SUMMARY1. The blockade by Mg2+ and intrinsic gating of the channel, which underlie the rectification of the inward rectifier K+ current, was investigated using the oil-gap voltage clamp method in isolated guinea-pig ventricular cells.2

The strong inward rectifier K + current, I K1 , shows significant outward current amplitude in the voltage range near the reversal potential and thereby causes rapid repolarization at the final phase of cardiac action potentials. However, the mechanism that generates the outward I K1 is not well understood. We recorded currents from the inside-out patches of HEK 293T cells that express the strong inward rectifier K + channel Kir2.1 and studied the blockage of the currents caused by cytoplasmic polyamines, namely, spermine and spermidine. The outward current-voltage (I-V ) relationships of Kir2.1, obtained with 5-10 µM spermine or 10-100 µM spermidine, were similar to the steady-state outward I-V relationship of I K1 , showing a peak at a level that is ∼20 mV more positive than the reversal potential, with a negative slope at more positive voltages. The relationships exhibited a plateau or a double-hump shape with 1 µM spermine/spermidine or 0.1 µM spermine, respectively. In the chord conductancevoltage relationships, there were extra conductances in the positive voltage range, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances, which generated most of the outward currents in the presence of 5-10 µM spermine or 10-100 µM spermidine, were quantitatively explained by a model that considered two populations of Kir2.1 channels, which were blocked by polyamines in either a high-affinity mode (Mode 1 channel) or a low-affinity mode (Mode 2 channel). Analysis of the inward tail currents following test pulses indicated that the relief from the spermine block of Kir2.1 consisted of an exponential component and a virtually instantaneous component. The fractions of the two components nearly agreed with the fractions of the blockages in Mode 1 and Mode 2 calculated by the model. The estimated proportion of Mode 1 channels to total channels was 0.9 with 0.1-10 µM spermine, 0.75 with 1-100 µM spermidine, and between 0.75 and 0.9 when spermine and spermidine coexisted. An interaction of spermine/spermidine with the channel at an intracellular site appeared to modify the equilibrium of the two conformational channel states that allow different modes of blockage. Our results suggest that the outward I K1 is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward I K1 , not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block. The outward current of the strong inward rectifier K + current, I K1 , plays an important role in the cardiac action potential. The outward I K1 is minimal at depolarized voltage levels, which is essential for generating long-lasting action potentials. On the other hand, the amplitude of the outward I K1 is large within the voltage range near the reversal potential (V rev ), and this outward I K1 produces rapid repolarization during the final phase of the cardiac action potentials (Luo & R...

1. The activation kinetics of the IRK1 channel stably expressed in L cells (a murine fibroblast cell line) were studied under the whole-cell voltage clamp. Without polyamines or Mg2+ in the pipettes, inward currents showed an exponential activation on hyperpolarization. The steep inward rectification of the currents around the reversal potential (Erev) could bedescribed by the open-close transition of the channel with first-order kinetics. 2. When the tetravalent organic cation spermine (Spm) was added in the pipettes, the activation kinetics changed; this was explicable by the increase in the closing rate constant.The activation of the currents observed without Spm or Mg2+ in the pipettes was ascribed to the unblocking of the 'endogenous-Spm block'.3. In the presence of the divalent cation putrescine (Put) (Hagiwara, Miyazaki & Rosenthal, 1976;Leech & dependent activation following an instantaneous current Stanfield, 1981;Kurachi, 1985). As the mechanism jump on hyperpolarization. The steep inward rectification accounting for this inward rectification, the block of the around the reversal potential (Erev), and the time-channel by an internal molecule has been proposed dependent property of the currents have been described (Hagiwara & Takahashi, 1974;Hille & Schwarz, 1978). The by the open-close transition of the activation gate, which intracellular cations Mg2+ (Horie,

We evaluated the significance of the host kallikrein-kinin system in tumor angiogenesis and tumor growth using two rodent models genetically deficient in a kallikrein-kinin system. Inoculation of Walker 256 carcinoma cells into the s.c. tissues of the back of normal Brown Norway Kitasato rats (BN-Ki rats) resulted in the rapid development of solid tumors with marked angiogenesis. By contrast, in kininogen-deficient Brown Norway Katholiek rats (BN-Ka rats), which cannot generate intrinsic bradykinin (BK), the weights of the tumors and the extent of angiogenesis were significantly less than those in BN-Ki rats. Daily administration of B(2) receptor antagonists significantly reduced angiogenesis and tumor weights in BN-Ki rats to levels similar to those in BN-Ka rats but did not do so in BN-Ka rats. Angiogenesis and tumor growth were significantly suppressed in B(2) receptor knockout mice bearing sarcoma 180 compared with their wild-type counterparts. Immunoreactive vascular endothelial growth factor (VEGF) was localized in Walker tumor stroma more extensively in BN-Ki rats than in BN-Ka rats, although immunoreactive B(2) receptor also was detected in the stroma to the same extent in both types of rats. Cultured stromal fibroblasts isolated from BN-Ki rats and BN-Ka rats produced VEGF in response to BK (10(-8)-10(-6) m), and this stimulatory effect of BK was abolished with a B(2) receptor antagonist, Hoe140 (10(-5) m). These results suggest that BK generated from kininogens supplied from the host may facilitate tumor-associated angiogenesis and tumor growth by stimulating stromal B(2) signaling to up-regulate VEGF production mainly in fibroblasts.

The outward component of the strong inward rectifier potassium current, I K1 , is significantly larger in ventricles than in atria of the heart, resulting in faster repolarization at the final phase of the action potential in ventricles. However, the underlying mechanism of the difference in I K1 remains poorly understood. I K1 channels are composed of subunits from the Kir2 subfamily, and I K1 amplitude is determined by the voltage-dependent blockade of the channel by the intracellular polyamines spermine and spermidine, and by Mg 2+ . Using a perforated patch-clamp method, which minimizes changes in the intracellular polyamine and Mg 2+concentrations, we detected repolarization-induced outward I K1 transients, which are caused by competition between Mg 2+ and spermine to block the channel, in ventricular but not in atrial myocytes from guinea-pig heart. The contribution of the Kir2.3 subunit to the I K1 channel was found to be minor in the guinea-pig heart, because the activation time course of the Kir2.3 currents was ∼10-fold slower than those of I K1 , and the marked external pH sensitivity of the Kir2.3 currents was not found in I K1 . Both the Kir2.1 and Kir2.2 currents recorded from inside-out patches exhibited outward transients similar to those of ventricular I K1 in the presence of 5-10 µM spermine and 0.6-1.1 mM Mg 2+ , and their amplitudes were diminished by increasing the spermine or spermidine concentrations. The total and free polyamine concentrations in guinea-pig cardiac tissues were higher in atria than ventricles. These results strongly suggest that different intracellular polyamine concentrations are responsible for the difference in atrial and ventricular I K1 of the guinea-pig heart.

No abstract

Outward currents through the inward rectifier K ϩ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg 2 ϩ (0.44-1.1 mM) or putrescine ( ‫ف‬ 0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K ϩ . Without internal Mg 2 ϩ , small outward currents flowed only at potentials between Ϫ 80 (the reversal potential) and ‫ف‬ Ϫ 40 mV during voltage steps applied from Ϫ 110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg 2 ϩ , small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between Ϫ 40 and 0 mV were concurrent with the contribution of Mg 2 ϩ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to Ϫ 50 mV after short depolarizing steps ( Ͼ 0 mV), a transient increase appeared in outward currents at Ϫ 50 mV. Since the peak amplitude depended on the fraction of Mg 2 ϩ -blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg 2 ϩ block, followed by a re-block of channels by spermine. Shift in the holding potential ( Ϫ 110 to Ϫ 80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg 2 ϩ -blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg 2 ϩ . When both spermine (1 M, an estimated free spermine level during whole-cell recordings) and putrescine (300 M) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg 2 ϩ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential. In the heart, large K ϩ conductance mediated by the inward rectifier K ϩ channel, i K1 , maintains the high negative value of the resting potential of ventricular cells and Purkinje fibers (Noble, 1984;Noma et al., 1984;Sakmann and Trube, 1984;Hume and Uehara, 1985). The property of this channel that strongly impedes the flow of outward currents (rectification) is important for the long plateau phase of the cardiac action potential (Hutter and Noble, 1960;Sakmann and Trube, 1984). It has also been suggested that the small outward i K1 currents that flow at potentials near the reversal potential (E rev ) 1 contribute to the final repolarization of the action potential (Giles and Imaizumi, 1988;Ibarra et al...

In heart cells, the catecholamine-activated cyclic AMP system regulates calcium and potassium channels. We report here a novel class of chloride channels that can be activated by adrenaline in mammalian ventricular cells. Like the agonist-activated Cl- channel currents of airway and colonic epithelial cells, the cardiac Cl(-)-channel current shows outward rectification. But the unit conductance of cardiac Cl- channels is smaller than that of epithelial Cl- channels. The cardiac Cl- channel is functionally voltage-independent, in contrast to the Cl- channel in colonic epithelial cells. This channel could be responsible for the beta-catecholamine-induced increase in cardiac membrane conductance that has been attributed to activation of a Cl- current. Thus, sympathetic control of cardiac electrical activity involves not only the voltage-dependent, excitation-related cation channels, but also anion channels that generate a steady current.