Extracellular potassium dependence of the Na⁺-K ⁺-ATPase in cardiac myocytes: Isoform specificity and effect of phospholemman

Cardiac Na⁺-K⁺-ATPase (NKA) regulates intracellular Na⁺, which in turn affects intracellular Ca²⁺ and contractility via the Na⁺/Ca²⁺ exchanger. Extracellular K⁺ concentration ([K⁺]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart. PLM reduces the intracellular Na⁺ affinity of NKA, an effect relieved by PLM phosphorylation. Here we tested whether the NKA α₁- vs. α₂- isoforms have different external K⁺ sensitivity and whether PLM and PKA activation affects the NKA affinity for K⁺ in mouse cardiac myocytes. We measured the external [K⁺] dependence of the pump current generated by the ouabain-resistant NKA isoform in myocytes from wild-type (WT) mice (i.e., current due to NKA-α₁) and mice in which the NKA isoforms have swapped ouabain affinities (α₁ is ouabain sensitive and α₂ is ouabain resistant) to assess current due to NKA-α₂. We found that NKA-α₁ has a higher affinity for external K⁺ than NKA-α₂ [half-maximal pump activation (K_0.5) = 1.5 ± 0.1 vs. 2.9 ± 0.3 mM]. The apparent external K⁺ affinity of NKA was significantly lower in myocytes from WT vs. PLM-knockout mice (K_0.5 = 2.0 ± 0.2 vs. 1.05 ± 0.08 mM). However, PKA activation by isoproterenol (1 μM) did not alter the K_0.5 of NKA for external K⁺ in WT myocytes. We conclude that 1) NKA-α₁ has higher affinity for K ⁺ than NKA-α₂ in cardiac myocytes, 2) PLM decreases the apparent external K⁺ affinity of NKA, and 3) phosphorylation of PLM at the cytosolic domain does not alter apparent extracellular K⁺ affinity of NKA.