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The outward component of the strong inward rectifier K+ current (IKir) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward IKir and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current–voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 μm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current–voltage relations showed one peak, like those of native IKir, outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K+] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward IKir. When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers.

The strong inward rectification of the whole cell Kir2.1 current, which is very similar to the cardiac inward rectifier K+ current (IK1), is caused by voltage-dependent blockade of outward currents by the intracellular polyamines spermine and spermidine. We recently showed that macroscopic Kir2.1 currents obtained from inside-out patches in the presence of various concentrations of cytoplasmic polyamines are well explained by the sum of the currents through two populations of channels that show differing susceptibilities to polyamine blockade. The outward currents obtained with 5–10 μm cytoplasmic spermine showed current–voltage relationships similar to those of IK1 and were considered to flow mostly through a small population of channels exhibiting lower spermine sensitivity. Here we used inside-out patches to examine the blockade of macroscopic Kir2.1 currents by cytoplasmic Mg2+ in the absence and presence of cytoplasmic spermine. Outward currents were blocked by 0.6 and 1.1 mm Mg2+ in a concentration-dependent manner, but a small fraction (∼0.1) of the macroscopic conductance was resistant to Mg2+ at those concentrations, suggesting there are two populations of Kir2.1 channels with different sensitivities to Mg2+. Furthermore, at those concentrations, Mg2+ blocked inward currents by inducing a shallow blocked state that differed from the deeper state causing the inward rectification. In the presence of 1.1 mm Mg2++ 5 μm spermine, Mg2+ blocked a substantial current component during depolarizing pulses and generated transient outward components, which is consistent with findings from earlier whole-cell experiments. In the steady state, Mg2+ blocked the currents at voltages around and negative to the reversal potential and induced sustained outward components. The steady-state and time-dependent current amplitudes and the fractional blockades caused by spermine and Mg2+ could be quantitatively explained by a model in which Mg2+ competes with spermine to block the high-affinity channel and induces three conductance states. The present results suggest that the outward IK1 flows through two populations of channels with different sensitivities to cytoplasmic blockers.

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 approximately 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 microM 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.

The strong inward rectifier K+ current, IK1, 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 IK1 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 5–10μm spermine or 10–100μm spermidine, were similar to the steady-state outward I–V relationship of IK1, showing a peak at a level that is ∼20mV 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 1μm spermine/spermidine or 0.1μm spermine, respectively. In the chord conductance–voltage 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 5–10μm spermine or 10–100μm 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 0.1–10μm spermine, 0.75 with 1–100μm 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 IK1 is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward IK1, not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.

Arginine residue at position 285 (R285) in the intracellular C-terminal domain of inward rectifier potassium channel Kir2.2 is conserved in many species, but missing in previously reported human Kir2.2 sequences. We here identified the human Kir2.2 gene in normal individuals, which contained R285 in the deduced amino-acid sequence (hKir2.2/R285). All 30 individuals we examined were homozygous for Kir2.2/R285 gene. The hKir2.2/R285 was electrophysiologically functional in both mammalian cells and Xenopus oocytes. However, the hKir2.2 missing R285 was functional only in Xenopus oocytes, but not in mammalian cells. Thus, R285 in Kir2.2 is important for its functional expression in mammalian cells.

Proinflammatory potency of the nonpeptide bradykinin (BK) B2 receptor agonist FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) was investigated. Intradermal injection of FR190997 (0.03–3 nmol site−1) into dorsal skin of rats increased vascular permeability in a dose-dependent manner. The effect was less than that of BK, but it was long-acting and was inhibited by treatment with FR173657 (3 mg kg−1, p.o.). Captopril (10 mg kg−1, i.p.) did not enhance the plasma extravasation by FR190997 (0.3 nmol site−1) in the presence of soybean trypsin inhibitor (SBTI, 30 μg site−1). Subcutaneous injection of FR190997 (3 nmol site−1) into the hindpaw of mice markedly induced paw swelling. The oedema lasted up to 3 h after the injection. Administration of indomethacin or NS-398 (10 mg kg−1, i.p.) significantly reduced it at 3 h after the injection. Simultaneous i.p. injection of prostaglandin (PG) E2 (1 nmol site−1) or beraprost sodium (0.5 nmol site−1) with FR190997 (5 nmol site−1) greatly enhanced frequency of writhing reactions in mice. FR190997 (0.3–30 nmol kg−1, i.v.) showed less increase in airway opening pressure (Pao) in the guinea-pig after i.v. injection. Furthermore, FR190997 (0.03–30 nmol) resulted in a very weak contraction of tracheal ring strips and lung parenchymal sections in vitro. In mice sponge implants, topical application of FR190997 increased angiogenesis and granulation with enhanced expressions of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNAs. These results indicate that FR190997 has proinflammatory long-lasting characteristics and it might be ‘a stable tool’ for studying the role of BK B2 receptor in vivo. British Journal of Pharmacology (2001) 133, 1296–1306; doi:10.1038/sj.bjp.0704208

1. Outward currents of the inwardly rectifying K+ current (IKir) in guinea-pig ventricular myocytes were studied in the presence of 1 mM intracellular free Mg2+ using the whole-cell patch-clamp technique. 2. During repolarizing voltage steps following a large depolarizing pulse (0 mV), outward IKir increased transiently at voltages positive to the K+ equilibrium potential (EK, -84 mV for 5.4 mM extracellular [K+]). The rising phase was almost instantaneous, while the decay was exponential. The decay rate was faster at voltages closer to EK (time constants, 33.9 +/- 9.8 and 4.8 +/- 1.4 ms at -30 and -50 mV, respectively). 3. The transient outward IKir was absent when the preceding depolarization was applied from -40 mV. Larger transient currents developed as the voltage before the depolarization was shifted to more hyperpolarized levels. 4. Shift of the depolarizing voltage from0 mV to more negative ranges diminished the amplitudes of transient outward IKir and instantaneous inward IKir during the subsequent repolarizing steps positive and negative to EK, respectively. Since blockage of IKir by internal Mg2+ occurs upon large depolarization, and the block is instantaneously relieved at voltages negative to EK, the rising phase of the transient outward IKir was attributed to the relief of Mg2+ block at voltages positive to EK. Transient outward IKir was absent when intracellular [Mg2+] was reduced to 10 microM or lower. 5. Prolongation of the repolarizing voltage step increased the amplitude of time-dependent inward IKir during the subsequent hyperpolarization, indicating the progress of a gating process (presumably the channel block by intracellular polyamine) during the decaying phase of outward IKir. 6. Progressive prolongation of the depolarizing pulse (0 mV) from 100 to 460 ms decreased the transient outward IKir amplitude during the subsequent repolarizing step due to slow progress of the gating (polyamine block) at0 mV. 7. Current-voltage relations measured using repolarizing ramp pulses (-3.4 mV ms-1) showed an outward hump at around -50 mV, the magnitude of which increased as the voltage before the conditioning depolarization (10 mV) was shifted to more negative levels. With slower ramp speeds (-1.5 and -0.6 mV ms-1), the hump was depressed at voltages near EK. 8. Our study suggests that the relief of Mg2+ block may increase outward IKir during repolarization of cardiac action potentials, and that the resting potential, the level/duration of action potential plateau and the speed of repolarization influence the outward IKir amplitude. 9. A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir.

Platelet-activating factor (PAF) is considered an important mediator in allergic and inflammatory reactions. To further investigate the role of PAF in allergic diseases, we examined the in vitro and ex vivo effect of anti-allergic drugs on PAF production by human neutrophils. Ketotifen and three Kampo medicines, which are widely used in the treatment of allergic diseases in Japan, inhibited PAF production by normal human neutrophils dose-dependently, whereas disodium chromoglycate (DSCG) and tranilast did not have any inhibitory effect. Ketotifen also suppressed ex vivo PAF production by neutrophils from healthy subjects. PAF production decreased from 40.5 +/- 5.0 units to 21.6 +/- 4.7 units after oral administration of 2 mg of ketotifen per day for one week. We also measured the concentration of lysoPAF in serum from patients with Japanese cedar pollinosis during the pollen season and followed the effect of ketotifen on lysoPAF levels in the serum from these patients. We found that the concentration of lysoPAF, a precursor and degradation product of PAF, in patients with cedar pollinosis (87.8 +/- 8.7 units) was significantly higher than in healthy subjects (54.7 +/- 7.7 units). After two weeks, lysoPAF levels decreased significantly (82.6 units to 41.3 units) only in the group treated with nasal aerosol and ketotifen. These results suggest that PAF may play a role in allergic diseases and that the clinical effects of anti-allergic drugs may at least partially be due to their anti-PAF action.

The inward rectifier K+ currents play an important role in cardiac repolarization. We have studied the blockage of Kir2 channels by polyamines and Mg2+ in detail to clarify the precise mechanisms that determine the voltage- and time-dependences of the cardiac strong inward rectifier K+ current, IK1, during repolarization. Our studies suggested that a large portion of the time-independent component of IK1, which flows at membrane potentials near the resting potential, is carried by a small (7–10%) conductance susceptible only to the low-affinity mode of blockage by intracellular polyamines. An “IK1 transient” caused by the relief of the Mg2+ block of IK1 during early phase 3 appeared to flow through the conductance susceptible to the high-affinity mode of the polyamine block. The fractional conductances showing distinct sensitivities to the polyamine blockage varied in the presence of different concentrations of polyamines, suggesting that IK1 is modulated by intracellular polyamines in a complicated manner. The D172N mutation in the transmembrane pore region of Kir2.1, which has been proposed to increase IK1, thereby causing the short QT syndrome, made almost all of the channel conductance susceptible only to the low-affinity block. Our study suggests that modulation of the fractions of IK1 exhibiting distinct sensitivities to the polyamine/Mg2+ block may be a potential pharmacological target for treating abnormal cardiac repolarization.