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 (Ipump) and NKA-mediated Na+ efflux (−d[Na]i/dt). Results:Ipump is composed of 88% α1-mediated Ipump (Iα1) and 12% α2-mediated Ipump (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+ (K1/2Na+ of 16.6±0.8 and 16.7±2.6 mmol/L respectively). Detubulation reduced (i) Ipump 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 Ipump 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