Dynamic Ca²⁺-Induced Inward Rectification of K⁺ Current During the Ventricular Action Potential

From the Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Milan, Italy.

Correspondence to Dr Antonio Zaza, Dipartimento di Fisiologia e Biochimica Generali, via Celoria 26, I-20133, Milan, Italy. E-mail zanto{at}imiucca.csi.unimi.it

	Abstract

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Abstract—Inward rectification, an important determinant of cell excitability, can result from channel blockade by intracellular cations, including Ca²⁺. However, mostly on the basis of indirect arguments, Ca²⁺-mediated rectification of inward rectifier K⁺ current (I_K1) is claimed to play no role in the mammalian heart. The present study investigates Ca²⁺-mediated I_K1 rectification during the mammalian ventricular action potential. Guinea pig ventricular myocytes were patch-clamped in the whole-cell configuration. The action potential waveform was recorded and then applied to reproduce normal excitation under voltage-clamp conditions. Subtraction currents obtained during blockade of K⁺ currents by either 1 mmol/L Ba²⁺ (I_Ba) or K⁺-free solution (I_0K) were used to estimate I_K1. Similar time courses were observed for I_Ba and I_0K; both currents were strongly reduced during depolarization (inward rectification). Blockade of L-type Ca²⁺ current by dihydropyridines (DHPs) increased systolic I_Ba and I_0K by 50.7% and 254.5%, respectively. ß-Adrenergic stimulation, when tested on I_0K, had an opposite effect; ie, it reduced this current by 66.5%. Ryanodine, an inhibitor of sarcoplasmic Ca²⁺ release, increased systolic I_Ba by 47.7%, with effects similar to those of DHPs. Intracellular Ca²⁺ buffering (BAPTA-AM) increased systolic I_Ba by 87.7% and blunted the effect of DHPs. Thus, I_K1 may be significantly reduced by physiological Ca²⁺ transients determined by both Ca²⁺ influx and release. Although Ca²⁺-induced effects may represent only a small fraction of total I_K1 rectification, they are large enough to affect excitability and repolarization. They may also contribute to facilitation of early afterdepolarizations by conditions increasing Ca²⁺ influx.

Key Words: inward rectifier K⁺ current • inward rectification • intracellular Ca²⁺ • action potential

	Introduction

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Several known K⁺ channels have a lower resistance to inward than to outward current flow, a property referred to as "inward rectification." Inward rectification, particularly pronounced in I_K1, is of pivotal importance in determining membrane excitability in cardiac myocytes.¹ I_K1 inward rectification is currently interpreted as a voltage-dependent blockade of the channel by intracellular cations.^{2 3 4 5} Small ions, such as Mg²⁺ and Ca²⁺, block the channel with fast kinetics; polyamines, such as spermine and spermidine, account for a slower component of rectification.^{4 6} Experiments performed in Mg²⁺-free conditions (excised inside-out patches) show that micromolar Ca²⁺ can block I_K1 from the intracellular side of the membrane at depolarized potentials.^{3 5} Moreover, in an elegant study on neonatal avian ventricular myocytes, Mazzanti and DeFelice⁷ reported that outward current through single I_K1 channels could be observed during the action potential if Ca²⁺ was omitted from the extracellular (pipette) solution. Nonetheless, a physiological relevance of Ca²⁺-induced rectification of I_K1 in the mammalian heart is either disputed or overlooked,^{3 8} mainly on the basis of the tenet that intracellular Mg²⁺ and polyamine concentrations are well above those required to saturate I_K1 rectification in excised-patch experiments.

The aim of the present study was to reconsider the role for Ca²⁺-induced I_K1 rectification in mammalian ventricular myocytes by adopting experimental conditions preserving cell integrity and simulating, as closely as possible, physiological electrical activity. The evidence obtained suggests that the transient rise in subsarcolemmal Ca²⁺, occurring during the plateau phase of the action potential as a consequence of both influx and release from the sarcoplasmic reticulum, may significantly contribute to I_K1 rectification.

	Materials and Methods

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Cell Isolation
Guinea pig ventricular myocytes were isolated by using a coronary perfusion method similar to the one described by Levi and Alloatti.⁹ In brief, guinea pigs weighing 200 to 300 g were anesthetized by exposure to a cotton wad soaked in tribromoethanol solution (200 mg of tribromoethanol in 10 mL of ether), killed through cervical dislocation, and exsanguinated. Hearts were quickly removed, and the ascending aorta was connected to the outlet of a Langendorff column, perfused with Tyrode's solution (37°C) containing (mmol/L) NaCl 154, KCl 4, CaCl₂ 2, MgCl₂ 1, HEPES-NaOH 5, and D-glucose 5.5, adjusted to pH 7.35, and equilibrated with 100% O₂. Perfusion with Tyrode's solution was maintained until vigorous mechanical activity resumed and blood was completely removed. The heart was then perfused, until arrest occurred, with a nominally Ca²⁺-free solution containing (mmol/L) NaCl 33.5, KCl 10, D-glucose 22, sucrose 132, KH₂PO₄ 1, MgSO₄ 5, HEPES KOH 10, and taurine 50, adjusted to pH 7.3, followed by the same solution to which 140 U/mL collagenase (Sigma type V), 2.5 U/mL trypsin (Sigma type III), and 1 mg/mL bovine serum albumin were added. When the effluent became slightly viscous, the heart was moved to Ca²⁺-free solution (0.1 mmol/L EGTA added), the atria were dissected, and the ventricles were chopped to 1-mm fragments. The fragments were exposed to gentle mechanical agitation in Ca²⁺-free solution with the addition of 0.3 U/mL protease (Sigma type XIV) and 1 mg/mL bovine serum albumin. Ten-milliliter samples of the suspension were collected every 5 minutes, filtered through a nylon mesh, and centrifuged at 750 rpm for 3 minutes. The pellets were resuspended in Tyrode's solution and stored in this solution at 4°C until use.

Rod-shaped Ca²⁺-tolerant myocytes were usually obtained after 10 to 15 minutes of mechanical agitation. Only quiescent myocytes with clear-cut striations were used for measurements; to limit the error generated by large currents through uncompensated series resistance, smaller cells were generally preferred.

Experimental Solutions and Recording Apparatus
The myocyte suspension was placed in a 30-mm polylysine-coated Petri dish with a plastic ring (to reduce total volume to 1 mL) and mounted on the stage of an inverted microscope. The dish was perfused at 2 mL/min with standard external Tyrode's solution containing (mmol/L) NaCl 154, KCl 4, CaCl₂ 2, MgCl₂ 1, HEPES-NaOH 5, and D-glucose 5.5, adjusted to pH 7.35. In a set of experiments testing the effect of low extracellular Ca²⁺, the concentration of this ion was reduced from 2 to 0.5 mmol/L; to compensate for the resulting decrease in surface charge shielding, extracellular Mg²⁺ was increased to 4 mmol/L. The cell under study was held within 300 µm from the tip (1 mm) of a thermostated multiline pipette, which was connected to solution reservoirs through electrically driven valves. This system allowed us to expose the cell to different solutions, with changes completed in 1 second. The solution temperature was monitored at the pipette tip with a fast-response digital thermometer (BAT-12, Physitemp) and kept at 35±0.1°C. Nifedipine (Sigma) and nisoldipine (Bayer) stock solutions were prepared by dissolving the substances in ethanol; the same ethanol concentration was present in all experimental solutions and did not exceed 0.05%. Nisoldipine was a generous gift from Bayer Italia.

Membrane potential and current were measured in the whole-cell configuration, under ruptured- and perforated-patch conditions (Axopatch 200-A, Axon Instruments), by borosilicate glass pipettes with tip resistances between 3 and 5 M. The pipette solution contained (mmol/L) potassium aspartate 110, KCl 23, CaCl₂ 0.4 (calculated free Ca²⁺, 10^-7 mol/L), MgCl₂ 3 (calculated free Mg²⁺, 0.1 mmol/L), HEPES KOH 5, EGTA KOH 1, GTP sodium salt 0.4, ATP sodium salt 5, and creatine phosphate 5, pH 7.3. In perforated-patch experiments, 130 µmol/L amphotericin B was added, whereas ATP, GTP, and creatine phosphate were omitted from the pipette solution. The composition of the pipette solution was also changed in selected experiments as specified in the appropriate section of "Results." Series resistances (<5 M in ruptured-patch and <10 M in perforated-patch experiments) and membrane capacitance were measured in every cell but were not compensated. An average junction potential of 5 mV, measured on moving the electrode tip from Tyrode's solution to "intracellular" (potassium aspartate) solution, was also left uncompensated.

The command potentials for the V-clamp amplifier were supplied through a 12-bit D/A converter (Labmaster TL-40) by an IBM-compatible PC (Pentium, 75 MHz) driven by custom-made software. Potential and current signals were filtered at 2 kHz and were tape-recorded, together with trigger signals, with an adapted VCR system. Recorded signals were later acquired through a 12-bit A/D (sampling rate, 5 kHz) for analysis.

AP-Clamp Procedure
Membrane currents were studied by the AP-clamp technique,^{10 11 12} as illustrated in Figure 1. Transmembrane potential was recorded (I-clamp configuration) during steady-state stimulation at a cycle length of 1 second in a single cell during superfusion with Tyrode's solution. The membrane potential waveform, corresponding to a single stimulation cycle, was digitized (5 kHz, 12-bit resolution) and stored in computer memory. After switching to V-clamp mode, the acquired waveform was used as the command signal to drive membrane potential in the same cell, thus allowing measurement of total membrane current during the cycle. Sustained activity was simulated by repeatedly applying the same waveform at a cycle length of 1 second. After a short stabilization period under AP-clamp conditions, total membrane current recorded during superfusion with Tyrode's solution (control) settled to a value close to zero, except for a short glitch corresponding to the stimulation artifact. The current recorded during subsequent exposure to a channel blocker (compensation current) provided a mirror image of the contribution of the blocked component to action potential.

View larger version (12K): [in this window] [in a new window]   Figure 1. AP-clamp technique. I-clamp mode: an action potential waveform was recorded during superfusion of Tyrode's solution (stimulation cycle length, 1 second). V-clamp mode: a, currents recorded while applying the action potential waveform during Tyrode superfusion (control current and current after return to control); except for a stimulation artifact, these currents approximate zero; b, current recorded during steady-state superfusion with the Ca2+ channel blocker nifedipine (compensation current); and c, blocker-sensitive current (Inif) obtained by the subtraction of compensation current from control current (a-b).

Recordings were considered acceptable only if (1) the current recorded in control conditions was negligible, indicating that reproduction of the spontaneous activity was satisfactory, and (2) washout of the blocker, performed after each exposure, was followed by reversal of the compensation current.

Identification of K⁺ currents required means to inhibit their conductance selectively. To compensate for the unavailability of blockers absolutely specific for I_K1, we used two different interventions that share a strong inhibitory effect on K⁺ conductances (particularly I_K1): blockade by 1 mmol/L Ba^{2+ 13} and removal of K⁺_o.^{12 14} The rationale was that if the observed effect was identically reproduced with both the approaches, it could be ascribed to a change in K⁺ currents with a reasonable confidence. Either Ba²⁺ blockade or removal of K⁺_o has been previously used to identify I_K1 in AP-clamp experiments^11,12; however, in previous studies, conductances other than I_K1 (I_Na, I_Ca, I_to, and I_NaK) were preventively blocked with the appropriate agents. Such an approach was not adopted in the present case, because I_Ca was an essential player and other contaminations, with the exception of I_NaCa, were substantially irrelevant to the interpretation of data (see "Discussion"). Interference by I_NaCa was evaluated in a specific set of experiments (see "Results").

Definitions
Throughout text and figures, data from AP-clamp experiments are presented in terms of "blocker-sensitive current" (I_Ba and I_0K). The latter was obtained by subtraction of compensation current traces from control ones. Current traces from three to five cycles at steady state in each condition were averaged before subtraction. Because of the complexity introduced by the subtraction procedure, the meaning of changes in "blocker-sensitive" currents is often not intuitive. It may help to consider that according to the procedure used in the present study, any current that is reduced by the blocker will contribute to the blocker-sensitive current with its original sign. For instance, if nifedipine selectively blocked I_CaL, the blocker-sensitive current I_nif (Figure 1, bottom panel) would be identical to true I_CaL. The opposite would be true for currents either directly or indirectly enhanced by the "blocker."

Currents measured during the activation cycle were divided, according to the cycle phase, into (1) diastolic current, measured during the diastolic interval; (2) systolic current, measured from the upstroke (excluding the artifact) to 50% of repolarization; and (3) peak current during fast repolarization. Whereas the latter represented a single point value, diastolic and systolic currents were measured as "average current"; this was done by integrating the current signal over the appropriate interval and dividing the result by the integration interval. Current density was estimated by dividing the current value by membrane capacitance. In all figures, membrane potential and current traces are aligned.

Statistical Analysis
Means were compared by Student t test for paired or unpaired observations as appropriate. ANOVA for paired measurements, in some cases with a grouping factor (two-way model), was performed whenever more than two means were compared. A probability level of P<0.05 was used to define significance throughout the study. In the text and figures, values are presented as mean±SEM.

	Results

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I-V Relations of I_Ba and I_0K
Quasi–steady-state I-V relations of I_Ba and I_0K were studied, under V-clamp conditions, by applying voltage ramps from -105 to +55 mV with a steepness of 0.03 V/s. The ramp protocol was applied in control conditions and during exposure to either 1 mmol/L Ba²⁺ (Figure 2a) or to K⁺-free solution (Figure 2b). I_Ba and I_0K were obtained by subtraction of the traces recorded in control and test solutions. In some of the experiments, potential contamination by I_CaL was prevented by performing the measurements in the presence of 0.2 µmol/L nisoldipine. Since the results obtained were superimposable, data obtained in the presence and absence of nisoldipine were pooled. As shown in Figure 2, the I-V relations of both I_Ba and I_0K displayed strong inward rectification up to -10 mV. Positive to this potential, while I_Ba began to rise again, I_0K underwent a further decay that, in a few cases, led to a small net inward current. Such mirror-like behavior of I_Ba and I_0K at positive potentials can be attributed to divergent effects of the two experimental interventions on I_K. I_K is reduced by Ba²⁺,¹ thus appearing at potentials positive to I_K threshold as outward I_Ba. Vice versa, as a consequence of the balance between incomplete depression of conductance of its slowly activating component (I_Ks) and the increased chemical gradient, I_K may be unchanged or even slightly increased by removal of extracellular K⁺.¹⁴ Thus, particularly under conditions in which I_Ks is larger (eg, during longer depolarizations), I_K might contribute to I_0K with a reversed sign, ie, as a small inward component. It is uncertain to which extent this may apply to I_0K recorded under AP-clamp conditions, because the proportion of I_K contributed by I_Ks during the action potential cannot be determined a priori. Nonetheless, a paradoxical increase in I_0K, but not in I_Ba, is expected from selective inhibition of any amount of I_Ks activated during the action potential.

View larger version (18K): [in this window] [in a new window]   Figure 2. A comparison between steady-state I-V relations of IBa and I0K. I-V relations were obtained by applying voltage ramps between -105 and 55 mV with a steepness of 0.03 V/s. a and b, Currents were recorded in control conditions (Tyrode's solution) and during exposure to 1 mmol/L Ba2+ () and K+-free solution (), respectively; since the effects of blockers were completely reversible on washout, the tracings shown in panels a and b were obtained from the same cell. I indicates current. c and d, IBa and I0K were obtained by digital subtraction of traces recorded in the presence of the blockers from control traces.

The reversal potential was slightly more positive for I_Ba (-83.1±0.89 mV, n=9) than for I_0K (-85.9±0.82 mV, n=9 [P<0.05 versus I_Ba]). When taking into account the offset introduced by the uncompensated junction potential (5 mV), the reversal potential was for both currents in reasonable agreement with the expected K⁺ equilibrium potential (-94 mV). The maximal conductance, estimated by fitting the linear portion of the I-V relation, was similar between I_Ba (0.65±0.13 nS/pF) and I_0K (0.61±0.13 nS/pF [P=NS versus I_Ba]). However, the maximal outward current, occurring in both cases between -70 and -60 mV, was smaller for I_Ba (2.41±0.23 pA/pF) than for I_0K (3.36±0.32 pA/pF [P<0.05 versus I_Ba]). Overall, these results might be interpreted as Ba²⁺ causing complete blockade of inward I_K1 but only submaximal inhibition of outward I_K1 (see "Discussion"); accordingly, I_K1 during the action potential might be underestimated by I_Ba.

I_Ba and I_0K During the Ventricular Action Potential
I_Ba and I_0K recorded during the ventricular action potential are compared in Figure 3; although with minor differences, both currents had a time course similar to the one predicted by numerical simulations of I_K1.¹⁵ The tracings in Figure 3 were selected from cells with relatively long action potentials, in which the contribution of I_Ks to I_K would be enhanced. Whereas I_Ba progressively increased during the plateau phase, the opposite was true for I_0K; this might suggest that I_K contamination may affect I_Ba and I_0K in opposite directions also under the conditions of AP-clamp measurements. As can be appreciated from the remaining figures, differences between I_0K and I_Ba were less obvious at shorter action potential durations, ie, in the majority of cases.

View larger version (10K): [in this window] [in a new window]   Figure 3. A comparison between IBa and I0K under AP-clamp conditions. a and b, Action potentials recorded from two different cells at a stimulation cycle length of 1 second. c and d, IBa and I0K recorded while applying the action potentials shown in the upper panels. Records were purposely selected to emphasize the differences between IBa and I0K; such differences were subtler in the majority of cells studied. See text for average results.

Although the time courses of I_Ba and I_0K were substantially similar, significant differences emerge from the quantitative analysis of currents during specific phases of the action potential. Average systolic and diastolic current densities and peak current density during fast repolarization are compared between I_Ba and I_0K in the Table. As predicted by the data presented in the previous section, I_0K was almost 2-fold larger than I_Ba over most of the excitation cycle, with the exception of the plateau phase (systolic current). This may be in apparent contrast with the larger I_Ba present at positive membrane potentials during steady-state depolarization (see Figure 2). However, it should be considered that activation of I_Ks, the current likely to account for the difference between I_Ba and I_0K at positive potentials (see above), would be largely incomplete during short-lasting action potentials. The average cell capacitance was 58.7±2.52 pF.

Effect of I_Ca Inhibition on I_Ba and I_0K During the Ventricular Action Potential
To minimize the possibility of effects resulting from ancillary drug properties,¹⁶ two DHPs with widely different potencies were used in assessing the effect of Ca²⁺ channel blockade in separate sets of experiments. Nifedipine and nisoldipine were applied at concentrations of 5 and 0.2 µmol/L, respectively, both sufficient to achieve almost complete blockade of I_CaL,¹⁷ as also verified in the setting of the present study (data not shown). The effect of inhibiting Ca²⁺ influx was also tested by lowering extracellular Ca²⁺ concentration from 2 to 0.5 mmol/L (low Ca²⁺ solution) and replacing it with Mg²⁺ (see "Materials and Methods").

Ca²⁺ channel blockade by either nisoldipine (Figure 4) or nifedipine (Figure 5) resulted in a significant increase of I_Ba. As also apparent from the dynamic I-V relations shown in panel c of each figure, such an effect was restricted to positive potentials, ie, those occurring during the action potential plateau and early repolarization phases; diastolic current and the peak current during fast repolarization were unmodified. Average data from the individual experimental groups are as follows: nisoldipine (n=8, Figure 4d) increased systolic I_Ba by 58.1±21.9% (0.689±0.066 pA/pF versus 0.502±0.082 pA/pF [P<0.05]); diastolic I_Ba (0.865±0.177 pA/pF versus 0.850±0.138 pA/pF [P=NS]) and peak I_Ba (2.756±0.212 pA/pF versus 2.768±0.213 pA/pF [P=NS]) remained unmodified. In the presence of nifedipine (n=11, Figure 5d) systolic I_Ba was increased by 50.7±14.6% (0.767±0.091 pA/pF versus 0.583±0.099 pA/pF [P<0.05]); diastolic I_Ba (0.583±0.106 pA/pF versus 0.559±0.093 pA/pF [P=NS]) and peak I_Ba (2.846±0.311 pA/pF versus 2.885±0.304 pA/pF [P=NS]) were unchanged by nifedipine.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of 0.2 µmol/L nisoldipine (nis) on IBa. a, Membrane potential. b, IBa recorded during the action potential shown in panel a in control conditions and during steady-state superfusion with nis. c, Dynamic I-V relation, obtained by plotting IBa (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) IBa density (see text for definition) recorded in control conditions and in the presence of nis. Average results are from 8 cells. *P<0.05 vs control.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effect of 5.0 µmol/L nifedipine (nif) on IBa. a, Membrane potential. b, IBa recorded during the action potential shown in panel a in control conditions and during steady-state superfusion with nif. c, Dynamic I-V relation, obtained by plotting IBa (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) IBa density (see text for definition) recorded in control conditions and in the presence of nif. Average results are from 11 cells. *P<0.05 vs control.

Whereas nifedipine-induced changes in I_Ba were fully and quickly reversible, the effect of nisoldipine reversed more slowly. Since the effects of the two DHPs were similar and complete washout could be more easily obtained with nifedipine, this drug was chosen to test the effect of Ca²⁺ channel blockade on I_0K (Figure 6).

View larger version (20K): [in this window] [in a new window]   Figure 6. Effect of 5.0 µmol/L nifedipine (nif) on I0K. a, Membrane potential. b, I0K recorded during the action potential shown in panel a in control conditions during steady-state superfusion with nif and after washout (return). c, Dynamic I-V relation, obtained by plotting I0K (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) I0K density (see text for definition) recorded in control conditions and in the presence of nif. Average results are from 6 cells. *P<0.05 vs control.

In the presence of nifedipine, systolic I_0K (n=6, Figure 6d) was reversibly increased by 254.5±66.6% (1.217±0.202 pA/pF versus 0.413±0.088 pA/pF [P<0.05]); diastolic I_0K (1.922±0.463 pA/pF versus 2.071±0.584 pA/pF [P=NS]) and peak I_0K (5.566±0.562 pA/pF versus 5.404±0.617 pA/pF [P=NS]) were not affected by nifedipine. Thus, albeit of larger magnitude, the effect of nifedipine on I_0K was qualitatively comparable to that exerted on I_Ba.

The changes in I_0K induced by a reduction in extracellular Ca²⁺ (Figure 7) were more complex than those of Ca²⁺ channel blockade. An increase of systolic I_0K was consistently observed; however, this was associated with a small reduction of peak I_0K and to relatively large, although inconsistent, variations of diastolic I_0K. In spite of such complexities, on average, the effect of low extracellular Ca²⁺ was consistent with that of Ca²⁺ channel blockade. In the presence of low Ca²⁺ (n=7, Figure 7d), systolic I_0K increased by 108.8±43.1% (0.454±0.072 pA/pF versus 0.255±0.049 pA/pF [P<0.05]). Diastolic I_0K, although obviously reduced in some cells, underwent opposite changes in others; thus, on average, this change did not achieve significance (-36.5±11.8%, 0.682±0.189 pA/pF versus 1.009±0.143 pA/pF [P=NS]). Peak I_0K during fast repolarization was reduced by 8.1±1.9% during low Ca²⁺ superfusion (2.806±0.201 pA/pF versus 3.075±0.244 pA/pF [P<0.05]). The effects of low Ca²⁺ superfusion were readily reversed on washout.

View larger version (20K): [in this window] [in a new window]   Figure 7. Effect of lowering extracellular Ca2+ from 2 mmol/L (control) to 0.5 mmol/L (low Ca2+) on I0K. a, Membrane potential. b, I0K recorded during the action potential shown in panel a in control conditions, during steady-state superfusion with low Ca2+ (), and after washout (return). c, Dynamic I-V relation, obtained by plotting I0K (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) I0K density (see text for definition) recorded in control conditions and in the presence of low Ca2+. Average results are from 7 cells. *P<0.05 vs control.

It can be anticipated that besides reducing K⁺ channel conductance, removal of K⁺_o will also inhibit the Na⁺-K⁺ pump,¹⁸ thus resulting in a reduction of I_NaK and in a progressive dissipation of the Na⁺ transmembrane gradient. A decrease in the Na⁺ gradient, in turn, will reduce the inward component of the current (I_NaCa) generated by the Na⁺-Ca²⁺ exchanger (ie, it will move the reversal potential of I_NaCa in the negative direction). As a consequence, I_0K might contain both I_NaK and I_NaCa; whereas the former would appear as an outward component, contamination by I_NaCa would shift I_0K in the inward direction at all potentials. Although DHP-induced enhancement of systolic I_0K could not be accounted for by an action on I_NaK (see "Discussion"), I_NaCa contamination might be seriously confusing. Indeed, a reduction in intracellular Ca²⁺ might cause I_NaCa to become less inward, thus potentially contributing to the extra outward I_0K appearing on Ca²⁺ channel blockade. To test this hypothesis, we studied the effect of nifedipine on I_0K in the presence of Na⁺-K⁺ pump inhibition by 20 µmol/L ouabain (added to all solutions). Immediately on ouabain superfusion, the compensation current became more inward over the whole cycle, a mirror image of the expected I_NaK time course¹⁵ ; more sustained ouabain superfusion caused the current to become progressively outward until a new, almost stable state was achieved. Under such a condition, further changes in current were minimal over the time required for I_0K measurements (full reversal of the effects of zero-K⁺ superfusion was obtained in each condition; see "Materials and Methods"). In the presence of ouabain (n=10, Figure 8), both systolic (0.18±0.04 pA/pF) and diastolic I_0K (0.36±0.22 pA/pF) were reduced with respect to control (P<0.05). Nifedipine (5 µmol/L) increased systolic I_0K to 0.53±0.071 pA/pF (467.8±157.7% [P<0.05]), a change larger that the one induced in the absence of ouabain (P<0.05, Figure 8d). Whereas, in the presence of ouabain, nifedipine also increased peak I_0K during fast repolarization by 18.1±5.3% (P<0.05), diastolic I_0K was not affected by the drug. Because of the instability generated by ouabain superfusion and the relatively long time required for washout, reversibility of the effects of nifedipine on I_0K could be tested in only 5 of 10 cells. In these cells, 68.9±12.9% of the nifedipine-induced increase of systolic I_0K (377.7±114.9% [P<0.05]) was reversed on washout (example in Figure 8). These experiments show that enhancement of systolic I_0K by Ca²⁺ channel blockade persisted in the presence of ouabain, thus arguing against the possibility that it originated from a change in either I_NaK or I_NaCa.

View larger version (23K): [in this window] [in a new window]   Figure 8. Effect of 5.0 µmol/L nifedipine (nif) on I0K during steady-state superfusion with 20 µmol/L ouabain. a, Membrane potential. b, I0K recorded during the action potential shown in panel a in control conditions, during superfusion with nif, and after washout (return). c, Dynamic I-V relation, obtained by plotting I0K (b) as a function of membrane potential (a). d, Comparison of the effect of nif (cont indicates absence of nif) on average systolic (syst) I0K density (see text for definition) in control conditions (data from Figure 6) and in the presence of ouabain (n=10). Ouabain reduced I0K in all conditions; the I0K change induced by nif was larger in the presence of ouabain than in control (see text for details). *P<0.05 within each experimental group (between-group significance was omitted for clarity). Statistical significance was tested by two-way ANOVA.

Effect of ß-Adrenergic Stimulation on I_0K
If inhibition of Ca²⁺ influx enhanced I_K1 during the action potential plateau phase, the opposite effect should be expected from an increase in Ca²⁺ influx. To test for this hypothesis I_0K was measured during ß-adrenergic stimulation by 0.1 µmol/L isoproterenol, an intervention known to increase I_CaL¹⁹ (Figure 9). Isoproterenol reversibly reduced systolic I_0K from 0.42±0.07 to 0.16±0.06 pA/pF (n=8 [P<0.05]); diastolic current and peak current during fast repolarization were unchanged by the agonist.

View larger version (18K): [in this window] [in a new window]   Figure 9. Effect of ß-adrenergic stimulation on I0K. a, Membrane potential. b, I0K recorded during the action potential shown in panel a in control conditions and during steady-state superfusion with 0.1 µmol/L isoproterenol (iso). c, Dynamic I-V relation, obtained by plotting I0K (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) I0K density recorded in control conditions and in the presence of iso. Average results are from 8 cells. *P<0.05 vs control.

I_Ba and Inhibition of Sarcoplasmic Reticulum Ca²⁺ Release
Rather high subsarcolemmal Ca²⁺ concentrations, on the order of 1 µmol/L, are required to induce I_K1 rectification in cell-excised membrane patches,^{3 5} thus suggesting the involvement of Ca²⁺ released by the sarcoplasmic reticulum. To test whether reticular Ca²⁺ release might contribute to I_K1 rectification, I_Ba was measured before and after sustained (5-minute) superfusion with 1 µmol/L ryanodine, an agent that causes depletion of intracellular Ca²⁺ stores.

Figure 10 shows that ryanodine superfusion was followed by an increase in systolic I_Ba qualitatively and quantitatively comparable to the one induced by Ca²⁺ channel blockade. Average results from five myocytes (Figure 10d) indicate that ryanodine treatment increased systolic I_Ba by 47.7±6.5% (0.51±0.1 pA/pF versus 0.35±0.076 pA/pF [P<0.05]) without altering either diastolic I_Ba (0.73±0.13 pA/pF versus 0.72±0.11 pA/pF [P=NS]) or the current peak during fast repolarization (1.88±0.33 pA/pF versus 1.99±0.32 pA/pF [P=NS]). The effect of ryanodine superfusion was only partially reversible over long recovery periods.

View larger version (20K): [in this window] [in a new window]   Figure 10. Effect of 1.0 µmol/L ryanodine (rya) on IBa. a, Membrane potential. b, IBa recorded during the action potential shown in panel a in control conditions and during steady-state superfusion (5 minutes) with rya. c, Dynamic I-V relation, obtained by plotting IBa (b) as a function of membrane potential (a). d, Comparison of the average systolic (syst) IBa density (see text for definition) recorded in control conditions and in the presence of rya. Average results are from 5 cells. *P<0.05 vs control.

Effect of Intracellular Ca²⁺ Buffering on I_Ba and on Its DHP-Induced Changes
A further proof of the contribution of subsarcolemmal Ca²⁺ to I_K1 rectification would be the observation of an increased systolic I_K1 following strong intracellular Ca²⁺ buffering. We initially performed such a test by removing Ca²⁺ from the pipette solution and replacing 1 mmol/L EGTA with 10 mmol/L BAPTA; although there was a trend to an increase in systolic I_Ba (0.63±0.04 pA/pF [n=7] versus 0.49±0.06 pA/pF [n=25] [P=NS]) the change was not statistically significant. However, such a negative finding might result from inadequate diffusion of BAPTA to subsarcolemmal spaces. Thus, in a further set of experiments, besides removing Ca²⁺ and including 10 mmol/L BAPTA in the pipette, cells were preincubated (>60 minutes) in a solution containing 10 µmol/L BAPTA-AM, a membrane-permeable analogue of BAPTA. In BAPTA-AM–treated cells (Figure 11), diastolic potential was depolarized (-63.7±1.1 versus -73.2±1.1 mV [P<0.05]), plateau potential was unchanged, and action potential duration, although apparently longer in some cells, was not, on average, significantly affected (229.4±25.3 versus 192.0±12.4 milliseconds [P=NS]). Compared with untreated cells, BAPTA-AM–treated cells had a larger diastolic I_Ba (1.04±0.09 versus 0.71±0.07 pA/pF [P<0.05]). The increase in diastolic I_Ba was similar to that measured from steady-state I-V relations of I_Ba (Figure 2c) for a change in membrane potential from -73.2 to -63.7 mV (1.75±0.14 versus 2.42±0.23 pA/pF, n=9); thus, changes in currents other than those reflected by I_Ba should account for the depolarization induced by BAPTA-AM (see above).

View larger version (19K): [in this window] [in a new window]   Figure 11. Effect of intracellular Ca2+ buffering on IBa and on its response to nifedipine (nif). The cell was preincubated with 10 µmol/L BAPTA-AM (Ca2+-free pipette solution containing 10 mmol/L BAPTA). a, Membrane potential. b, IBa recorded during the action potential shown in panel a in control conditions and during steady-state superfusion with 5 µmol/L nif (). c, Dynamic I-V relation, obtained by plotting IBa (b) as a function of membrane potential. d, Comparison between average systolic (syst) IBa density between BAPTA-AM–treated and untreated (intracellular pCa 7) cells. e, Comparison between average syst IBa density, recorded from BAPTA-AM–treated cells, in control conditions and in the presence of 5 µmol/L nif. *P<0.05 vs control.

Consistent with an inhibition of I_K1 by subsarcolemmal Ca²⁺ transients, systolic I_Ba was increased in BAPTA-AM–treated cells (0.92±0.10 pA/pF, n=10 [P<0.05]) (Figure 11d).

In the presence of BAPTA-AM, nifedipine failed to increase average systolic I_Ba (+18.45±7%, n=9 [P=NS]) (Figure 11). Although nifedipine still induced an outward shift of I_Ba, this was limited to the early portion of the plateau phase, when I_Ba was initially smaller; in some cells, as the one shown in Figure 11, late-systolic I_Ba was even reduced below control values by nifedipine (see "Discussion"). Similar to the effect of low extracellular Ca²⁺ on I_0K, nifedipine reduced diastolic I_Ba and peak I_Ba during fast repolarization; however, because of the inhomogeneity of effects among cells, significance was achieved only for the change in diastolic current (0.82±0.08 versus 1.00±0.09 pA/pF, n=9 [P<0.05]).

Dependence of I_Ba Modulation on Intracellular Mg²⁺ Activity
This set of experiments was designed to verify whether the effect of Ca²⁺ channel blockade on I_Ba might be affected by the intracellular levels of Mg²⁺, the other divalent cation known to contribute to physiological inward rectification of I_K1. To this end, I_Ba and its modulation by nifedipine were evaluated in the presence of pipette Mg²⁺ activities of 0.1 mmol/L (n=9) and 1 mmol/L (n=9), respectively. Average results from these experiments are shown in Figure 12. Whereas in control conditions I_Ba was reduced by higher intracellular Mg²⁺, the nifedipine-induced increase in systolic I_Ba was enhanced by the same intervention (Figure 12a). Increasing intracellular Mg²⁺ from 0.1 to 1 mmol/L reduced systolic I_Ba from 0.563±0.088 to 0.347±0.022 pA/pF (P<0.05). Nifedipine increased I_Ba by 0.209±0.026 pA/pF (54.7±16.9% [P<0.05]) in 0.1 mmol/L Mg²⁺ and by 0.313±0.037 pA/pF (93.6±11.8% [P<0.05]) in 1 mmol/L Mg²⁺. Both absolute and percent nifedipine–induced changes of systolic I_Ba were significantly larger in the presence of higher intracellular Mg²⁺ activity. As shown in panels b and c of Figure 12, diastolic and peak I_Ba also tended to be reduced in the presence of higher intracellular Mg²⁺; however, such a change did not achieve statistical significance (see "Discussion").

View larger version (13K): [in this window] [in a new window]   Figure 12. Dependence of IBa, and of its nifedipine (nif)-induced changes, on intracellular Mg2+ activity (, 0.1 mmol/L; , 1 mmol/L). a, Average systolic IBa density. b, Average diastolic IBa density. c, Peak IBa density during fast repolarization. *P<0.05 vs control (within group); P<0.05 vs 1 mmol/L Mg2+ (between groups). In panel a, nif-induced change is larger (P<0.05) in 1 mmol/L than in 0.1 mmol/L Mg2+ (significance not shown for clarity). Statistical significance was tested by two-way ANOVA.

In order to verify whether inhibition of Ca²⁺ influx might also affect I_K1 under conditions preserving an intact intracellular environment, the effects of nifedipine on I_Ba were tested by the perforated-patch technique in 5 cells. Under such conditions, systolic I_Ba was increased from 0.69±0.22 to 1.01±0.23 pA/pF, a change (+71.17±20.5%) similar to the one observed, under ruptured-patch conditions, with 1 mmol/L intracellular Mg²⁺.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study show that inhibition of either Ca²⁺ influx or Ca²⁺ release from the sarcoplasmic reticulum may substantially increase both I_Ba and I_0K during the plateau phase of the guinea pig ventricular action potential. Opposite changes were observed during ß-adrenergic stimulation, probably as a result of enhanced Ca²⁺ influx.

Although I_K1 is the main constituent of I_Ba and I_0K, other channels may contribute, obviously to different extents, to each of the blocker-sensitive currents. Thus, the interpretation of the present findings depends on whether they could be accounted for by changes in conductances other than I_K1.

Do Changes in I_Ba and I_0K Reflect Changes in I_K1?
Ba²⁺-Sensitive Current, I_Ba
At the concentration used, Ba²⁺ may inhibit other K⁺ currents, such as I_K and I_to^{13 20} and, possibly, I_CaL; thus, all these currents might contaminate I_Ba. If I_Ba included I_CaL, block by DHPs might result in an outward shift of I_Ba, possibly similar to the one observed. However, the effect of DHPs on systolic I_Ba was reproduced by ryanodine and intracellular Ca²⁺ buffering (interventions that should not affect I_CaL channels directly) and was qualitatively very similar to the one exerted on I_0K, for which a contamination by I_CaL is very unlikely. On the other hand, both I_to and I_K should be reduced by DHPs as a consequence of either direct channel blockade^{21 22} or reduced intracellular Ca²⁺.^{23 24} Thus, the effects expected from DHP-induced changes in these currents are opposite those observed in I_Ba.

Zero-K⁺–Sensitive Current, I_0K
Removal of K⁺_o may decrease the conductance of all K⁺ channels, inhibit the Na⁺-K⁺ pump, and, secondary to the latter effect, change the Na⁺ gradient driving the Na⁺-Ca²⁺ exchanger (see "Results"). Thus, I_0K putative contaminants include I_to, I_K, I_NaK, and, indirectly, I_NaCa. For what concerns I_to contamination, the same reasoning exposed for I_Ba applies to I_0K. However, decreased Ca²⁺ influx should selectively depress I_Ks, thus possibly inducing a paradoxical increase in I_0K independent of changes in I_K1 (see below).

Either direct or indirect inhibition of I_NaK by DHPs would reduce systolic I_0K, an effect opposite the one observed. Conversely, as already discussed in "Results," I_NaCa contamination might well account for the observed changes. However, the persistence of systolic I_0K enhancement by nifedipine in the presence of a large concentration of ouabain, a condition in which I_0K contamination by both I_NaK and I_NaCa should be largely removed, argues against this interpretation. In the presence of ouabain, Ca²⁺ extrusion through the sarcolemma should be inhibited,²⁵ thus resulting in the persistence of elevated subsarcolemmal Ca²⁺. This might account for the extension of nifedipine-induced enhancement of I_0K to the fast repolarization phase (see Figure 8).

In light of these considerations and of the previously described effects of intracellular Ca²⁺ on the current,^{3 5 7} the most likely explanation of the effects of all the interventions tested on I_Ba and I_0K is a removal of Ca²⁺-induced rectification of I_K1.

Differences Between I_Ba and I_0K and in Their Response to DHPs
During diastole and fast repolarization, I_0K was significantly larger than I_Ba; moreover, although the two systolic currents were similar in control conditions, DHP-induced enhancement was larger for I_0K than for I_Ba. A possible interpretation of these findings is a more complete inhibition of outward I_K1 by K⁺_o removal than by Ba²⁺, whose blocking effect on outward I_K1 appeared to be incomplete. A dependence of blocking potency on the direction of current is a common property of multi-ion pores and has been previously described for blockade of I_K1 by monovalent and divalent cations.^{1 2} The different sensitivity of systolic I_Ba and I_0K to DHPs might reflect selective inhibition of contaminating I_Ks by reduced intracellular Ca²⁺,²³ an effect that might enhance drug-induced increase in I_0K only (see above). Thus, the effect of subsarcolemmal Ca²⁺ on I_K1 might be slightly overestimated by the effect of DHP on I_0K and underestimated by those on I_Ba.

Effects of DHPs in the Presence of Intracellular Ca²⁺ Buffering
The concentrations of intracellular Ca²⁺ reported to inhibit I_K1 in excised membrane patches vary considerably (0.1 to 10 µmol/L at plateau potentials, Mg²⁺-free conditions, 20°C) and may be higher than those measured in the cytoplasm at the peak of physiological Ca²⁺ transients (1 µmol/L).^{3 5 26} However, during electrical activity, Ca²⁺ concentrations in the restricted subsarcolemmal space may exceed those in the bulk cytoplasm. This would be consistent with the evidence suggesting that Ca²⁺-induced rectification may actually be a very localized phenomenon that would require I_K1 channels to be near Ca²⁺ influx and release sites. In chick myocytes, outward elementary I_K1, measured in the cell-attached configuration during a stimulated action potential, was affected by Ca²⁺ concentration in the cell-attached pipette only; the persistence of Ca²⁺ influx through the rest of the cell membrane appeared as irrelevant.⁷ In the present study, diffusion of a Ca²⁺ chelator (BAPTA-AM) through the whole membrane surface was required to increase systolic I_Ba and to blunt its nifedipine-induced changes. Moreover, even in the presence of the chelator, nifedipine still caused an early increase in I_Ba, thus suggesting that at the time of peak Ca²⁺ influx, buffering might have still been incomplete. At this concern, it should also be considered that the effects of Ca²⁺ buffering might have been partly offset by increased Ca²⁺ influx, due to lesser inactivation of I_CaL²⁷ and reverse operation of the Na⁺-Ca²⁺ exchanger.^{28 29} Later on during the course of action potential, I_Ba was even reduced by nifedipine in some cells; this reduction was perhaps due to drug actions unmasked by the removal of Ca²⁺-mediated effects. On the other hand, in the presence of extensive intracellular Ca²⁺ buffering, similar to what observed during exposure to low Ca²⁺ (see Figure 7), the effects of nifedipine on I_Ba were not limited to the plateau phase. Thus, under conditions in which the supply of Ca²⁺ from other sources (intracellular stores, Na⁺-Ca²⁺ exchanger) might also be reduced, the effects of I_CaL blockade on I_Ba might be more complex than usual, and their interpretation might be more difficult.

Dependence of the Effects of DHPs on Free Intracellular Mg²⁺
An increase in intracellular Mg²⁺ from 0.1 to 1 mmol/L was associated with a reduction of systolic I_Ba. The decrease in I_Ba did not reach significance during diastole or fast repolarization (Figure 12b and 12c). However, it should be stressed that the blocking effect of Mg²⁺ may be easier to detect at depolarized potentials³; moreover, smaller effects at diastolic potentials might be overlooked (because of the lower power of the statistics) when internal comparisons are impossible. This finding suggests that Mg²⁺-induced I_K1 rectification might still be incomplete at 0.1 mmol/L, a concentration two orders of magnitude above the K_d measured in cell-excised patches at plateau potentials (3 µmol/L).³ This suggests that Mg²⁺ affinity for I_K1 channels may be affected by experimental conditions, such as cell integrity and temperature.

Inhibition of Ca²⁺ influx had a larger effect on I_Ba at 1 than at 0.1 mmol/L intracellular Mg²⁺. This would be incompatible with the competition of Ca²⁺ and Mg²⁺ ions for a common binding site on the channel. On the other hand, previous data show that Ca²⁺-induced, but not Mg²⁺-induced, I_K1 rectification might be abolished by disruption of the cytoskeleton,³⁰ an observation also consistent with the two cations acting on different sites. As an alternative, the rectifying effect of Ca²⁺ observed in the present study may not represent a simple blockade, such as the one described in Mg²⁺-free conditions,^{3 5} but may be a novel, possibly more complex, effect.

Functional Implications of Ca²⁺-Induced Rectification of I_K1
According to the results of the present study, removal of Ca²⁺-induced rectification may increase systolic I_K1 from 2- to 4-fold, corresponding to increases of 0.2 to 0.8 pA/pF in average systolic current. As shown by the effect of isoproterenol, opposite changes in I_K1 can result from enhancement of Ca²⁺ influx. In ventricular myocytes with a capacitance of 60 pF, membrane input resistances in the order of 30 to 50 M can be measured during the action potential plateau (authors' unpublished data, 1997). Thus, shifts of several millivolts in plateau potentials might conceivably result from changes in the extent of Ca²⁺-induced I_K1 rectification. This may be relevant to the role of Ca²⁺ influx/release in the modulation of action potential duration. For instance, changes in I_K1 might contribute to the marked action potential shortening induced by Ca²⁺ channel blockers or to action potential prolongation often resulting from exposure to catecholamines.³¹

Ca²⁺-induced I_K1 rectification might reduce the amount of inward current necessary to the induction of early afterdepolarizations, thus contributing to the facilitation of these phenomena by catecholamines. Increased subsarcolemmal Ca²⁺ may also lead to enhancement of I_K²³; thus, in normal conditions, the effects of Ca²⁺ on I_K1 may be offset by those on I_K. However, either drug-induced or genetic impairment of I_K conductance^{32 33 34} might unmask the arrhythmogenic potential of the Ca²⁺-induced decrease in I_K1.

Even if Ca²⁺-induced rectification may be important functionally, it represents only a small fraction of total I_K1 rectification. Indeed, on the basis of the conductance measured from the linear portion of the I_0K I-V relation, unrectified I_K1 should amount to >70 pA/pF at plateau potentials compared with <1 pA/pF measured in the presence of Ca²⁺ channel blockade. Thus, the effect of Ca²⁺ observed in the present study may account for <2% of total I_K1 rectification, with the remaining part being attributable to Mg²⁺ and polyamines. Nonetheless, whereas Mg²⁺ and polyamine-induced rectification is present to the same extent during the whole cardiac cycle, Ca²⁺-induced rectification may represent a dynamic phenomenon exquisitely sensitive to modulation. The small proportion of total I_K1 rectification accounted for by Ca²⁺ influx may also explain why this phenomenon could not be observed in single-channel studies on guinea pig ventricular myocytes.³

	Selected Abbreviations and Acronyms

 

AP-clamp = action potential clamp DHP = dihydropyridine I-clamp = current clamp I-V = current-voltage I0K = current sensitive to K+o removal IBa = Ba2+-sensitive current ICa = Ca2+ current ICaL = L-type Ca2+ current IK = delayed rectifier K+ current IK1 = inward rectifier K+ current IKs = slowly activating K+ current INa = Na+ current INaCa = Na+-Ca2+ exchanger current INaK = Na+-K+ pump current Inif = nifedipine-sensitive current Ito = transient outward current K+o = extracellular K+ V-clamp = voltage clamp

	Acknowledgments

 
The present study was partly supported by Telethon grant No. 396 and by Ministero Università Ricerca Scientifica e Tecnologica funding. We are grateful to Dario DiFrancesco and Michele Mazzanti for reading the manuscript and providing constructive criticism and to Gaspare Mostacciuolo for expert technical assistance.

Received November 25, 1997; accepted March 4, 1998.

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