Regulation of the cardiac delayed rectifier K^+ channel by membrane phosphoinositides

应用<ks>全细胞膜片钳技术，观察了膜磷脂磷脂酰肌醇4，5-二磷酸（PtdIns（4，5）P_2）对豚鼠心房肌细胞延迟整流钾电流（I_2）慢激活成分的调节作用。通过记录移液管在细胞内应用50 μM渥曼青霉素引起I_2在<ks>10-15分钟内逐渐增加到初始幅度的208.5±14.6%（n = 9），当通过测量在2秒去极化到+30 mV后返回到-50 mV的保持电位时引起的尾电流幅度来评估时，在膜片膜破裂后不久获得。胞内应用抗PtdIns（4，5）P_2单克隆抗体也使尾电流幅值<ks>增加198.4±19.9%（n = 5）。而用外源性PtdIns（4，5）P_2（100μM）负荷心房细胞，则使I_2尾电流幅值显著下降<ks>，为44.8±8.2%（n = 5）。这些结果表明，内源性膜PtdIns（4，5）P_2对I_2通道有明显的抑制作用<ks>。细胞内应用新霉素（50 μM）或铝（50 μM）可使尾电流幅值分别<ks>增加161.0±13.5%（n = 4）和150.0±8.2%（n = 4），表明<ks>膜PtdIns（4，5）P_2通过与PtdIns（4，5）P_2上带负电荷的头基的静电相互作用抑制I_2通道。外源<ks>性PtdIns（4，5）P_2几乎完全抑制50 μM ATP刺激P_2Y受体引起的I_（10）增加。这一结果表明，PtdIns（4，5）P_2的耗竭主要参与了<ks>刺激P_2Y受体引起的豚鼠心房细胞I_2的增强。

Regulation of the slowly activating component of delayed rectifier K^+ current (I_<ks>) by the membrane phospholipid phosphatidylinositol 4, 5-bisphosphate (PtdIns(4, 5)P_2) was examined in guinea-pig atrial cells using whole-cell patch-clamp method. Intracellular application of 50 μM wortmannin through a recording pipette evoked progressive increase in I_<ks> over a 10-15 min to 208.5±14.6% (n = 9) of initial magnitude obtained shortly after rupture of the patch membrane, when assessed by measuring the amplitude of tail current elicited upon return to a holding potential of -50 mV following a 2-s depolarization to +30 mV. Intracellular application of anti-PtdIns(4, 5)P_2 monoclonal antibodies also increased the amplitude of I_<ks> tail current to 198.4±19.9% (n = 5). In contrast, intracellular loading of atrial cells with exogenous PtdIns(4, 5)P_2 (100μM) produced a marked decrease in the amplitude of I_<ks> tail current to 44.8±8.2% (n = 5). These results strongly suggest that endogenous membrane PtdIns(4, 5)P_2 produces a potent inhibitory action on I_<ks> channels. Intracellular application of neomycin (50 μM) or aluminum (50 μM) evoked an increase in the amplitude of I_<ks> tail current to 161.0±13.5% (n = 4) and 150.0±8.2% (n = 4), respectively, which suggests that I_<ks> channel is inhibited by membrane PtdIns(4, 5)P_2 through electrostatic interactions with negatively charged head group on PtdIns(4, 5)P_2. Enhancement of I_<ks> by P2Y receptor stimulation with 50 μM ATP was almost totally abolished by exogenously applied PtdIns(4, 5)P_2. This result suggests that depletion of membrane PtdIns(4, 5)P_2 is primarily involved in an enhancement of I_<ks> evoked by stimulation of P2Y receptor in guinea-pig atrial cells.