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

采用全细胞膜片钳法研究了膜磷脂磷脂酰肌醇4,5 -二磷酸（PtdIns(4,5)P_2）对豚鼠心房细胞延迟整流K^+电流慢激活组分（I_<ks>）的调节作用。通过记录移液管在细胞内施加50 μM wortmannin，在10-15分钟内，I_<ks>逐渐增加到膜片膜破裂后不久的初始幅度的208.5±14.6% (n = 9)，这是通过测量在2秒退极化到+30 mV后返回到-50 mV保持电位时引起的尾电流幅度来评估的。细胞内应用抗ptdins (4,5)P_2单克隆抗体也使I_<ks>尾电流幅值增加到198.4±19.9% （n = 5）。相比之下，外源性PtdIns(4,5)P_2 （100μM）在心房细胞内负荷时，I_<ks>尾电流幅值显著降低至44.8±8.2% （n = 5）。这些结果强烈提示内源性膜PtdIns(4,5)P_2对I_<ks>通道具有有效的抑制作用。细胞内应用新霉素（50 μM）和铝（50 μM）可使I_<ks>通道尾电流幅值分别增加到161.0±13.5% （n = 4）和150.0±8.2% (n = 4)，表明膜PtdIns(4,5)P_2通过与PtdIns(4,5)P_2上带负电荷的头基团静电相互作用抑制I_<ks>通道。外源性PtdIns(4,5)P_2几乎完全消除了50 μM ATP刺激P2Y受体对I_<ks>的增强作用。提示P2Y受体刺激豚鼠心房细胞引起的I_<ks>增强主要参与了P2Y受体刺激引起的膜PtdIns(4,5)P_2的耗竭。

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.