Stanley Christopher M, Gagnon Dominique G, Bernal Adam, Meyer Dylan J, Rosenthal Joshua J, Artigas Pablo
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas.
Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas; Department of Physics, Texas Tech University, Lubbock, Texas.
Biophys J. 2015 Nov 3;109(9):1852-62. doi: 10.1016/j.bpj.2015.09.015.
Cardiac cells express more than one isoform of the Na, K-ATPase (NKA), the heteromeric enzyme that creates the Na(+) and K(+) gradients across the plasmalemma. Cardiac isozymes contain one catalytic α-subunit isoform (α1, α2, or α3) associated with an auxiliary β-subunit isoform (β1 or β2). Past studies using biochemical approaches have revealed minor kinetic differences between isozymes formed by different α-β isoform combinations; these results make it difficult to understand the physiological requirement for multiple isoforms. In intact cells, however, NKA enzymes operate in a more complex environment, which includes a substantial transmembrane potential. We evaluated the voltage dependence of human cardiac NKA isozymes expressed in Xenopus oocytes, and of native NKA isozymes in rat ventricular myocytes, using normal mammalian physiological concentrations of Na(+)o and K(+)o. We demonstrate that although α1 and α3 pumps are functional at all physiologically relevant voltages, α2β1 pumps and α2β2 pumps are inhibited by ∼75% and ∼95%, respectively, at resting membrane potentials, and only activate appreciably upon depolarization. Furthermore, phospholemman (FXYD1) inhibits pump function without significantly altering the pump's voltage dependence. Our observations provide a simple explanation for the physiological relevance of the α2 subunit (∼20% of total α subunits in rat ventricle): they act as a reserve and are recruited into action for extra pumping during the long-lasting cardiac action potential, where most of the Na(+) entry occurs. This strong voltage dependence of α2 pumps also helps explain how cardiotonic steroids, which block NKA pumps, can be a beneficial treatment for heart failure: by only inhibiting the α2 pumps, they selectively reduce NKA activity during the cardiac action potential, leading to an increase in systolic Ca(2+), due to reduced extrusion through the Na/Ca exchanger, without affecting resting Na(+) and Ca(2+) concentrations.
心脏细胞表达不止一种钠钾ATP酶（NKA）同工型，这种异聚酶可在质膜上形成钠（Na⁺）和钾（K⁺）梯度。心脏同工酶包含一种与辅助β亚基同工型（β1或β2）相关联的催化α亚基同工型（α1、α2或α3）。过去使用生化方法的研究揭示了由不同α-β同工型组合形成的同工酶之间存在微小的动力学差异；这些结果使得理解多种同工型的生理需求变得困难。然而，在完整细胞中，NKA酶在更复杂的环境中发挥作用，其中包括显著的跨膜电位。我们使用正常哺乳动物生理浓度的细胞外钠（Na⁺ₒ）和细胞外钾（K⁺ₒ），评估了非洲爪蟾卵母细胞中表达的人心脏NKA同工酶以及大鼠心室肌细胞中天然NKA同工酶的电压依赖性。我们证明，尽管α1和α3泵在所有生理相关电压下都具有功能，但α2β1泵和α2β2泵在静息膜电位时分别被抑制约75%和约95%，并且仅在去极化时才会明显激活。此外，磷蛋白（FXYD1）抑制泵功能但不会显著改变泵的电压依赖性。我们的观察结果为α2亚基（约占大鼠心室总α亚基的20%）的生理相关性提供了一个简单的解释：它们作为一种储备，在持久的心脏动作电位期间被招募来进行额外的泵浦活动，此时大部分Na⁺进入细胞。α2泵的这种强烈电压依赖性也有助于解释强心甾类药物（可阻断NKA泵）为何可作为心力衰竭的有益治疗方法：通过仅抑制α2泵，它们在心脏动作电位期间选择性地降低NKA活性，由于通过钠/钙交换器的排出减少，导致收缩期Ca²⁺增加，而不影响静息时的Na⁺和Ca²⁺浓度。
