Differential distribution and regulation of mouse cardiac Na⁺/K⁺-ATPase α₁ and α₂ subunits in T-tubule and surface sarcolemmal membranes

Objectives: Two Na⁺/K⁺-ATPase (NKA) α-subunit isoforms, α₁ and α₂, are expressed in the adult mouse heart. The subcellular distribution of these isoforms in T-tubule and surface sarcolemmal (SSL) membranes and their regulation by cAMP-dependent protein kinase (PKA) is unclear. Methods: We used formamide-induced detubulation of mouse ventricular myocytes to investigate differential functional distribution and regulation by PKA of α₁ and α₂ in T-tubule versus SSL membranes by measuring NKA current (I_pump) and NKA-mediated Na⁺ efflux (- d[Na]_i/dt). Results: I_pump is composed of 88% α₁-mediated I_pump (Iα₁) and 12% α₂-mediated I_pump (Iα₂). α₁ and α₂ subunits demonstrate distinct ouabain affinities (105 ± 6 and 0.3 ± 0.1 μmol/L respectively) but similar affinity for intracellular Na⁺ (K_1/2Na⁺ of 16.6 ± 0.8 and 16.7±2.6 mmol/L respectively). Detubulation reduced (i) I_pump density (1.42 ± 0.1 to 1.20 ± 0.04 pA/pF), (ii) cell capacitance (181 ± 12 to 127±17 pF), and (iii) Iα₂ contribution (12 to 6%). Total I_pump density was ∼ 60% higher in T-tubule (1.94 pA/pF, derived) vs. SSL membranes. Although T-tubule membranes represent only 30% of total surface area, they generate ∼ 70% of Iα₂ and ∼ 37% of Iα₁. Iα₁ density was substantially higher than Iα₂ in SSL (Iα₁:Iα₂ = 16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 μmol/L) significantly increased α₁-mediated NKA Na⁺ affinity (from 16.6 ± 0.8 to 13.3 ± 1.4 mmol/L) and caused a small increase in maximal NKA Na⁺ efflux rate. ISO had no effect on α₂-mediated NKA activity. Conclusion: These data suggest that NKA α₁ and α₂ subunits are differentially localised and regulated by PKA in T-tubule and SSL membranes and may have distinct regulatory roles in cardiac excitation-contraction coupling.