus was observed with concentrations of 4-AP as high as 10 mmol/L. The mean reduction in I_sus (measured during the second voltage pulse of a two-pulse protocol) following exposure to 10 mmol/L 4-AP was 70.4±6.27% (n=5). This is in marked contrast to previous reports in which the noninactivating current measured after the decay of I_to was completely abolished by 2 mmol/L 4-AP.¹⁶

View larger version (14K): [in this window] [in a new window]   Figure 1. Effects of 4-AP on isolated human atrial myocytes. Currents were elicited by voltage protocol shown in inset. A, Currents recorded before (control) and 5 minutes after a steady state reduction in current amplitude had been achieved after exposure to 10 mmol/L 4-AP in the bath solution. Current measured during the second voltage pulse was reduced by 48%. B, Mean concentration-response curve. Current amplitudes were measured at the end of the second voltage pulse. Data represent mean±SEM (n=3 to 5). Line is a fit of the following equation: B(%)=100/[1+(IC50/D)n], where B(%) is the percent current block at a drug concentration D, IC50 is the concentration of 4-AP that causes 50% block, and n is the Hill coefficient.

Although unable to abolish 100% of the current, relatively low concentrations of 4-AP (ie, <100 µmol/L) did markedly reduce I_sus, suggesting the presence of a highly 4-AP–sensitive component (Fig 1B). Therefore, we examined the kinetics and current-voltage relation of the 4-AP–sensitive current to determine its similarities to the Kv1.5-like current previously described in human atrium.^{5 16} Fig 2A shows a typical family of currents recorded in the presence of pipette and bath K⁺. Currents were elicited by a two-pulse protocol in which the amplitude of the second pulse varied from -80 mV to +80 mV (see Fig 2, inset). Currents illustrated are those elicited during the second series of voltage pulses. Interestingly, in the absence of 4-AP (control), the current-voltage relation reversed polarity at -20 mV (Fig 2A, bottom), suggesting that not all of the sustained current is carried by K⁺-selective channels. After the addition of 10 mmol/L 4-AP (Fig 2B), the outward current was markedly reduced, with relatively little effect on the inward current. The 4-AP difference current (Fig 2C) reveals a current that is outwardly rectifying and has an E_rev near -70 mV, very similar to that described for the Kv1.5-like current in human atrial cells.¹⁶ The current that remained after exposure to 10 mmol/L 4-AP (Fig 2B) was also rapidly activating and noninactivating, and it showed outward rectification. However, the current-voltage relation indicated that this current reversed polarity near 0 mV using physiological K⁺ concentrations (see "Materials and Methods"). The mean current amplitude measured at +80 mV after the addition of 10 mmol/L 4-AP was 2.36±0.10 pA/pF (n=5) compared with 5.19±0.91 pA/pF (n=4) recorded before the addition of 10 mmol/L 4-AP.

View larger version (28K): [in this window] [in a new window]   Figure 2. Current-voltage relations before and after the administration of 10 mmol/L 4-AP. Current families were elicited by the voltage protocol shown in inset. Currents shown were elicited by the second series of voltage pulses. The current-voltage relation for each family of currents is shown directly below. A, Currents recorded before addition of 4-AP. Vm indicates membrane voltage. B, Currents from the same cell after addition of 10 mmol/L 4-AP to the bath. C, 4-AP (10 mmol/L)–sensitive current measured as currents before drug minus those after drug addition.

Experiments in K⁺-Free Internal Solutions
The fact that 10 mmol/L 4-AP failed to abolish all of the sustained current and that the remaining current had an E_rev near 0 mV suggested that this outwardly rectifying current did not reflect a previously described K⁺ current. We next investigated whether this current could be blocked by replacement of internal K⁺ with Cs⁺. The results of an experiment examining the effects of replacement of cytoplasmic K⁺ by perfusion with a Cs⁺-substituted (K⁺-free) internal solution are shown in Fig 3. Application of a voltage pulse to +60 mV immediately after rupture of the cell membrane before appreciable perfusion occurred elicited a classic I_to and steady state current similar to those observed with normal K⁺-containing internal solution (compare Fig 3A with Fig 1A). However, after 5 minutes of perfusion with a Cs⁺ internal solution, I_to was completely abolished, and only a noninactivating current that was 40% of the amplitude of the initial outward sustained current recorded at the onset of dialysis remained. This suggests that a substantial portion of the outward current elicited by depolarizing pulses in human atrial myocytes is carried through channels that are neither K⁺ selective nor blocked by Cs⁺. The possibility that the remaining current may reflect I_sus, which remained as a result of poor dialysis, is unlikely, since the amplitude of the remaining current did not change over the time course of a typical experiment (Fig 3B) and I_to was completely abolished.

View larger version (25K): [in this window] [in a new window]   Figure 3. Currents recorded in a human atrial myocyte in the presence of pipette Cs+. A, Currents recorded immediately after rupture of the membrane (upper trace) and 5 minutes after rupture (lower trace). Pipette K+ had been replaced with Cs+. Pulse protocol shown in inset. B, Bar graph illustrating reduction in current measured at the end of the voltage pulse as a function of time after seal rupture. Currents measured immediately after seal rupture were taken as 100%. Error bars are mean±SEM (n=5).

The current recorded in the presence of pipette Cs⁺ appeared as a rapidly activating or nearly instantaneous current that showed outward rectification and reversed polarity at a voltage between -10 and 0 mV (Fig 4A and 4B), characteristics very similar to those for the current recorded in the presence of pipette K⁺ and 10 mmol/L 4-AP (Fig 2B). This current was seen in 100% of all cells tested (n=142) from 17 different hearts from subjects of different ages (see "Materials and Methods"). The amplitude measured at the end of an 800-millisecond pulse to +80 mV in myocytes isolated from young human atria (age, 1 day to 15 months) was 5.54±1.04 pA/pF (n=9), which was not significantly different from that measured in cells isolated from adult atria (6.31±1.35 pA/pF, n=8). Upon addition of 1 mmol/L Zn²⁺ to the bath solution, the amplitudes of this current measured at -100 mV and +60 mV were reduced by 33.6±5.3% and 48.1±4.0%, respectively (n=5 or 6, P<.05) (Fig 4C). On average, the current amplitude was reduced by 41.8±2.9% over the voltage range of +80 to +40 mV and by 32.3±0.5% over the voltage range of -100 mV to -60 mV. A 10-fold increase in the concentration of Zn²⁺ (10 mmol/L) produced a 57.2±5.5% (n=4) decrease in the current amplitude measured at +60 mV, whereas a concentration of 100 µmol/L Zn²⁺ was without effect (2.9±0.3% decrease at +60 mV, n=3). In contrast, both Ba²⁺ (1.8 mmol/L) and 4-AP (10 mmol/L) added to the bath solution were without significant effect on current amplitude measured in the presence of internal Cs⁺ (percent reduction, 1.9±6.2% and 5.6±2.7%, respectively) (n=3 to 5) (test potential, +60 mV; holding potential, -40 mV).

View larger version (21K): [in this window] [in a new window]   Figure 4. Currents recorded in pipette Cs+. A, Family of current traces recorded with a pipette solution in which K+ had been replaced by Cs+. Pulse protocol shown in inset. B, Current-voltage relation for current traces illustrated in panel A. Currents were measured at the end of the voltage pulse. Vm indicates membrane voltage. C, Current-voltage relation before and after addition of 1 mmol/L Zn2+ to the solution bathing six cells. Currents were elicited by a series of 800-millisecond pulses from -100 to +80 mV from a holding potential of -40 mV. Error bars represent mean±SEM.

Effects of Changes in External Cl^-
Since the E_rev of this current was near 0 mV, very similar to that predicted by the Nernst equation for a Cl^--selective channel (-0.9 mV with [Cl]_i of 142 mmol/L and [Cl]_o of 147 mmol/L), we next tested whether this current may be carried by Cl^-. When the Cl^- equilibrium potential was markedly altered (from -0.9 to +67.7 mV) by virtual elimination of extracellular Cl^- by replacement of 137 mmol/L NaCl with 137 mmol/L sodium isethionate (n=6), the observed E_rev and amplitude of the current were not significantly changed (Fig 5A). In addition, the voltage dependence of this current was not linear when external and internal Cl^- concentrations were very similar (147 and 142 mmol/L, respectively) (Fig 5A), as has been shown for other Cl^- currents.^{18 19} Exposure of cells to the anion conductance blocker DIDS (200 µmol/L) had little effect on this current, producing only a 3.0±9.5% decrease in current amplitude (test potential, +60 mV) (n=5). In contrast to the isoproterenol-activated Cl^- current described in guinea pig ventricle,^{18 19} this current was unresponsive to 1 µmol/L isoproterenol (6.45±5.84% increase, n=4) (Fig 5B), although 1 µmol/L isoproterenol was observed to produce a threefold increase in the amplitude of the L-type Ca²⁺ current in cells bathed in Cd²⁺-free external solution (data not shown). Taken together, these results indicate that this current is not carried by Cl^-.

View larger version (15K): [in this window] [in a new window]   Figure 5. Cl- dependence of current. A, Current-voltage relation in the presence of 147 mmol/L Cl- or 10 mmol/L Cl- in the bath solution. Cl- was reduced by replacing NaCl with sodium isethionate. Pulse protocol is shown in inset. Symbols represent mean±SEM (n=5 to 9). Vm indicates membrane voltage. B, Lack of effect of isoproterenol on novel ion current. Currents were elicited by a 320-millisecond voltage pulse to +60 from a holding potential of -40 mV. Currents were recorded at room temperature before and 5 minutes after the addition of 1 µmol/L isoproterenol to the bath solution.

Effects of Changes in Internal and External Cations
Cation channels that conduct current in the presence of Cs⁺ have recently been described in rat neuronal²⁰ and atrial²¹ preparations. Since the outwardly rectifying current described here was recorded in the presence of 140 mmol/L Cs⁺ in the pipette solution, we examined the possibility that this current may reflect a cation conductance. The E_rev and voltage dependence of this current (Fig 6A) suggest that it may be carried by multiple ions. Therefore, we tested the hypothesis that the inward component of this current is carried by the major extracellular ion (Na⁺) and the outward component is carried by the major intracellular ion (Cs⁺). If this hypothesis is true, then reducing extracellular Na⁺ with a bulky nonpermeant substitute (eg, NMDG) should shift the E_rev to more hyperpolarized potentials and reduce the amplitude of the inward current while not markedly affecting outward current. Likewise, reducing intracellular Cs⁺ should shift the E_rev to more depolarized potentials and decrease the amplitude of the outward current while producing little effect on inward current. Consistent with this hypothesis, reducing the extracellular Na⁺ concentration from 137 to 3 mmol/L by replacing NaCl with NMDG shifted the E_rev of this current by 20 mV, from -2 to -21 mV, and significantly reduced the amplitude of the current measured at -100 mV, from -1.47±0.27 to -0.81±0.12 pA/pF (P<.05) (n=6) (compare Fig 6A and 6B). However, the amplitudes of the currents measured at +60 mV were very similar (1.97±0.53 pA/pF in 137 mmol/L Na⁺ versus 2.13±0.42 pA/pF in 3 mmol/L Na⁺). As illustrated in Fig 6C, reducing intracellular Cs⁺ concentration from 140 to 20 mmol/L by replacement with 120 mmol/L TEA shifted the E_rev to more depolarized potentials (+12 mV) and dramatically reduced the amplitude of the outward current measured at +60 mV, from 1.97±0.53 to 0.73±0.14 pA/pF (P<.05) (n=5 or 6) while not altering the amplitude of the current measured at -100 mV (-1.47±0.27 versus -1.63±0.21 pA/pF, respectively). Replacement of all (137 mmol/L) extracellular Na⁺ (with 137 mmol/L NMDG) and all intracellular Cs⁺ (140 mmol/L) with 140 mmol/L TEA nearly abolished this current, with the current amplitudes at +60 mV and -100 mV being reduced to 0.31±0.03 and -0.37±0.06 pA/pF, respectively (n=5) (Fig 6D). These results strongly suggest that the outward and inward components of this current are carried predominantly by the cations Cs⁺ and Na⁺, respectively.

View larger version (24K): [in this window] [in a new window]   Figure 6. Cation dependence of current. Currents were elicited by a series of 800-millisecond pulses from -100 to +80 mV from a holding potential of -40 mV. Illustration shown above each current-voltage relation indicates the concentration of pipette Cs+ and bath Na+. Arrow indicates Erev. Currents were measured at the end of the voltage pulse. Error bars represent mean±SEM (n=5 to 9). A, Current-voltage relation recorded in 140 mmol/L pipette Cs+ and 137 mmol/L bath Na+. The current reverses near 0 mV. Vm indicates membrane voltage. B, Current-voltage relation recorded in 140 mmol/L pipette Cs+ and 3 mmol/L bath Na+. C, Current-voltage relation recorded in 20 mmol/L pipette Cs+ and 137 mmol/L bath Na+. D, Current-voltage relation for cells recorded in the absence of pipette Cs+ and bath Na+. Cs+ and Na+ were replaced by equimolar concentrations of TEA (140 mmol/L) and NMDG (137 mmol/L), respectively. The pH levels of the internal and external solutions were adjusted with tetramethylammonium hydroxide.

P_Na/P_Cs was calculated according to the following equation:

where F, R, T, and z have their usual meanings. When [Na]_o is 137 mmol/L, [Na]_i is 8 mmol/L, [Cs]_o is 4 mmol/L, [Cs]_i is 140 mmol/L, and E_rev is -2 mV (Fig 6A), the above equation predicts P_Na/P_Cs of 0.94.

Lack of Effect of Ouabain and Nisoldipine
To further characterize this current, we examined the possibility that this current may reflect either an efflux of Cs⁺ ions through unblocked L-type Ca²⁺ channels or that this current is in part carried by the Na⁺,K⁺-ATPase. Fig 7A is a family of current traces elicited from a holding potential of -40 mV before addition of 10 µmol/L nisoldipine. Fig 7B represents currents recorded from the same cell from a holding potential of 0 mV after the addition of 10 µmol/L nisoldipine. The fact that this current could be recorded in the presence of 10 µmol/L nisoldipine and from a depolarized holding potential of 0 mV, which completely inactivates L-type Ca²⁺ channels recorded in human atrial myocytes (half-inactivation potential, -18.6±1.6 mV; n=11; authors’ unpublished data, 1995), indicates that the outward limb of this current is not due to Cs⁺ efflux through Ca²⁺ channels.²² Furthermore, in the presence of 10 µmol/L ouabain²³ and 10 µmol/L nisoldipine, neither the amplitude nor the E_rev of this current is changed (Fig 7C). This further indicates that this current is not carried by the Na⁺,K⁺-ATPase.

View larger version (22K): [in this window] [in a new window]   Figure 7. Lack of effect of nisoldipine and ouabain. Currents were elicited by pulse protocol in Fig 6. A, Family of currents elicited from a holding potential of -40 mV before addition of drug. B, Same cell after addition of 10 µmol/L nisoldipine in the bath solution. Currents were elicited from a holding potential of 0 mV. C, Current-voltage relation measured in the control condition and after the addition of 10 µmol/L nisoldipine and 10 µmol/L ouabain to the bath solution. Vm indicates membrane voltage. All currents were elicited from a holding potential of -40 mV. Error bars are mean±SEM (n=5).

Lack of Effect of Cell Swelling
The possibility that this current may have been activated by cell stretch or swelling before or during patch formation was examined by exposing cells to an external solution supplemented with 75 mmol/L mannitol. This concentration of mannitol has been shown to produce a marked (threefold) reduction in the amplitude of swelling-induced currents in canine atria,²⁴ but produced only a small reduction (8.2±4.3%, n=5) in the amplitude of the current reported in the present study.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates for the first time the presence of a nonselective cation current in human heart that is carried by Na⁺ and K⁺ under physiological conditions. This is in marked contrast to previous reports indicating that most if not all of the outward current measured in human atrial myocytes is carried by the K⁺-selective channel Kv1.5.¹⁶ It is not clear why the present results are different, but these results may reflect differences in patient characteristics and/or isolation procedures. The similarity of the currents recorded with K⁺ and those recorded with pipette Cs⁺ (in regard to their E_rev, insensitivity to 4-AP, and kinetics) strongly suggests that these currents are conducted through a single channel that is permeable to Cs⁺, K⁺, and Na⁺.

Comparison With Previously Described Currents
Recently, Wang et al¹⁶ described a rapidly activating delayed rectifier current in human atrial myocytes. This current, referred to as I_sus or I_kur, is believed to be carried by the protein product of the K⁺ channel gene Kv1.5⁵ and has been shown to be carried by K⁺, since substitution of pipette K⁺ with Cs⁺ completely abolished this current. In addition, I_sus was very sensitive to the K⁺ channel blocker 4-AP, with an IC₅₀ of 50 µmol/L. The fact that the current described in the present study could be recorded after replacement of pipette K⁺ with Cs⁺ (Figs 3 and 4) and is not abolished by 10 mmol/L 4-AP (Fig 2) makes it very unlikely that the current described here reflects a Kv1.5-like current.

In 1993, Backx and Marban²⁵ described a rapidly activating current (I_kp) active at plateau potentials in guinea pig ventricular myocytes. The current described in the present study is different from I_kp in its E_rev (0 mV versus -30 mV for I_kp) and sensitivity to Ba²⁺. The current described in the present study is unaffected by the addition of 1.8 mmol/L Ba²⁺ to the bath solution, whereas I_kp is profoundly blocked by 1 mmol/L Ba²⁺.²⁵

Cs⁺ efflux through L-type Ca²⁺ channels has been reported in cardiac cells. However, the insensitivity of this current to blockers of L-type Ca²⁺ channels (200 µmol/L Cd²⁺ or 10 µmol/L nisoldipine) and the ability to record currents at holding potentials that completely inactivate L-type Ca²⁺ channels in human atrial myocytes (half-inactivation potential, -18.6±1.6 mV) strongly suggests that this current is not carried by Cs⁺ efflux through L-type Ca²⁺ channels.

Hagiwara et al²⁶ recently described a nonselective cation current present in rabbit sinoatrial node and, to a lesser extent, in atrial myocytes. This current had a linear current-voltage relation and a E_rev recorded in 150 mmol/L bath Na⁺ and 150 mmol/L pipette Cs⁺ between -10 mV and 0 mV and was sensitive to changes in either bath Na⁺ or pipette Cs⁺. Furthermore, similar to the current reported in the present study, the cation current described in rabbit atrium was insensitive to 1 mmol/L Ba²⁺. The similarities in the biophysical and pharmacological properties of the current described in human atrium and that in rabbit atrium suggest that both currents may be conducted through the same, or a similar, channel.

Limitations
Although seal resistances were typically >10 G, passage of ions through a pathway between the cell membrane and the pipette tip (eg, seal leak) is undoubtedly present, resulting in a finite contribution by a seal leak current to the observed macroscopic current. However, we feel that it is unlikely that the passage of ions through a leak pathway is responsible for the majority of the current observed in the present study for the following reasons: (1) As indicated in Fig 6, the E_rev of this current is sensitive to changes in internal Cs⁺ and external Na⁺. Changes in E_rev due to selective substitution of these cations with NMDG or TEA are consistent with a membrane conductance. (2) The inward and outward limbs of this current are markedly reduced by external Zn²⁺ (Fig 4C) but not external Ba²⁺. These results are most consistent with selective block by Zn²⁺ of a membrane conductance (ie, through an ion channel).

A fundamental assumption of the present study is that the blockers used to eliminate other currents (ie, Cd²⁺, nisoldipine, and Ba²⁺) are effective over the voltage range studied. Nisoldipine and Cd²⁺ were used to eliminate L-type Ca²⁺ currents, and Ba²⁺ was used to block I_K1. The concentrations of blockers used in the present study have been shown to effectively inhibit these currents.^{16 22} For instance, Cd²⁺ at a concentration of 200 µmol/L has been shown to effectively eliminate inward Ca²⁺ current through L-type Ca²⁺ channels,¹³ whereas higher concentrations of dihydropyridine antagonists have been used to eliminate outward Cs⁺ current through these channels.²² Similarly, over the voltage range of -100 to -50 mV, 1 mmol/L Ba²⁺ has been shown to be effective in abolishing I_K1 in humans.¹⁶

The P_Na/P_Cs derived for this current in the presence of [Na]_o of 137 mmol/L, [Na]_i of 8 mmol/L, [Cs]_o of 4 mmol/L, and [Cs]_i of 140 mmol/L was predicted to be 0.94. If this permeability ratio is assumed, the predicted E_rev for this current when external Na⁺ is lowered from 137 to 3 mmol/L is -100 mV. This is dramatically different from the E_rev of -21 mV observed experimentally when the external Na⁺ concentration is reduced (see Fig 6). One possible explanation for the disparity in predicted and observed E_rev is that NMDG is substantially permeable through this conductance pathway. A P_NMDG/P_Na ratio of 0.44 would be required to explain the observed shift in E_rev seen upon changing external Na⁺ (Fig 6). Similarly, TEA may be assumed likewise to be substantially permeable through this conductance pathway. A similar low ion selectivity has been reported for the nicotinic acetylcholine receptor and voltage-sensitive Cl^- channels, which are permeable to several organic ions and molecules.^{27 28 29} Although a finite permeability cannot be ruled out, a substantial permeability of NMDG and TEA for the present channel (on the order of 50% of that predicted for Na⁺ and Cs⁺) is inconsistent with the results illustrated in Fig 6D, where current is almost completely absent upon complete replacement of all Cs⁺ and Na⁺ for TEA and NMDG. An alternative explanation for the disparity in predicted and observed E_rev is that this channel may become nonselective in the presence of high internal Cs⁺. A similar ion-modulated selectivity has been reported for Na⁺ channels in sympathetic neurons.³⁰ Further experiments must be performed to test these hypotheses.

Potential Significance
The physiological role of this nonselective cation current is at present unknown. Considering the voltage dependence and rapid activation kinetics of this current, we speculate that this current can provide an important influence on all phases of the action potential as well as contribute to the resting membrane potential in human atrium. Further elucidation of the biophysical and pharmacological properties of this nonselective cation current may provide a more complete understanding of the physiology and pharmacology of human atrial repolarization as well as identify a new potential target for antiarrhythmic therapy.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine Erev = reversal potential IK1 = inward rectifier K+ current Isus = sustained current Ito = transient outward K+ current NMDG = N-methyl-D-glucamine PNa, PCs, and PNMDG = permeability coefficients TEA = tetraethylammonium chloride

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-36096 and K04 HL-02520. The authors wish to thank Dr Theresa Roca and Mary Richardson, RN, for technical assistance and Dr Arthur Pickoff for helpful discussion.

Received December 15, 1994; accepted July 11, 1995.

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