Lu Fang-Min, Hilgemann Donald W
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX.
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
J Gen Physiol. 2017 Jul 3;149(7):727-749. doi: 10.1085/jgp.201711780. Epub 2017 Jun 12.
Decades ago, it was proposed that Na transport in cardiac myocytes is modulated by large changes in cytoplasmic Na concentration within restricted subsarcolemmal spaces. Here, we probe this hypothesis for Na/K pumps by generating constitutive transsarcolemmal Na flux with the Na channel opener veratridine in whole-cell patch-clamp recordings. Using 25 mM Na in the patch pipette, pump currents decay strongly during continuous activation by extracellular K (τ, ∼2 s). In contradiction to depletion hypotheses, the decay becomes stronger when pump currents are decreased by hyperpolarization. Na channel currents are nearly unchanged by pump activity in these conditions, and conversely, continuous Na currents up to 0.5 nA in magnitude have negligible effects on pump currents. These outcomes are even more pronounced using 50 mM Li as a cytoplasmic Na congener. Thus, the Na/K pump current decay reflects mostly an inactivation mechanism that immobilizes Na/K pump charge movements, not cytoplasmic Na depletion. When channel currents are increased beyond 1 nA, models with unrestricted subsarcolemmal diffusion accurately predict current decay (τ ∼15 s) and reversal potential shifts observed for Na, Li, and K currents through Na channels opened by veratridine, as well as for Na, K, Cs, Li, and Cl currents recorded in nystatin-permeabilized myocytes. Ion concentrations in the pipette tip (i.e., access conductance) track without appreciable delay the current changes caused by sarcolemmal ion flux. Importantly, cytoplasmic mixing volumes, calculated from current decay kinetics, increase and decrease as expected with osmolarity changes (τ >30 s). Na/K pump current run-down over 20 min reflects a failure of pumps to recover from inactivation. Simulations reveal that pump inactivation coupled with Na-activated recovery enhances the rapidity and effectivity of Na homeostasis in cardiac myocytes. In conclusion, an autoregulatory mechanism enhances cardiac Na/K pump activity when cytoplasmic Na rises and suppresses pump activity when cytoplasmic Na declines.
几十年前，有人提出心肌细胞中的钠转运是由受限的肌膜下空间内细胞质钠浓度的大幅变化所调节的。在此，我们通过在全细胞膜片钳记录中使用钠通道开放剂藜芦碱产生组成性跨肌膜钠通量，来探究钠钾泵的这一假说。在膜片吸管中使用25 mM钠时，泵电流在细胞外钾持续激活期间强烈衰减（τ，约2秒）。与耗尽假说相反，当泵电流因超极化而减小时，衰减变得更强。在这些条件下，钠通道电流几乎不受泵活性的影响，相反，幅度高达0.5 nA的持续钠电流对泵电流的影响可忽略不计。使用50 mM锂作为细胞质钠类似物时，这些结果更加明显。因此，钠钾泵电流衰减主要反映了一种使钠钾泵电荷运动固定化的失活机制，而非细胞质钠耗尽。当通道电流增加超过1 nA时，具有不受限制的肌膜下扩散的模型准确预测了通过藜芦碱打开的钠通道观察到的电流衰减（τ约15秒）以及钠、锂和钾电流的反转电位变化，以及在制霉菌素通透的心肌细胞中记录到的钠、钾、铯、锂和氯电流。吸管尖端的离子浓度（即接入电导）几乎无延迟地跟踪由肌膜离子通量引起的电流变化。重要的是，根据电流衰减动力学计算的细胞质混合体积随渗透压变化如预期那样增加和减少（τ>30秒）。钠钾泵电流在20分钟内的衰减反映了泵无法从失活状态恢复。模拟结果表明，泵失活与钠激活的恢复相结合可增强心肌细胞中钠稳态的快速性和有效性。总之，一种自动调节机制在细胞质钠升高时增强心脏钠钾泵活性，而在细胞质钠下降时抑制泵活性。
