Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes

AbstractArginine 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

ｶｴﾙ心臓八木式灌流法の定量的実験への改良 ―心拍出量測定から心電図同時記録まで―

伝統的な生理学実習項目の一つであるｶｴﾙ心臓の八木式灌流実験法に改良を加え､心室拍動をより定量的に観察記録できるようにした｡すなわち､収縮･拡張ｻｲｸﾙ中の心室の容積変化を気圧変化に変換して捉える簡便な装置(連通標本槽)を考案し､圧変化を拍出量の指標として連続描記できるようにした｡連通標本槽は心室を浸す槽と圧変化を発生する気密な槽とを連結したものである｡この方法により､前負荷の増減に適応する心拍出量の増減､すなわちﾌﾗﾝｸ･ｽﾀｰﾘﾝｸﾞ機構を明確に示すことができる｡また､大動脈内静水圧を増加させる装置を考案した｡これを用いることにより､後負荷増加に対応する心室拍動動態のﾕﾆｰｸな変化を観察できる｡さらに､この実験系における心電図の安定した記録法を考案した｡これにより心臓の機械的活動と電気的活動とを同時に対応させることができる｡われわれの新しい実験方法は､学生実習において学生に心臓の生理学･薬理学的性質を学ばせるために大変有用であろう｡The Yagi\u27s perfusion system has widely been used to study the beats of excised frog heart. Here, we describe a new method to obtain a measure of cardiac output of the frog heart perfused with the Yagi\u27s system. In the new method, the changes in ventricular volume during systole and diastole are converted to the changes in air pressure by using the communicating vessel chamber in which the tissue chamber bathing the ventricle is connected via a communicating tube to the air-tight chamber for pressure detection. With this method, the Frank-Starling mechanism associated with the changes in preload is clearly demonstrable. With an additional apparatus to increase the hydrostatic pressure within the aorta, it is possible to show the unique changes in ventricular dynamics accompanying the increases in afterload. In addition, a method to stably record the ECG simultaneously with the above measurement is described. Our new methods may be useful for the student practice in which students should learn the physiological as well as pharmacological properties of heart

Rapidly and slowly activating components of delayed rectifier K+ current in guinea-pig sino-atrial node pacemaker cells

The components and properties of the delayed rectifier K+ current (IK) in isolated guinea-pig sino-atrial (SA) node pacemaker cells were investigated using the whole-cell configuration of the patch-clamp technique. An envelope of tails test was conducted by applying depolarizing pulses from a holding potential of −50 mV to +30 mV for various durations ranging from 40 to 2000 ms. The ratio of the tail current amplitude elicited upon return to the holding potential to the magnitude of the time-dependent outward current activated during depolarizing steps was dependent on the pulse duration, while after exposure to the selective IKr inhibitor E-4031 (5 μm) this current ratio became practically constant irrespective of the pulse duration. These observations are consistent with the presence of the E-4031-sensitive, rapidly activating and E-4031-resistant, slowly activating components of IK (IKr and IKs, respectively) in guinea-pig SA node cells. The activation range for IKr, defined as the E-4031-sensitive current (half-maximal activation voltage (V1/2) of −26.2 mV) was much more negative than that for IKs, defined as the E-4031-resistant current (V1/2 of +17.2 mV). IKr exhibited a marked inward rectification at potentials positive to −50 mV, whereas IKs showed only a slight rectification. In the current-clamp experiments, bath application of E-4031 (0.5 and 5 μm) initially slowed the repolarization at potentials negative to approximately −30 mV and produced a significant depolarization of the maximum diastolic potential, followed by the arrest of electrical activity, thus indicating that the late phase of the repolarization leading to the maximum diastolic potential at around −60 mV in spontaneous action potentials is primarily produced by IKr in guinea-pig SA node cells. External application of the selective IKs inhibitor 293B (30 μm) also delayed the repolarization process at potentials negative to about −20 mV and induced moderate depolarization of the maximum diastolic potential leading to the arrest of the spontaneous activity. These results provide evidence to suggest that both IKr and IKs are present and play crucial roles in the spontaneous electrical activity of guinea-pig SA node pacemaker cells

A repolarization-induced transient increase in the outward current of the inward rectifier K+ channel in guinea-pig cardiac myocytes

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.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).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.Shift of the depolarizing voltage from > 0 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 μm or lower.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.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) at > 0 mV.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.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.A kinetic model incorporating a competition between polyamine block and Mg2+ block was able to account for the time dependence of outward IKir