Transient outward current carried by inwardly rectifying K+ channels in guinea pig ventricular myocytes dialyzed with low-K+ solution

Pavel Zhabyeyev, Tatsuya Asai, Sergey Missan, and Terence F. McDonald

Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

Submitted 3 November 2003 ; accepted in final form 26 June 2004


    ABSTRACT
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 ABSTRACT
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There have been periodic reports of nonclassic (4-aminopyridine insensitive) transient outward K+ current in guinea pig ventricular myocytes, with the most recent one describing a novel voltage-gated inwardly rectifying type. In the present study, we have investigated a transient outward current that overlaps inward Ca2+ current (ICa,L) in myocytes dialyzed with 10 mM K+ solution and superfused with Tyrode’s solution. Although depolarizations from holding potential (Vhp) –40 to 0 mV elicited relatively small inward ICa,L in these myocytes, removal of external K+ or addition of 0.2 mM Ba2+ more than doubled the amplitude of the current. The basis of the enhancement of ICa,L was the suppression of a large transient outward K+ current. Similar enhancement was observed when Vhp was moved to –80 mV and test depolarizations were preceded by short prepulses to –40 mV. Investigation of the time and voltage properties of the outward K+ transient indicated that it was inwardly rectifying and unlikely to be carried by voltage-gated channels. The outward transient was attenuated in myocytes dialyzed with high-Mg2+ solution, accelerated in myocytes dialyzed with 100 µM spermine solution, and abolished with time in myocytes dialyzed with ATP-free solution. These and other findings suggest that the outward transient is a component of classic "time-independent" inwardly rectifying K+ current.

L-type calcium current; magnesium; spermine; ATP


REPOLARIZATION OF THE CARDIAC ACTION POTENTIAL is driven by a number of outward K+ currents, including the transient outward current (Ito); two delayed-rectifier currents, termed rapidly activating (IKr) and slowly activating (IKs) (3, 5, 7, 23, 41, 53); and inwardly rectifying K+ current, termed IK1 (10, 15, 26, 29, 36, 47). The relative densities of these repolarizing K+ currents varies with species and tissue region, and this accounts for the diversity of action potential configuration in mammalian ventricular tissue (7, 20, 27, 48, 52). For example, the repolarizing thrust of 4-aminopyridine (4-AP)-sensitive Ito ensures that the action potential plateau in rat ventricular myocytes is much shorter than the plateau in Ito-deficient guinea pig ventricle (3, 13, 14, 20, 25, 48).

Despite the apparent absence of classic Ito in guinea pig ventricular myocytes, there have been sporadic accounts of "transient outward" K+ currents in these cells. Twenty years ago, Matsuda and Noma (34) recorded a time-dependent outward current at plateau potentials in myocytes dialyzed with a low-K+ (10 mM) pipette solution. The current overlapped with inward L-type Ca2+ current (ICa,L), was suppressed on removal of external K+ (K) or addition of Ba2+, and was attributed to intrinsic voltage-dependent gating of IK1. Later, Inoue and Imanaga (16) described an "A-type" transient outward K+ current that developed upon removal of external divalent cations; however, follow-up investigation by others established that this transient is caused by the outward movement of monovalent cations through L-type Ca2+ channels (9, 30). A time-dependent, Ba2+-sensitive, "patch-duration-dependent," AP-insensitive transient outward K+ current [IK(PDD)] was described by Martin et al. (31). This current had a linear dependence on voltage and was prominent only in myocytes that were dialyzed for prolonged time periods. More recently, Li and colleagues (24, 25) described a 4-AP-insensitive transient outward current that was not dependent on prolonged dialysis, termed Ito,ir. This current was suppressed by removal of Kor addition of 0.2 mM Ba2+, two interventions that are commonly used to suppress IK1. However, Ito,ir differed from IK1 in that it displayed prominent time dependence, relatively weak rectification, and insensitivity to dialysate Mg2+ and spermine. For these reasons, Ito,ir was thought to be carried by a novel voltage-gated K+ channel.

During the course of studying the effects of low-K+ dialysates on membrane currents in guinea pig ventricular myocytes, we observed a 4-AP-insensitive, ICa,L-overlapping, time-dependent outward current that appeared to be similar to the current described by Matsuda and Noma (34). The present study was undertaken to investigate the properties and identity of the overlapping current.


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All procedures were performed in accordance with Canadian national and Dalhousie University regulations on the care and treatment of laboratory animals.

Ventricular Myocytes

Male guinea pigs (250–350 g) were killed by cervical dislocation, and single ventricular myocytes were enzymatically isolated as described previously (38). The excised hearts were mounted on a Langendorff column and perfused through the aorta with Ca2+-free Tyrode’s solution (37°C) containing collagenase (0.1–0.15 mg/ml; Yakult Pharmaceutical, Tokyo, Japan) for 10–15 min. The cells were dispersed and stored at 22°C in a high-K+, low-Na+ solution supplemented with 50 mM glutamic acid and 20 mM taurine. A few drops of the cell suspension were placed in a 0.3-ml perfusion chamber mounted on an inverted microscope stage. After the cells had settled to the bottom, the chamber was perfused (2 ml/min) with normal Tyrode’s solution at 35–36°C. Normal Tyrode’s solution contained (in mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4 with NaOH). Na+-free solution contained N-methyl-D-glucamine (NMDG+) instead of Na+, and K+-free solution contained NMDG+ instead of K+. Each of these solutions contained 3 µM E4031 to block rapidly activating IKr and 3 µM glibenclamide to suppress any contribution of ATP-sensitive K+ current to net membrane current.

Whole cell membrane currents were recorded using an EPC-7 amplifier (List Electronic, Darmstadt, Germany). Recording pipettes were fabricated from thick-walled borosilicate glass capillaries (H15/10/137; Jencons Scientific, Leighton Buzzard, UK) and filled with a Na+-free solution to minimize contributions from Na+-K+ pump, Na+/Ca2+ exchanger, and other Na+-dependent currents. The standard filling solution contained (in mM) 10 KCl, 20 CsCl, 110 CsOH, 110 aspartic acid, 5 MgATP, 5 ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 5 HEPES (pH 7.2 with KOH). NMDG+-based filling solution was made by substituting NMDG+ for Cs+, and sucrose-based solution was made by replacing CsCl and Cs-aspartate with 265 mM sucrose. In some experiments, the calculated free Mg2+ concentration of the standard pipette solution was modified from normal 0.6 mM to 1 µM or 7 mM; in others, the MgATP of standard solution was decreased from 5 to 0 mM with compensatory addition of 0.7 mM MgCl2 and 4 mM Cs aspartate. The pipettes had resistances of 2–3 M{Omega} when filled with pipette solution; series resistance ranged between 3 and 7 M{Omega} and was compensated by 60–80%. Membrane current signals were filtered at 3 kHz and digitized with an A/D converter (Digidata 1200A; Axon Instruments, La Jolla, CA) and pCLAMP software (Axon Instruments) at a sampling rate of 8 kHz before analysis.

Drugs

E4031 (Eisai, Tokyo, Japan) and glibenclamide (Sigma, St. Louis, MO) were dissolved in the bathing solution. Nisoldipine was kindly provided by Bayer (Etobicoke, ON, Canada), dissolved in dimethyl sulfoxide (0.1 M stock solution; Sigma), and stored in the dark at –20°C. Appropriate amounts of stock solution were freshly added to bathing solutions, and these were protected from the light during experiments. Spermine (Sigma) was dissolved in the pipette solution.

Statistics

Results are expressed as means ± SE, and differences were tested for significance using Student’s t-test or a Student-Newman-Keuls multiple comparisons test. Differences were considered to be statistically significant at P < 0.05.


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Time-Dependent Outward Current Overlapping ICa,L

Effects of removal of external K+ and addition of Ba2+. Ventricular myocytes were superfused with 5.4 mM K+ Tyrode’s solution and patched with pipettes that contained 10 mM K+ solution. At patch breakthrough, IK1 at the holding potential (Vhp) of –40 mV was a sizable (~0.5 nA) outward current; during the next 5 min, the current declined in amplitude and then reversed in direction. These changes indicate that dialysis decreased intracellular K+ concentration ([K+]i) and shifted the K+ equilibrium potential (EK) to a voltage above –40 mV.

Under steady-state conditions, 100-ms depolarizations to 0 mV elicited inward ICa,L, and subsequent repolarizations to –40 mV elicited currents that relaxed (half time ~20 ms) in the inward direction (and therefore were not due to deactivation of a voltage dependent K+ current) (Fig. 1A, left). Replacement of 5.4 mM K+ superfusate with K+-free superfusate both suppressed steady-state inward IK1 and increased the amplitude of peak ICa,L from control 0.59 ± 0.05 to 1.35 ± 0.1 nA (n = 9) (P < 0.001) (e.g., Fig. 1A, right). Matsuda and Noma (34) reported a similar response; recordings in their Fig. 7 show that peak ICa,L (+15 mV) increased from 0.6 to 1 nA after the removal of 2.7 mM K.



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Fig. 1. Modulation of current overlapped with inward L-type Ca2+ current (ICa,L) by overlapping time-dependent outward current. Myocytes dialyzed with low-K+ solution were bathed with normal Tyrode’s solution (Ctl, 5.4 mM K+), held at –40 mV, and pulsed to 0 mV for 100 ms at 0.2 Hz. A and B: increase in peak inward ICa,L after removal of external K+ (0 mM K+) or addition of 0.2 mM Ba2+. Recordings were obtained just before and 3 min after the interventions. C and D: unmasking of ICa,L outward current by application of 1 mM Cd2+. The recordings were obtained at 5-min intervals in the following order: control, Cd2+, and Cd2+ plus 0 mM K+ or 0.2 mM Ba2+. Horizontal dashed lines indicate zero-current levels.

 


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Fig. 7. Rundown of time-dependent outward current, inward tail current, and inward IK1 in a myocyte dialyzed with ATP-free solution. The myocyte was superfused with Cd2+-Tyrode’s solution and pulsed from –40 to 0 mV except for determination of I-V relationships. A: currents elicited by depolarizing ramps (100 mV/s) applied at 5 and 20 min after patch breakthrough; each ramp was preceded by a 200-ms hyperpolarization to –110 mV. B: currents elicited by depolarizing pulses applied just before the ramps.

 
The effects of 0.2 mM Ba2+ on membrane currents in myocytes superfused with 5.4 mM K+ solution were similar to those caused by removal of K, i.e., suppression of inward IK1 and tail current at –40 mV and increase in the amplitude of peak ICa,L from 0.64 ± 0.06 to 1.55 ± 0.15 nA (n = 7) (P < 0.001) (e.g., Fig. 1B).

It seemed likely that the enhancement of ICa,L caused by the removal of Kor the addition of Ba2+ was related to inhibition of an outward K+ current that overlapped ICa,L. To unmask the overlapping outward K+ current, ICa,L was blocked by application of 1 mM Cd2+. As illustrated by the superimposed recordings in Fig. 1C, the Cd2+-unmasked current had the appearance of a rapidly activating transient outward current. Although the current was insensitive to 2 mM 4-AP (n = 5, data not shown), both its time-dependent and steady-state components were almost fully suppressed by removal of K(Fig. 1C) and addition of 0.2 mM Ba2+ (Fig. 1D).

To evaluate the impact of K-sensitive overlapping outward current on peak ICa,L at 0 mV, we measured the effects of Kremoval on membrane currents at a fixed depolarization time (3 ms at 0 mV) both before and after the application of Cd2+. In six myocytes, brief removal of Kincreased ICa,L by 0.79 ± 0.08 nA and decreased Cd2+-unmasked outward current by 0.68 ± 0.07 nA. A similar correlation was found in four experiments with 0.2 mM Ba2+; addition of the K+-channel blocker increased ICa,L by 0.93 ± 0.09 nA and decreased unmasked outward current by 0.78 ± 0.07 nA.

Effects of increasing Vhp from –40 to –80 mV. A surprising finding in experiments with myocytes with Vhp fixed at –80 mV was that ICa,L elicited by depolarization from prepulse potential –40 mV was substantially larger than ICa,L recorded in myocytes with Vhp fixed at –40 mV. To investigate this phenomenon, myocytes held at –40 mV for ~10 min after patch breakthrough were subsequently held at –80 mV for several minutes. In response to the change in Vhp, peak ICa,L upon depolarizations from –40 to 0 mV increased with time and reached a new steady-state amplitude after 2–3 min (Fig. 2, A and B); at 3 min, the increase in ICa,L was 133 ± 9% (n = 20 myocytes). The stimulation was not due to a Vhp-induced increase in the availability of Ca2+ channels, because there was no detectable effect of Vhp when myocytes were pretreated with 0.2 mM Ba2+ (Fig. 2C); i.e., peak ICa,L were 1.37 ± 0.14 nA when Vhp was –40 mV and 1.49 ± 0.14 nA when Vhp was changed to –80 mV (n = 9 myocytes).



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Fig. 2. Effects of changing the holding potential (Vhp) on ICa,L and other membrane currents. Myocytes bathed in normal Tyrode’s solution were depolarized from –40 mV (Vhp or prepulse potential) to 0 mV for 100 ms at 0.2 Hz except during current-voltage (I-V) determinations. A: recordings showing the effects of changing Vhp from –40 to –80 mV for 3 min. The change in Vhp changed the direction of inwardly rectifying K+ current (IK1) at –40 mV and markedly increased the amplitude of peak ICa,L at 0 mV. B: time plot of the amplitudes of peak ICa,L (0 mV) and steady-state IK1 (–40 mV) (reference point, zero current) during changes in Vhp. C: occlusion by Ba2+ of the stimulatory effect of Vhp –80 mV on ICa,L. The Vhp –40 mV record was obtained 3 min after the addition of Ba2+, and the Vhp –80 mV record 3 min after the subsequent change in Vhp. D: Ba2+-sensitive current [control – Ba2+ (3 min)] obtained from a Cd2+-treated myocyte that was held at –80 mV and depolarized to the prepulse potential of –40 mV and then to test potential 0 mV. The large outward transient at –40 mV was followed by a relatively small one at 0 mV. E: normalized mean I-V relationships determined from 5 myocytes held at –40 mV and then at –80 mV for 3–5 min. Filled symbols represent amplitudes at the end of 100-ms pulses; open symbols represent amplitudes of peak inward-directed current. Normalization: current amplitudes on Vhp –40 and –80 mV runs were normalized to the amplitude of the peak inward current at 0 mV (=1.00) on the Vhp –40 mV run. The horizontal dashed lines on the recordings in A, C, and D indicate zero-current levels.

 
The occlusionary effect of Ba2+ (Fig. 2C) indicated that Vhp-induced enhancement of ICa,L was related to a decrease in overlapping outward current. To examine outward current under Vhp –80 mV conditions, we pretreated myocytes with 1 mM Cd2+ to block ICa,L and Na+ current (INa) (49) and recorded membrane current before and after the addition of 0.2 mM Ba2+. As illustrated by the recording of Ba2+-sensitive current shown in Fig. 2D, the prepulses from Vhp –80 to –40 mV elicited large time-dependent outward currents, whereas the test pulses from –40 to 0 mV elicited relatively small currents. Similar results were obtained from four myocytes in which INa and ICa,L were suppressed by using NMDG+ superfusate that contained 10 µM nisoldipine.

An important observation in the Vhp trials was that the direction of IK1 at –40 mV was dependent on Vhp; at steady state, the current was inward when Vhp was –40 mV and outward when Vhp was –80 mV (Fig. 2E). Because inwardly rectifying channels are strongly selective for K+ and do not conduct Cs+ ions (33), these changes in current direction can be attributed to changes in EK. EK estimated from the reversal potential of Ba2+-sensitive current was –30 ± 3 mV when Vhp was –40 mV and –55 ± 5 mV when Vhp was –80 mV (n = 5). The estimated EK for Vhp –40 mV corresponds to a mean [K+]i of 17 mM, a value higher than the [K+] (10 mM) of the dialysate. A similar discrepancy was noted by Matsuda and Noma (34), and the likely reason is that K+ "efflux" from cytoplasm to pipette failed to keep pace with K+ influx generated by steady-state inward IK1. The imbalance increased as a consequence of larger inward IK1 when Vhp was changed to –80 mV (e.g., Fig. 2, A and B), resulting in an increase in calculated mean [K+]i to 43 mM.

Time- and Voltage-Dependent Properties of the Outward and Inward K+ Currents

The voltage dependencies of peak and steady-state outward currents were determined by measuring Ba2+-K-sensitive currents in Cd2+-treated myocytes that were pulsed from Vhp –40 mV to higher potentials. The representative recordings and average current-voltage (I-V) relationships shown in Fig. 3 indicate that the peak current increased to a saturating level at 0 mV, whereas the steady-state current increased up to –10 mV and declined at higher potentials. We note that these results should be interpreted with caution because they may have been affected by a voltage-dependent block of outward current by intracellular Cs+ (see Ref. 33). On the other hand, similar results were obtained when myocytes were dialyzed with either NMDG+- or sucrose-based low-K+ solution (n = 2 each; data not shown) instead of the standard Cs+-based solution.



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Fig. 3. Voltage dependence of the outward K+ current. Myocytes were superfused with Tyrode’s solution that contained 1 mM Cd2+ and depolarized for 200 ms from Vhp –40 mV to more positive potentials at 0.1 Hz. A, left: control recordings; middle: recordings obtained after application of K+-free solution that contained 0.2 mM Ba2+; right: difference currents obtained after subtraction of middle traces from those at left. The arrowheads between the zero-current dashed line and the dashed lines extending from the difference trace at –20 mV illustrate how measurements of peak (Ip) and steady-state (Iss) difference currents were obtained. For reference, the –30-mV difference trace is at bottom, and the +40-mV trace is the innermost rapidly decaying one. B: voltage dependence of Ip and Iss difference currents; n = 9 myocytes.

 
To investigate the relationship between the time-dependent outward current and the inward tail current, myocytes were depolarized to 0 mV for a variable time and then repolarized to –40 mV. The superimposed recordings in Fig. 4A indicate that longer depolarizations produced larger decays of the outward current and that these were followed by larger inward tail currents. In five experiments of this type, there was a close correspondence between the time course of the decay of the outward current ({tau} = 22 ± 2 ms) and the time course of the increase in the amplitude of the inward tail current ({tau} = 22 ± 4 ms).



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Fig. 4. Time- and voltage-dependent properties of outward and inward K+ currents. Myocytes superfused with Cd2+-Tyrode’s solution were held at –40 mV, depolarized to 0 mV, and repolarized to more negative potentials. A: inward tail currents elicited after depolarizations of varying duration. Note the correspondence between the time course of the outward current and the time course of the amplitudes of the inward tail currents. B: voltage dependence of the tail current.

 
It is important to consider whether the inward tail currents were due to depletions of Kthat accumulated during the preceding depolarizations. Suppose that steady-state EK = –30 mV just before depolarization from –40 to 0 mV (see above) and that the time-dependent decay of outward current during the depolarization was due to a current-dependent accumulation of Kthat decreased effective EK to, for example, –10 mV (which would explain the relative amplitudes of peak and steady-state currents at 0 mV in Fig. 3B). If so, the inward driving force would be 30 mV at the moment of repolarization to –40 mV and would decline to its steady-state value of 10 mV at some point thereafter. However, this is an unlikely scenario, because it is apparent from data at voltages more negative than EK in Fig. 2E that any postaccumulation reequilibration of Kduring repolarization would generate inward currents that relaxed in the outward direction, i.e., the very opposite of that which we observed. That being said, the delayed dipping of the tails below the steady-state current level at –40 mV in Fig. 4A suggests that slight accumulations may indeed have occurred during the depolarizations.

As noted earlier (see also DISCUSSION), the inward tail currents cannot have been due to deactivation of a voltage-dependent conductance activated on depolarization. Rather, the results in Fig. 4A suggest that the tail current was due to a time-dependent recovery from rectification that occurred during the preceding depolarization to 0 mV. Further information on the recovery process was obtained from myocytes that were repolarized to voltages between –30 and –70 mV after 200-ms depolarizations to 0 mV. The recordings in Fig. 4B indicate that both the amplitude and the rate of rise of the tail current increased with negative voltage in a manner consistent with earlier findings regarding the "activation" of guinea pig IK1 at voltages negative to EK (19, 22, 32).

Modulation by Mg2+, Spermine, and ATP

For additional information regarding the K+ current under investigation, we evaluated the effects of established modulators of inwardly rectifying K+ currents, including intracellular Mg2+ (18, 19, 44, 45), spermine (6, 8, 18), and ATP (21, 42, 46).

Modulation by Mg2+. Cd2+-treated myocytes were dialyzed with a solution that had a free Mg2+ concentration of 1 µM, 0.6 mM, or 7 mM and pulsed from Vhp –40 to 0 mV for measurement of peak (5 ms) and steady-state (200 ms) outward current amplitudes (Fig. 5). After 10 min of dialysis, the amplitudes of the peak currents were 0.74 ± 0.06 nA in the 1 µM trials (n = 18), 0.56 ± 0.07 nA in the 0.6 mM trials (n = 16), and 0.37 ± 0.05 nA in the 7 mM trials (n = 6) (P < 0.05 or 0.01 for each pair of mean values). Similarly, the amplitudes of the steady-state currents declined with higher Mg2+ concentration (P < 0.01 between 1 µM and 0.6 or 7 mM) (Fig. 5C). Additional observations were that the high-Mg2+ dialysate had little effect on steady-state inward IK1 but slowed the time course of the inward tail current (Fig. 5, A and B).



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Fig. 5. Effects of dialysate free Mg2+ concentration on outward and inward K+ currents. Myocytes were superfused with Cd2+-Tyrode’s solution and pulsed from Vhp –40 to 0 mV. A and B: results from representative myocytes dialyzed with solution that contained 1 µM (A) and 7 mM (B) free Mg2+. Left: time plots of the amplitudes of Ip outward current at 0 mV (measured at 5 ms), Iss outward current at 0 mV (measured at 200 ms), and steady-state inward IK1 at –40 mV (measured just before depolarizing pulses); right: sample recordings obtained at 10 min postpatch. The calibration bar indicates 0.5 nA. C: summary of Ip and Iss data obtained from myocytes dialyzed with 1 µM (n = 18), 0.6 mM (n = 16), and 7 mM (n = 6) free Mg2+ solution. All measurements were obtained after 10 min of dialysis.

 
Modulation by spermine. To determine the effects of intracellular spermine, membrane currents recorded from myocytes dialyzed with 100 µM (n = 6) or 1 mM (n = 3) spermine solution were compared with those recorded from 10 "matched" control myocytes. The 100 µM spermine dialysate decreased the amplitudes of peak (5 ms) and steady-state (200 ms) outward currents at 0 mV (P < 0.05 or 0.01 at ≥7 min postpatch) and increased the amplitude of the inward tail current at –40 mV (Fig. 6, A and B). For the trials with 1 mM spermine, the tip of the pipette was filled with control solution to delay the onset of spermine action. The representative data shown in Fig. 6C indicate that high spermine markedly inhibited outward current at 0 mV and inward current at –40 mV.



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Fig. 6. Effects of spermine pipette solution on outward and inward K+ currents. Myocytes were superfused with Cd2+-Tyrode’s solution, dialyzed with control or spermine-containing 0.6 mM Mg2+ solution, and pulsed from Vhp –40 to 0 mV. A and B, left: time plots of the amplitudes of peak outward current at 0 mV (Ip), steady-state outward current at 0 mV (Iss), and steady-state inward IK1 at –40 mV in myocytes dialyzed with control solution (n = 10) (A) and 100 µM spermine solution (n = 6) (B); right: example recordings obtained after 10 min of dialysis. C: results obtained from a myocyte that was patched with a pipette that, except for the very tip, was filled with 1 mM spermine solution. The recordings (right) were obtained at the times indicated on the time plot.

 
Modulation by ATP. IK1 in guinea pig ventricular myocytes is dependent on cell energy metabolism, and ATP-free intracellular solution promotes rundown of the current (21, 46). To determine whether the membrane currents that we investigated were affected by decreasing intracellular ATP concentration, four myocytes were dialyzed with ATP-free solution and pulsed from Vhp –40 to 0 mV, except for periodic determinations of the I-V relationship. As illustrated by the recordings shown in Fig. 7, dialysis with ATP-free solution resulted in a progressive rundown of peak and steady-state outward current at 0 mV, inward tail current at –40 mV, and IK1 at potentials between –110 and +20 mV.


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 ABSTRACT
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Matsuda and Noma (34) measured membrane currents in guinea pig ventricular myocytes that were dialyzed with a low-K+ Cs+ solution and found that ICa,L at plateau potentials was distorted by the presence of an overlapping, time-dependent outward current. The results of the present study confirm and extend this finding and provide new information on the time-dependent outward current. We discuss the overlap, identify the contaminant outward current as time-dependent IK1, and then consider how the latter "transient outward current" relates to transient outward currents previously described in guinea pig ventricular myocytes.

ICa,L and Overlapping Time-Dependent Outward IK1

The amplitude of peak ICa,L on depolarizations from –40 to 0 mV was markedly increased by the following three interventions: removal of K, addition of 0.2 mM Ba2+, and changing of Vhp from –40 to –80 mV. The sensitivity of peak ICa,L to the first two of these interventions suggested a mechanism that involved an ICa,L-overlapping K+ current, and this was confirmed by the unmasking of a time-dependent outward current when ICa,L was blocked by the application of Cd2+. The time course of the unmasked current resembled that of a rapidly activating transient outward current, and this raises the question of whether the overlapping current was carried by one or more of the voltage-dependent K+ channels (Kv1.4, Kv4.2, Kv4.3) responsible for transient outward currents in cardiac cells (4, 35, 50). Although involvement of Kv channels in the overlapping current is not excluded by the finding that the overlapping current was sensitive to Kand Ba2+ (39), it can be ruled out on other grounds. First, a recent study of Kv-subunit mRNA and protein in rabbit and guinea pig ventricular tissue concluded that Kv1.4, Kv4.2, and Kv4.3 are not expressed in guinea pig tissue (54). Second, the effects of changing Vhp from –40 to –80 mV on the overlapping current (Fig. 2) argue against involvement of rapidly activating/inactivating Kv current of any origin. As indicated by the marked increase in ICa,L at 0 mV, the more negative Vhp resulted in a marked decrease of the overlapping outward current, which in turn was linked to the decay of a large time-dependent outward current during the prepulse from Vhp –80 to –40 mV. The latter decay was not due to voltage-dependent inactivation because orders-longer conditioning at –40 mV (e.g., when Vhp was set to –40 mV) resulted in larger (not smaller) overlapping outward current at 0 mV, even in the face of the marked decline in outward K+ driving force that occurred when Vhp was –40 mV.

The anomalous behavior of overlapping outward current when Vhp was changed is most easily explained by identifying the overlapping current as time-dependent outward IK1. Then, the decay of time-dependent outward current during the prepulse to –40 mV was due to a strong time-dependent rectification that partially occluded time-dependent rectification on the subsequent test pulse to 0 mV; a similar "conditioning" at –40 mV did not occur when Vhp was –40 mV, because EK, a major determinant of the rectification of IK1 (28, 29), was now more positive than –40 mV (Fig. 2B).

There were a number of other findings that support the view that the overlapping outward current was time-dependent IK1. First, the current was completely suppressed by two interventions known to inhibit IK1, removal of Ko+ and addition of Ba2+. Second, repolarizations from 0 mV elicited tail currents whose configuration and dependence on duration of the preceding depolarization were consistent with those expected for IK1 recovering from rectification. Third, the overlapping outward current responded to dialysates with modified Mg2+, spermine, and ATP in the manner expected for IK1. For example, low-Mg2+ dialysates enhanced the current, whereas high-Mg2+ and spermine dialysates inhibited it (see below).

Transient Outward Currents in Guinea Pig Ventricular Myocytes

After the Matsuda and Noma (34) report of a time-dependent outward current that overlapped ICa,L, Saigusa and Matsuda (40) studied myocytes bathed with high-K+ solution and observed rapidly decaying outward transients ({tau} ~5 ms at EK + 15 mV) whose amplitude was linearly dependent on voltage. Subsequently, Ishihara et al. (19) investigated K+ currents in myocytes using the oil gap voltage-clamp method and observed rapidly decaying outward transients ({tau} ~3 ms at EK + 15 mV; <1 ms at EK + 40 mV) that had a linear I-V relationship; experimental problems noted by these authors included the presence of restricted oil membrane extracellular spaces, K-insensitive outward transients, and rapid washout of ICa,L (19). Later, Martin et al. (31) described IK(PDD), which developed after prolonged dialysis via large-tipped (≤1 M{Omega}) pipettes. In contrast to the time-dependent outward current described in the present study, IK(PDD) had a linear I-V relationship, was unaffected by variation (1 nM–1 mM) of free Mg2+ in the dialysate, and was not observed when 10 mM Cs+ was included in the dialysate.

More recently, Li et al. (25) investigated a transient outward current, Ito,ir, that was independent of patch duration. Ito,ir was insensitive to 4-AP, inhibited by the removal of Kor the addition of 0.2 mM Ba2+, and had a peak amplitude that saturated near EK +60 mV. In other words, Ito,ir had properties similar to those attributed to time-dependent outward IK1 in the present study. However, Li et al. (25) had several grounds for concluding that Ito,ir was most likely a novel, long-overlooked, voltage-dependent current and not IK1. These grounds were as follows: 1) Ito,ir was insensitive to intracellular Mg2+, 2) Ito,ir was insensitive to intracellular spermine, and 3) Ito,ir was a time-dependent weakly rectifying current, whereas outward IK1 has long been described as a virtually time-independent, strongly rectifying current (12, 37). These grounds and their relationship to the present findings are considered below.

Responses to Mg2+. Li et al. (25) evaluated the sensitivity of Ito,ir to intracellular Mg2+ by comparing the amplitude of Ito,ir just after patch breakthrough with that measured after 10-min dialysis with Mg2+-free solution. They found that there was no significant difference between these values in four test myocytes and concluded that Ito,ir was insensitive to Mg2+. That was not the case for the time-dependent outward current that we examined in the present study; its amplitude was significantly increased by low (1 µM) Mg2+ solution, and significantly decreased by high (7 mM) Mg2+ solution. High Mg2+ also induced a marked slowing of the inward tail at –40 mV, an effect that may be due to a negative shift in the "activation" time constant (17, 4345).

Responses to spermine. Li et al. (25) used a 10 µM spermine dialysate to evaluate whether the polyamine had an inhibitory effect on Ito,ir and concluded that it did not. However, the following considerations suggest that the 10 µM concentration they used for this test may have been too low: 1) Stadnicka et al. (43) found that a 10-min dialysis of guinea pig ventricular myocytes with 25 µM spermine solution had no effect on classic outward IK1, 2) the normal cytoplasmic concentration of free spermine estimated for various cell types ranges from 10 to 75 µM (2, 6, 51), 3) free spermine in the dialysate may be lower than total spermine because of binding by ATP (51), and 4) intracellular spermine is buffered by nucleic acids and proteins (2, 6, 51). It seems likely that these are among the reasons why investigators of spermine action on whole cell Kir2.1 current (17, 18) and other inwardly rectifying currents (1) have used concentrations between 100 µM and 1 mM. In the present study, 100 µM spermine solution had little effect on steady-state inward IK1 at –40 mV; however, it partially inhibited time-dependent outward current at 0 mV (suggesting enhanced block) and increased the amplitude of the inward tail current at –40 mV (suggesting recovery from larger block). These results, as well as the pronounced inhibition of all current components by 1 mM spermine dialysate, are consistent with earlier findings regarding Kir2.1 current (11, 17, 28).

Time and voltage dependence of IK1. Li et al. (25) pointed out that Ito,ir is a time-dependent current, whereas outward IK1 is a time-independent current. Clearly, this distinction between the two currents depends on whether outward IK1 really is a time-independent current. It is important to remember that the (now entrenched) description of outward IK1 is based on a theoretical formulation of an instantaneously rectifying current that, at the time, provided a useful framework for interpretation of membrane currents in cardiac Purkinje fibers (37). Thereafter, the true time- and voltage-dependent nature of the current is likely to have gone unnoticed in studies that used "unfavorable" experimental protocols/conditions such as voltage-ramp commands, depolarizing steps from holding potentials above EK, nonblocked INa, and nonblocked ICa,L. We have elucidated some of the time- and voltage-dependent properties of outward IK1, and the similarity of these properties to those ascribed to Ito,ir (25) is noteworthy.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by the Canadian Institutes of Health Research.


    ACKNOWLEDGMENTS
 
We are grateful to Gina Dickie for excellent technical assistance.

Current address of T. Asai: Department of Information Science, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. F. McDonald, Dept. of Physiology and Biophysics, Dalhousie Univ., 5850 College St., Sir Charles Tupper Medical Bldg., Rm. 3B-1, Halifax, NS, Canada B3H 4H7 (E-mail: terence.mcdonald{at}dal.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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