INTRODUCTION

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BARIUM is one of the frequently used divalent cations in patch-clamp studies. In cardiac research, it is probably most well known as a blocker of inward rectifier K⁺ currents (I_K1) (5, 11, 14, 20). Some of the altered electrophysiological properties of the heart, such as membrane depolarization and spontaneous activity induced by Ba²⁺, can be attributed to its ability to inhibit I_K1 (5, 11). As a matter of fact, Ba²⁺ has also been shown to block several other K⁺ channels and currents, including the ATP-sensitive K⁺ current (16), ACh-induced K⁺ current (8), Ca²⁺-activated K⁺ current (15), delayed rectifier K⁺ current (1), cloned Shaker K⁺ channels (7), and M-like K⁺ channels of rod photoreceptors of tiger salamanders (21). Ba²⁺ has actually been a useful probe for exploring the mechanisms of ion conduction (4, 5).

However, whether Ba²⁺ can also block the cardiac transient outward K⁺ current (I_to) is still unclear. Although there is a report indicating that Ba²⁺ blocked I_to in rat ventricular cells at high concentrations (9), no aimed study in this regard has been detailed in the literature. We therefore carried out experiments to investigate specifically the effects of Ba²⁺ on I_to in isolated single canine ventricular and atrial myocytes.

	METHODS

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Cell isolation. Single ventricular and atrial myocytes were isolated from the left ventricular epicardium and the right atrium, respectively, from adult mongrel dogs (20-26 kg) of either sex with previously described Langendorff perfusion techniques (12, 13, 17, 18). The preparation was perfused with Ca²⁺-containing Tyrode solution at 37^°C until the effluent was clear of blood, and the perfusate was then switched to Ca²⁺-free Tyrode solution for 20 min at a constant rate of 12 ml/min, followed by perfusion with the same solution containing collagenase (110 U/ml CLS II collagenase; Worthington Biochemical, Freehold, NJ) and 0.1% BSA (Sigma Chemical, St. Louis, MO). The dispersed cells were stored in Kraftbrühe (KB) medium at 4°C for later electrophysiological experiments.

Patch-clamp recording. Patch-clamp recording techniques used in this study have been described in detail elsewhere (12, 13, 17, 18). Ionic currents were recorded with the whole cell voltage-clamp methods, using an Axopatch 200B amplifier (Axon Instruments, Burlingame, CA). Borosilicate glass electrodes (1 mm outer diameter) had tip resistances of 1-3 M when filled with pipette solution. Junction potentials were zeroed before formation of the membrane-pipette seal in Tyrode solution. Mean seal resistance averaged 15 ± 1 G. Several minutes after seal formation, the membrane was ruptured by gentle suction to establish the whole cell configuration. The capacitance and series resistance were electrically compensated to minimize the duration of the capacitive surge on the current recording and the voltage drop across the clamped cell membrane. Leak currents were linearly subtracted. Cells with changing leak current (indicated by >10-pA changes in holding current at 50 mV) were rejected. Experiments were conducted at 36 ± 1°C unless otherwise specified.

Data analysis. Group data are expressed as means ± SE. Statistical comparisons among groups were performed by ANOVA. If significant effects were indicated by ANOVA, a t-test with Bonferroni correction or a Dunnett's test was used to evaluate the significance of differences between individual means. Otherwise, baseline and drug data were compared by Student's t-test. A two-tailed P < 0.05 was taken to indicate a statistically significant difference. A nonlinear least-squares curve-fitting program (Clampfit in pCLAMP 6.0 or GraphPad Prism) was used to perform curve-fitting procedures.

	RESULTS

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Depolarizing pulses activated a rapidly activating and inactivating outward current, known to be a 4-AP-sensitive I_to, in both ventricular and atrial myocytes. Exposure of the cells to Ba²⁺ markedly diminished the current, and complete reversion of the effects was achieved on washout of Ba²⁺. Figure 1A displays a typical example from a representative ventricular myocyte. Ba²⁺ at a concentration of 100 µM produced ~60% reduction in the current amplitude. Notice that in the absence of Ba²⁺, there is a time-independent outward component, presumably caused by overlapping I_K1. This component was indeed depressed by Ba²⁺.

View larger version (39K): [in this window] [in a new window]   Fig. 1.   Representative experiments showing Ba2+ block of cardiac transient outward K+ current (Ito) and inward rectifier K+ currents (IK1). Ito in canine ventricular (A) or atrial (B) myocytes were activated with voltage protocols shown in inset. Pulse duration was 200 ms, and interpulse interval was 10 s. For the sake of clarity, only current traces of the first 100 ms are shown. C: inward rectifier K+ currents (IK1) elicited by voltage steps ranging from 100 mV to 0 mV from a holding potential of 50 mV. Dashed lines indicate zero current levels. Only current traces obtained at 100 and 60 mV are shown for the sake of clarity and better comparison of voltage dependence of Ba2+ action.

The inhibition of I_to seen with Ba²⁺ could have been confounded by potential contamination caused by concomitant blockade of I_K1. To exclude this possibility, effects of Ba²⁺ on I_to in atrial myocytes were also evaluated because atrial I_K1 is known to have a negligible outward component (20, 23). An example of such experiments is shown in Fig. 1B. Atrial I_to was diminished to the same extent as ventricular I_to by 100 µM Ba²⁺ in the bathing solution. Similar results were obtained from another three atrial myocytes.

Ba²⁺ is known to be a blocker of I_K1. To compare the relative potency of Ba²⁺ on I_to and I_K1, effects of Ba²⁺ on ventricular I_K1 were assessed. The results from a representative experiment are shown in Fig. 1C. Because Ba²⁺ blockade of I_K1 is voltage dependent, currents recorded at test potentials of 100 and 60 mV are displayed for comparison. These two voltages were chosen because an inward current was elicited at 100 mV and the maximum outward I_K1 was seen at 60 mV. As expected, Ba²⁺ at a concentration of 100 µM nearly abolished the inward I_K1 at 100 mV, whereas the effect was considerably attenuated with less negative potentials, in this case at 60 mV (Fig. 1C, right).

To investigate whether the inhibition of I_to by Ba²⁺ was also voltage dependent, mean data of current-voltage relationships of ventricular I_to before and after Ba²⁺ are presented in Fig. 2A. Ba²⁺ blocked I_to equally at various voltages tested without showing voltage dependence (P > 0.05, F-test). For instance, the percent inhibition was 58% at 10 mV and 60% at +40 mV (P > 0.05, t-test). In addition, Ba²⁺ did not alter the inactivation kinetics either. For example, under control conditions, the inactivation time constants calculated by the double-exponential fits to the decaying phase of I_to were 3.8 ± 0.1 and 19.0 ± 2.2 ms for the rapid and slow components, respectively. The same analysis yielded time constants of 3.6 ± 0.3 and 20.7 ± 2.7 ms, respectively, in the presence of 100 µM Ba²⁺.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Mean data on Ba2+ blockade of Ito and IK1. A: current-voltage relationships (I-V curves) of Ito. B: dose-response curves of Ito and IK1 blockade by Ba2+ determined with 3 different voltage protocols. VIto, ventricular Ito; VIK1, ventricular IK1. Symbols represent averaged experimental data [n = 6 for VIto (Ba2+), n = 8 for VIto (4-AP), n = 4 for VIK1] at varying concentrations; lines represent best fits to Hill equation: B = 100/[1 + (IC50/D)nH ], where B is percent change of Ito or IK1 at a drug concentration D, IC50 is Ba2+ concentration that produces 50% inhibition of current, and nH is Hill coefficient. IC50 was determined with a test potential of +50 mV for Ito. A test potential of 100 mV was used to evaluate Ba2+ effects on inward IK1, and a test potential of 60 mV was used to assess inhibition of outward IK1. [Drug], drug concentration.

Concentration-dependent blockade of I_to as well as I_K1 by Ba²⁺ was analyzed using the Hill equation, and the data are shown in Fig. 2B. The IC₅₀ (concentration needed for half-maximal inhibition) for I_to inhibition in ventricular cells was 41.7 ± 1.6 µM (Hill coefficient = 0.6). Whereas Ba²⁺ blocked the inward I_K1 recorded at 100 mV with greater potency, as indicated by the smaller IC₅₀ (2.6 ± 0.6 µM; Hill coefficient = 0.7), a much higher concentration of Ba²⁺ was required to achieve the same degree of block of the outward I_K1 elicited at 60 mV (IC₅₀ = 26.5 ± 2.2 µM; Hill coefficient = 0.8).

4-AP is considered to be a selective blocker of I_to. To compare the relative potency of 4-AP with that of Ba²⁺, additional experiments were performed with serial concentrations of 4-AP. Our results demonstrated that 4-AP was ~10 times less potent (IC₅₀ = 425.4 ± 38.6 µM; Hill coefficient =0.6) than Ba²⁺ in blocking I_to under the same conditions (Fig. 2B). 4-AP at 2 mM completely suppressed the portion of I_to that was not inhibited by 100 µM Ba²⁺, as well as I_Kur.d (data not shown).

It was noticed that, as shown in Fig. 1B, although Ba²⁺ produced significant inhibition of I_to, it left I_Kur.d unaltered in atrial myocytes, as suggested by the lack of change in the sustained component remaining after complete inactivation of I_to. However, the analysis of this current was complicated by the overlapping I_to in these experiments. To clarify this issue, we conducted additional experiments at 22°C. Voltage protocols shown in Fig. 3, inset, activated a rapidly activating and slowly inactivating delayed rectifier current with typi cal characteristics of I_Kur.d (23, 24). Bath application of Ba²⁺ failed to alter I_Kur.d even at elevated concentrations of 1 mM or higher.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Lack of effects of Ba2+ on ultra-rapid delayed rectifier K+ current (IKur.d) in canine atrial myocytes. Voltage protocols are shown in inset. To inactivate Ito, a 200-ms prepulse to +50 mV was added 10 ms before each test pulse. Experiments were conducted at 22°C to provide better resolution of the initial activation phase of the current. Similar results were obtained from a total of 5 cells.

	DISCUSSION

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We have demonstrated in this study that Ba²⁺ produced significant inhibition of I_to, in addition to its well-known effects on I_K1, in cardiac myocytes. Noticeably, the potency of I_to blockade was comparable to inhibition of the outward I_K1 at 60 mV, and I_to was actually more sensitive to Ba²⁺ than to 4-AP. On the other hand, Ba²⁺ did not affect I_Kur.d.

Although Ba²⁺ has been shown to block several types of K⁺ currents (1, 5, 7, 8, 11, 14-16, 20, 21), only one report (9) has presented one example showing the inhibition of rat ventricular I_to by a high concentration of Ba²⁺ (3.6 mM). To date, no aimed and detailed characterization of Ba²⁺ blockade of I_to has yet been documented despite the fact that Ba²⁺ is used so extensively for patch-clamp studies. This report represents the first aimed study of Ba²⁺ blockade of I_to. It is unlikely that the Ba²⁺ block of I_to seen in our experiments was caused simply by surface charge effects because Ba²⁺ did not alter the voltage-dependent steady-state inactivation of I_to (data not shown). In addition, the effects of Ba²⁺ on I_K1 were also consistent with previous reports (5, 11, 14, 20) in terms of the voltage and time dependence. There should have been minimal contamination of I_to measurement by other currents because Na⁺ current, Ca²⁺ current, and ATP-sensitive K⁺ current were suppressed throughout the experiments. In addition, with the concentrations of Ba²⁺ used in our study, I_Kur.d was not affected. Only I_to currents elicited at +50 mV were used for evaluating the concentration-dependent Ba²⁺ effects because at less positive voltages (between 40 and +10 mV) there are outward I_K1 currents, and effects of Ba²⁺ on these currents could complicate the analysis of I_to.

Our finding bears several important implications. An interesting finding is that Ba²⁺ blocks I_to with ~10 times higher potency than 4-AP (IC₅₀ = 40 µM vs. 430 µM for Ba²⁺ vs. 4-AP, respectively), a compound commonly used as a selective I_to blocker (9, 10, 18, 19, 23). This indicates that Ba²⁺ could be used as an I_to blocker in some applications when I_to needs to be removed to minimize overlapping currents. This is particularly true when studying I_Kur.d. To date, there are no pharmacological tools for separating I_Kur.d and I_to from each other because 4-AP at concentrations that block I_Kur.d also significantly inhibit I_to (19, 23). Our finding that Ba²⁺ at 1 mM completely suppresses I_to and I_K1 but does not alter I_Kur.d at all implies that Ba²⁺ provides an ideal tool for studying I_Kur.d in a condition free of contaminating currents.

Moreover, when the potency was compared between I_to and I_K1, it was found that although the inward I_K1 at 100 mV was more sensitive to Ba²⁺ than was I_to, the sensitivity of the outward I_K1 at 60 mV was in the same range as that of I_to. It has been demonstrated that Ba²⁺ could alter cardiac electrical activities such as membrane depolarization and induction of pacemaker activity, changes presumably caused by reduction in outward currents. These Ba²⁺-induced alterations have been interpreted as a result of I_K1 inhibition. Our finding here suggests that Ba²⁺ blockade of I_to may also contribute to the electrophysiological effects of Ba²⁺ in the heart. Future studies are certainly required to test this possibility.

Our finding also suggests that Ba²⁺ can be used to explore the structure-function relationships of A-type K⁺ channels in the light of its ability to block I_to potently. In fact, Ba²⁺ has been used as a probe for investigating the pore properties of Shaker K⁺ channels (6, 7), and two external Ba²⁺ binding sites were identified. It has been strongly suggested that Kv4.2 and Kv4.3 channels are the major components of the native I_to (2, 3), and detailed studies on the interactions of Ba²⁺ with these channels may help us understand mechanisms of Ba²⁺ blockade of I_to. However, whether Ba²⁺ also blocks these channels is still unknown, and future studies are absolutely needed to address this interesting and important issue in detail.

Finally, our study also suggests that caution must be exercised when Ba²⁺ is used to block I_K1 to allow the study of I_to because, as has actually happened in many studies (10, 22), the interpretation and conclusion can be profoundly obscured by the concomitant inhibition of I_to as the result of a significant underestimation of I_to magnitude. Thus the use of Ba²⁺ cannot be easily validated and justified for experiments involving I_to.